Compositions, systems, and methods for epigenetic regulation of lipoprotein a (LPA) gene expression

Abstract

Provided in some aspects are epigenetic -modifying DNA-targeting systems, such as systems containing a CRISPR-Cas / guide RNA (gRNA) DNA-binding domain and a transcriptional repressor domain, for the transcriptional repression of Lipoprotein(a) (Lp(a)). In some embodiments, the systems can be multiplexed systems for additional transcriptional repression of proprotein convertase subtilisin / kexin type 9 (PCSK9). Also provided herein are methods and uses related to the provided epigenetic -modifying DNA targeting systems in connection with treatments for cardiovascular disease.

Description

Attorney No. 224742004040 COMPOSITIONS, SYSTEMS, AND METHODS FOR EPIGENETIC REGULATION OF LIPOPROTEIN A (LPA) GENE EXPRESSIONCross-Reference to Related Applications

[0001] This application claims priority from U. S. Provisional Application No. 63 / 749, 965 filed January 27, 2025, U. S. Provisional Application No. 63 / 799,289 filed May 02, 2025, and U. S. Provisional Application No. 63 / 886,065 filed September 22, 2025, the contents of each are incorporated by reference in their entireties.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 224742004040SeqList.xml, created January 26, 2026, which is 728,368 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to epigenetic-modifying DNA-targeting systems, such as systems containing a CRISPR-Cas / guide RNA (gRNA) DNA-binding domain and a transcriptional repressor domain, for the transcriptional repression of Lipoprotein(a) (Lp(a)). In some embodiments, the systems can be multiplexed systems for additional transcriptional repression of proprotein convertase subtilisin / kexin type 9 (PCSK9). In some aspects, the present disclosure also provides methods and uses related to the provided epigenetic-modifying DNA targeting systems in connection with treatments for cardiovascular disease.Background

[0004] Lipoprotein(a) (Lp(a)) is a risk factor for heart disease, including atherosclerotic cardiovascular disease (ASCVD) and calcific aortic valve stenosis. Lp(a) is a lipoprotein that can accumulate to form arterial plaques and increases inflammation and calcification in heart cells. There is a dearth of effective treatment options for lowering Lp(a) and many current treatments for cardiovascular disease, including administration of statins and antibody therapy, face challenges including toxicity and high cost associated with repeated drug administration. There is a need for new and improved methods to overcome these challenges. The present disclosure addresses these and other needs.1MF-366426018Attorney No. 224742004040 Summary

[0005] In some aspects, provided herein is a plurality of polynucleotides encoding an epigenetic-modifying DNA-targeting system comprising: a) a polynucleotide encoding a fusion protein comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional repressor domain; and b) at least one gRNA that targets a target site of Lp(a) located between the (i) hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782; and / or (ii) the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973In some of any of the provided embodiments, the plurality of polynucleotides further comprise: c) at least one gRNA that targets a target site of PCSK9 located within 500bp of the hg38 genomic coordinate chr1:55,039,548. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,800 and chr6: 160,679,650. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,280 and chr6: 160,679,602.

[0006] In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,000 and chr6: 160,679,350. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 000-160,679,200; chr6: 160, 679, 200-160,679,400; chr6:160, 679, 400-160,679,600; or chr6:160, 679, 500-160,679,700.

[0007] In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35,256-258, and 418-424, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35,256-258, and 418-424. In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 258, and 418-424, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 258, and 418-424. In some of any of the provided embodiments, the target site forLp(a) is located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973.2MF-366426018Attorney No. 224742004040

[0008] In some of any of the provided embodiments, the target site for PCSK9 is located within 110 bp of the hg38 genomic coordinate chr1:55,039,548. In some of any of the provided embodiments, the target site for PCSK9 is within the coordinates chr1: 55,039,438-55,039,658. In some of any of the provided embodiments, the target site for PCSK9 is located within 80 bp of the hg38 genomic coordinate chr1: 55,039,548. In some of any of the provided embodiments, the target site for PCSK9 is within the coordinates chr1: 55,039,470-55,039,597.

[0009] In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58 or 89-100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58, 89-100, and 409-412, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58, 89-100, and 409-412. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs:: 48, 49, 52 and 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs:: 48, 49, 52 and 89. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58 or 89-10. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48.

[0010] In some of any of the provided embodiments, the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas protein is a dCas12 protein.

[0011] In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 126, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 is set forth in SEQ ID NO: 126.3MF-366426018Attorney No. 224742004040

[0012] In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 127. In some of any of the provided embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 is set forth in SEQ ID NO: 128.

[0013] In some of any of the provided embodiments, each gRNA comprises a gRNA spacer sequence that is complementary to the target site of the respective gene. In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40 or 253-255, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40,253-255, 417, and 425-430, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40,253-255, 417, and 425-430. In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 255, 417, and 425-430.

[0014] In some of any of the provided embodiments, the at least one gRNA that targets a target site of PCSK9 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS:59-71 or 101-112, or a contiguous portion thereof of at least 14 nt; optionally wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:61 or a contiguous portion of at least 14 nt. In some of any of the provided embodiments, the at least one gRNA that targets a target site of PCSK9 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 59-71, 101-112, and 413-416, or a contiguous portion thereof of at least 14 nt; optionally wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:61 or a contiguous portion of at least 14 nt. In some of any of the provided embodiments, the at least one gRNA that targets a target site of PCSK9 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 59-71, 101-112, and 413-416, optionally wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:61.. In some of any of the provided embodiments, the at least one gRNA that targets a target site of PCSK9 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 61, 62, 65 and 101.

[0015] In some of any of the provided embodiments, each gRNA independently comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided 4MF-366426018Attorney No. 224742004040 embodiments, each gRNA independently comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0016] In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 36-40 or 253-255. In some of any of the provided embodiments, the at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 59-71 or 101-112; optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:61.

[0017] In some of any of the provided embodiments, each gRNA further comprises a scaffold sequence set forth in SEQ ID NO:85. In some of any of the provided embodiments, each gRNA further comprises a scaffold sequence set forth in SEQ ID NO:85 or SEQ ID NO: 259. In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) comprises the sequence set forth in any one of SEQ ID NOS:41-45 or 250-252. In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) comprises the sequence set forth in any one of SEQ ID NOS:41-45, 247-252, and 439-449. In some of any of the provided embodiments, at least one gRNA that targets a target site of PCSK9 comprises the sequence set forth in any one of SEQ ID NOS:72-84 or 113-124; optionally wherein gRNA comprises the sequence set forth in SEQ ID NO: 74. In some of any of the provided embodiments, at least one gRNA that targets a target site of PCSK9 comprises the sequence set forth in any one of SEQ ID NOS: 72-84, 113-124, and 431-438; optionally wherein gRNA comprises the sequence set forth in SEQ ID NO: 74 or 431. In some of any of the provided embodiments, the at least one gRNA that targets a target site of Lp(a) is set forth in any one of SEQ ID NOS:41-45 or 250-252. In some of any of the provided embodiments, the at least one gRNA that targets a target site of PCSK9 is set forth in any one of SEQ ID NOS: 72-84 or 113-124, optionally wherein the gRNA is set forth in SEQ ID NO:74. In some of any of the provided embodiments, at least one gRNA comprises modified nucleotides for increased stability.

[0018] In some of any of the provided embodiments, the at least one transcriptional repressor domain is capable of reducing transcription of Lp(a). In some of any of the provided embodiments, the at least one transcriptional repressor domain is capable of reducing transcription of PCSK9.

[0019] In some of any of the provided embodiments, the at least one transcriptional repressor domain is a single transcriptional repressor domain. In some of any of the provided embodiments, the single transcriptional repressor domain comprises a catalytically inactive DNA methyltransferase domain or a functional portion thereof. In some of any of the provided embodiments, the catalytically inactive DNA methyltransferase domain or a functional portion thereof is capable of recruiting domains with DNA methyltransferase activity. In some of any of the provided embodiments, the length of the single transcriptional repressor domain is less than 750 amino acids, 600 amino acids, 510 amino acids, 400 amino acids, or 300 amino in length. In some of any of the provided embodiments, the single5MF-366426018Attorney No. 224742004040 transcriptional repressor domain comprises a DNMT3L domain or functional portion thereof. In some of any of the provided embodiments, the DNMT3L domain or a functional portion thereof is the only transcriptional repressor domain present. In some of any of the provided embodiments, the single transcriptional repressor domain consists of a DNMT3L domain or functional portion thereof. In some of any of the provided embodiments, the polynucleotides are devoid of any additional transcriptional repressor domains, including but not limited to DNMT3 A, KRAB domains, SID domains, or other heterochromatin-inducing domains.

[0020] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a Krüppel-associated box (KRAB) domain. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase (DNMT).

[0021] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a histone methyltransferase, or a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase and a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase and a histone methyltransferase. In some of any of the provided embodiments, one or more of the at least one transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3 A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3 A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxil repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a KRAB domain, a DNMT3 A domain, a DNMT3L domain, or a combination of any of the foregoing.

[0022] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 130, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0023] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNMT3 A domain or a variant or portion thereof that exhibits transcriptional 6MF-366426018Attorney No. 224742004040 repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 132, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0024] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNMT3L domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 134, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0025] In some of any of the provided embodiments, the at least one transcriptional repressor domain is a DNMT3 A-DNMT3L fusion protein domain or a variant thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 136 or SEQ ID NO: 138, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0026] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a sequence selected from any one of SEQ ID NOS: 130, 132, 134, 136, and 138-146, or a domain thereof, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0027] In some of any of the provided embodiments, the at least one transcriptional repressor domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain. In some of any of the provided embodiments, the fusion protein further comprises one or more nuclear localization signals (NLS). In some of any of the provided embodiments, the fusion protein further comprises one or more linkers connecting two or more of: the DNA-binding domain, the at least one transcriptional repressor domain, and the one or more nuclear localization signals.

[0028] In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in any one of SEQ ID NOS: 88, 148, 150, or 152, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, optionally wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 150.

[0029] In some of any of the provided embodiments, the polynucleotide encoding the fusion protein is mRNA. In some of any of the provided embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 156, or a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. In some of any of the provided embodiments, the mRNA has the sequence is set forth in SEQ ID NO: 153.7MF-366426018Attorney No.224742004040

[0030] In some aspects, provided herein is an epigenetic -modifying DNA-targeting system for repressing transcription of Lipoprotein(a) (Lp(a)), comprising a fusion protein comprising: (a) a DNA-binding domain for targeting to a target site of Lp(a), wherein the target site for Lp(a) is located between the (i) hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782; and / or (ii) the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973, and (b) at least one transcriptional repressor domain.

[0031] In some of any of the provided embodiments, the DNA-targeting system does not introduce a genetic disruption or a DNA break. In some of any of the provided embodiments, the DNA-binding domain is selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I- Seel enzyme or a variant thereof, optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing. In some of any of the provided embodiments, the DNA-binding domain is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and the system further comprises a gRNA for targeting the DNA-binding domain to a target site of Lp(a).

[0032] In some aspects, provided herein is an epigenetic -modifying DNA-targeting system for repressing transcription of Lipoprotein(a) (Lp(a)), comprising: (a) a fusion protein comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional repressor domain; and (b) a gRNA that targets a target site of Lp(a), wherein the target site for Lp(a) is located between the (i) hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782; and / or (ii) the hg38 genomic coordinates club: 160,519,674 and club:160,520,973.

[0033] In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,000 and chr6: 160,679,350. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 000-160,679,200; chr6: 160, 679, 200-160,679,400; chr6:160, 679, 400-160,679,600; or chr6:160, 679, 500-160,679,700.

[0034] In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35.8MF-366426018Attorney No. 224742004040

[0035] In some of any of the provided embodiments, the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas protein is a dCas12 protein.

[0036] In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 126, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 is set forth in SEQ ID NO: 126.

[0037] In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 127. In some of any of the provided embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 is set forth in SEQ ID NO: 128.

[0038] In some of any of the provided embodiments, the gRNA comprises a gRNA spacer that is complementary to the target site of the gene. In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments, the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0039] In some of any of the provided embodiments, the gRNA comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 36-40. In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:85. In some of any of the provided embodiments, the gRNA comprises the sequence set forth in any one of SEQ ID NOS:41-45. In some of any of the provided embodiments, the gRNA is set forth in any one of SEQ ID NOS:41-45. In some of any of the provided embodiments, the gRNA comprises modified nucleotides for increased stability.

[0040] In some of any of the provided embodiments, the at least one transcriptional repressor domain is capable of reducing transcription of Lp(a). In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a Krüppel-associated box (KRAB) domain. In some9MF-366426018Attorney No. 224742004040 of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase (DNMT).

[0041] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a histone methyltransferase, or a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase and a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase and a histone methyltransferase.

[0042] In some of any of the provided embodiments, the transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3 A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3 A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxil repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing. In some of any of the provided embodiments, the transcriptional repressor domain is a KRAB domain, a DNMT3 A domain, a DNMT3L domain, or a combination of any of the foregoing.

[0043] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 130, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0044] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNMT3 A domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 132, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0045] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNMT3L domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 134, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.10MF-366426018Attorney No.224742004040

[0046] In some of any of the provided embodiments, the at least one transcriptional repressor domain is a DNMT3 A-DNMT3L fusion protein domain or a variant thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 136 or SEQ ID NO: 138, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0047] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a sequence selected from any one of SEQ ID NOS: 130, 132, 134, 136, and 138-146, or a domain thereof, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some of any of the provided embodiments, the at least one transcriptional repressor domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain.

[0048] In some of any of the provided embodiments, the fusion protein further comprises one or more nuclear localization signals (NLS). In some of any of the provided embodiments, the fusion protein further comprises one or more linkers connecting two or more of: the DNA-binding domain, the at least one transcriptional repressor domain, and the one or more nuclear localization signals. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in any one of SEQ ID NOS: 88, 148, 150, or 152, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, optionally wherein the fusion protein is set forth in SEQ ID NO: 150.

[0049] In some of any of the provided embodiments, transcription of Lp(a) is repressed by a log2 fold-change of at or lesser than -1.0. In some of any of the provided embodiments, repressed transcription of Lp(a) in a cell or population of cells leads to a reduction of Lp(a). In some of any of the provided embodiments, in some of any of the provided embodiments, repressed transcription of Lp(a) in a cell or population of cells leads to a reduction of low-density lipoprotein (LDL), optionally wherein the reduction of LDL occurs extracellularly. In some of any of the provided embodiments, the cell or population of cells is a liver cell or comprises liver cells. In some of any of the provided embodiments, the cell or population of cells is in a subject. In some of any of the provided embodiments, the reduction of LDL occurs in the subject or a fluid, tissue, or organ thereof. In some of any of the provided embodiments, the reduction of LDL occurs in the blood of a subject.

[0050] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising a plurality of DNA-targeting modules for repressing transcription of a plurality of genes, wherein the plurality of DNA-targeting modules comprises: (a) a first DNA-targeting module for repressing transcription of Lipoprotein (a) (Lp(a)), wherein the first DNA-targeting module comprises (i) a fusion protein comprising a DNA-binding domain for targeting to a target site for Lp(a), wherein the 11MF-366426018Attorney No.224742004040 target site for Lp(a) is located between the (A) hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782; and / or (B) the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973, and (ii) at least one transcriptional repressor domain, and (b) a second DNA-targeting module for repressing transcription of Proprotein convertase subtilisin / kexin type 9 (PCSK9), wherein the second DNA-targeting module comprises (i) a fusion protein comprising a DNA-binding domain for targeting to a target site for PCSK9, wherein the target site for PCSK9 is located within 500bp of the hg38 genomic coordinate chrl:55,039,54, and (ii) at least one transcriptional repressor domain.

[0051] In some of any of the provided embodiments, the DNA-targeting system does not introduce a genetic disruption or a DNA break. In some of any of the provided embodiments, the fusion protein of each DNA-targeting module comprises a DNA-binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof, optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing.

[0052] In some of any of the provided embodiments, the first and second DNA-targeting modules comprise the same fusion protein. In some of any of the provided embodiments, the first and second DNA-targeting modules comprise different fusion proteins.

[0053] In some of any of the provided embodiments, the first DNA-targeting module comprises a first targeting polynucleotide for targeting to the target site of Lp(a) and the second DNA-targeting module comprises a second targeting polynucleotide for targeting to the target site of PCSK9, wherein the first and second targeting polynucleotides complex with the DNA-binding domain of the fusion protein. In some of any of the provided embodiments, the DNA-binding domain is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and the first and second targeting polynucleotides comprise a first gRNA and a second gRNA, respectively.

[0054] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system for repressing transcription of Lipoprotein (a) (Lp(a)) and Proprotein convertase subtilisin / kexin type 9 (PCSK9) comprising: (a) a fusion protein comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional repressor domain; (b) at least a first gRNA that targets a target site of LP(a) located between (i) the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782; and / or (ii) the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973; and (c) at least a second gRNA that targets a target site of PCSK9 located within 500bp of the hg38 genomic coordinate chr1:55,039,54.

[0055] In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160,12MF-366426018Attorney No. 224742004040 679,000 and chr6: 160,679,350. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 000-160,679,200; chr6: 160, 679, 200-160,679,400; chr6: 160, 679, 400-160,679,600; or chr6: 160, 679, 500-160,679,700.

[0056] In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35 or 256-258, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58 or 89-100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0057] In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35 or 256-258. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58 or 89-10. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48.

[0058] In some of any of the provided embodiments, the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas protein is a dCas12 protein.

[0059] In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 126, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 is set forth in SEQ ID NO: 126.

[0060] In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10 A and H840A, with reference to numbering of positions of SEQ ID NO: 127. In some of any of the provided embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%,13MF-366426018Attorney No. 224742004040 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 is set forth in SEQ ID NO: 128.

[0061] In some of any of the provided embodiments, each gRNA comprises a gRNA spacer sequence that is complementary to the target site of the respective gene. In some of any of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of: (a) a gRNA targeting a target site of Lp(a) comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40 or 253-255, or a contiguous portion thereof of at least 14 nt; and (b) a gRNA targeting a target site of PCSK9 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS:59-71 or 101-112, or a contiguous portion thereof of at least 14 nt; optionally wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:61 or a contiguous portion of at least 14 nt.

[0062] In some of any of the provided embodiments, each gRNA independently comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided embodiments, each gRNA independently comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of: (a) a gRNA targeting a target site of Lp(a) comprising the gRNA spacer sequence set forth in any one of SEQ ID NOS: 36-40 or 253-255; and (b) a gRNA targeting a target site in PCSK9 comprising the gRNA spacer sequence set forth in any one of SEQ ID NOS: 59-71 or 101-112; optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:61.

[0063] In some of any of the provided embodiments, each gRNA further comprises a scaffold sequence set forth in SEQ ID NO:85. In some of any of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of: (a) a gRNA targeting a target site in Lp(a) comprising the sequence set forth in any one of SEQ ID NOS:41-45 or 250-252; and (b) a gRNA targeting a target site in PCSK9 comprising the sequence set forth in any one of SEQ ID NOS:72-84 or 113-124; optionally wherein gRNA comprises the sequence set forth in SEQ ID NO: 74.

[0064] In some of any of the provided embodiments, the first gRNA and the second gRNA are selected from two different members of the group consisting of: (a) a gRNA targeting a target site in Lp(a) set forth in any one of SEQ ID NOS:41-45 or 250-252; and (b) a gRNA targeting a target site in PCSK9 set forth in any one of SEQ ID NOS: 72-84 or 113-124, optionally wherein the gRNA is set forth in SEQ ID NO:74. In some of any of the provided embodiments, at least one gRNA comprises modified nucleotides for increased stability.

[0065] In some of any of the provided embodiments, the at least one transcriptional repressor domain is capable of reducing transcription of Lp(a) and PCSK9. In some of any of the provided 14MF-366426018Attorney No. 224742004040 embodiments, the at least one transcriptional repressor domain comprises a Krüppel-associated box (KRAB) domain. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase.

[0066] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a histone methyltransferase, or a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNA methyltransferase and a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase. In some of any of the provided embodiments, at least one transcriptional repressor domain comprises a DNA methyltransferase and a histone methyltransferase.

[0067] In some of any of the provided embodiments, one or more of the at least one transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3 A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxil repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD 1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a KRAB domain, a DNMT3 A domain, a DNMT3L domain, or a combination of any of the foregoing.

[0068] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 130, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0069] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNMT3 A domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 132, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0070] In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises a DNMT3L domain or a variant or portion thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 134, a portion thereof, or an amino acid15MF-366426018Attorney No. 224742004040 sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0071] In some of any of the provided embodiments, the at least one transcriptional repressor domain is a DNMT3 A-DNMT3L fusion protein domain or a variant thereof that exhibits transcriptional repressor activity. In some of any of the provided embodiments, the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 136 or SEQ ID NO: 138, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0072] In some of any of the provided embodiments, at least one transcriptional repressor domain comprises a sequence selected from any one of SEQ ID NOS: 130, 132, 134, 136, and 138-146, or a domain thereof, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some of any of the provided embodiments, the at least one transcriptional repressor domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain.

[0073] In some of any of the provided embodiments, the fusion protein further comprises one or more nuclear localization signals (NLS). In some of any of the provided embodiments, the fusion protein further comprises one or more linkers connecting two or more of: the DNA-binding domain, the at least one transcriptional repressor domain, and the one or more nuclear localization signals. In some of any of the provided embodiments, the fusion protein comprises the sequence set forth in any one of SEQ ID NOS: 88, 148, 150, or 152, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, optionally wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 150.

[0074] In some of any of the provided embodiments, transcription of Lp(a) and PCSK9 is repressed by a log2 fold-change of at or lesser than -1.0. In some of any of the provided embodiments, repressed transcription of Lp(a) and PCSK9 in a cell or population of cells leads to a reduction of Lp(a). In some of any of the provided embodiments, the reduction of Lp(a) is greater than the reduction of Lp(a) resulting from comparable repressed transcription of Lp(a) or PCSK9 alone. In some of any of the provided embodiments, repressed transcription of Lp(a) and PCSK9 in a cell or population of cells leads to a reduction of low -density lipoprotein (LDL). In some of any of the provided embodiments, the reduction of LDL is greater than the reduction of LDL resulting from comparable repressed transcription of Lp(a) or PCSK9 alone.

[0075] In some of any of the provided embodiments, reduction of LDL occurs extracellularly. In some of any of the provided embodiments, the cell or population of cells is a liver cell or comprises liver cells. In some of any of the provided embodiments, the cell or population of cells is in a subject. In some of any of the provided embodiments, the reduction of LDL occurs in the subject or a fluid, tissue, or 16MF-366426018Attorney No. 224742004040 organ thereof. In some of any of the provided embodiments, the reduction of LDL occurs in the blood of a subject.

[0076] In some aspects, provided herein is a guide RNA (gRNA) that targets a target site of Lp(a), wherein the target site of Lp(a) is located between (i) the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782; and / or (ii) the hg38 genomic coordinates chr6: 160,519,674 and club:160,520,973.

[0077] In some of any of the provided embodiments, the target site for Lp(a) is located between (i) the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650; and / or (ii) the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,000 and chr6: 160,679,350. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700. In some of any of the provided embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160, 679, 000-160,679,200; club: 160, 679, 200-160,679,400; chr6:160, 679, 400-160,679,600; or chr6:160, 679, 500-160,679,700.

[0078] In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35 or 256-258, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any of the provided embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35 or 256-258.

[0079] In some of any of the provided embodiments, the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40 or 253-255, or a contiguous portion thereof of at least 14 nt. In some of any of the provided embodiments,the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any of the provided embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any of the provided embodiments, the gRNA comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 36-40 or 253-255.

[0080] In some of any of the provided embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 85 or 259. In some of any of the provided embodiments, the gRNA comprises the sequence set forth in any one of SEQ ID NOS:41-45 or 250-252. In some of any of the provided embodiments, the gRNA is set forth in any one of SEQ ID NOS:41-45 or 250-252. In some of any of the provided embodiments, the gRNA comprises modified nucleotides for increased stability.

[0081] In some aspects, provided herein is a plurality of gRNAs comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a target site of Lp(a) located between (i) the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782; and / or (ii) the hg38 genomic coordinates17MF-366426018Attorney No. 224742004040 chr6: 160,519,674 and chr6: 160,520,973, and the second gRNA targets a target site of PCSK9 located within 500bp of the hg38 genomic coordinate chr1:55,039,548.

[0082] In some of any of the provided embodiments, the first gRNA is selected from any of the provided gRNAs. In some of any of the provided embodiments, the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58 or 89-100; optionally wherein the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48. In some of any of the provided embodiments, the second gRNA: a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 59-71 or 101-112; optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:61; and / orb) is set forth in any one of SEQ ID NOS:72-84 or 113-124; optionally wherein the gRNA is set forth in SEQ ID NO: 74.

[0083] In some aspects, provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof; and (b) any of the provided gRNAs or any of the provided pluralities of gRNAs.

[0084] In some of any of the provided embodiments, the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein. In some of any of the provided embodiments, the dCas protein lacks nuclease activity. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any of the provided embodiments, the dCas protein is a dCas12 protein.

[0085] In some of any of the provided embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any of the provided embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125. In some of any of the provided embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 126, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSaCas9 is set forth in SEQ ID NO: 126.

[0086] In some of any of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any of the provided embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10 A and H840A, with reference to numbering of positions of SEQ ID NO: 127. In some of any of the provided embodiments, the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the provided embodiments, the dSpCas9 is set forth in SEQ ID NO: 128.

[0087] In some aspects, provided herein is a polynucleotide encoding any of the provided epigenetic-modifying DNA-targeting systems, any of the provided gRNAs, any of the provided pluralitieses of gRNAs, any of the provided Cas-gRNA combinations, or a portion or a component of any of the foregoing. In some aspects, provided herein is a polynucleotide encoding any of the provided 18MF-366426018Attorney No.224742004040 epigenetic -modifying DNA-targeting systems. In some aspects, provided herein is a polynucleotide encoding any of the provided gRNAs. In some aspects, provided herein is a polynucleotide encoding any of the provided pluralities of gRNAs. In some aspects, provided herein is a polynucleotide encoding any of the provided Cas-gRNA combinations.

[0088] In some aspects, provided herein is a plurality of polynucleotides encoding any of the provided epigenetic-modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, or a portion or a component of any of the foregoing. In some aspects, provided herein is plurality of polynucleotides encoding any of the provided epigenetic-modifying DNA-targeting systems. In some aspects, provided herein is a plurality of polynucleotides encoding any of the provided pluralities of gRNAs. In some aspects, provided herein is a plurality of polynucleotides encoding any of the provided Cas-gRNA combinations.

[0089] In some aspects, provided herein is plurality of polynucleotides comprising: a) a polynucleotide encoding the fusion protein of any of the provided epigenetic -modifying DNA-targeting systems; and the gRNA of any of the provided epigenetic -modifying DNA-targeting systems. In some of any of the provided embodiments, the polynucleotide encoding the fusion protein is mRNA. In some of any of the provided embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 156, or a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. In some of any of the provided embodiments, the mRNA has the sequence is set forth in SEQ ID NO: 153.

[0090] In some aspects, provided herein is a vector comprising any of the provided polynucleotides. In some aspects, provided herein is a vector comprising any of the provided pluralities of polynucleotides.

[0091] In some of any of the provided embodiments, the vector is a viral vector. In some of any of the provided embodiments, the vector is an adeno-associated virus (AAV) vector. In some of any of the provided embodiments, the vector is selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some of any of the provided embodiments, the vector is a lentiviral vector.

[0092] In some of any of the provided embodiments, the vector is a non-viral vector. In some of any of the provided embodiments, the non-viral vector is selected from: a lipid nanoparticle, a liposome, an exosome, or a cell penetrating peptide. In some of any of the provided embodiments, the non-viral vector is a lipid nanoparticle. In some of any of the provided embodiments, the lipid nanoparticle comprises an amino sugar derivative of galactose, optionally an N-Acetylegalactosamine (GalNAc) moiety. In some of any of the provided embodiments, the vector exhibits hepatocyte tropism.

[0093] In some aspects, provided herein is a lipid nanoparticle comprising any of the provided polynucleotides. In some aspects, provided herein is a lipid nanoparticle comprising any of the provided pluralities of polynucleotides.19MF-366426018Attorney No.224742004040

[0094] In some aspects, provided here is a method of decreasing transcription of Lp(a) in a cell or population of cells, the method comprising administering to a cell or population of cells any of the provided epigenetic-modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, any of the provided polynucleotides, any of the provided pluralities of polynucleotides, any of the provided vectors, any of the provided lipid nanoparticles, or a portion or a component of any of the foregoing.

[0095] In some of any of the provided embodiments, Lp(a) is epigenetically modified. In some of any of the provided embodiments, the transcription of Lp(a) is decreased in comparison to a comparable cell or population of cells not subjected to the method. In some of any of the provided embodiments, the transcription of Lp(a) is reduced by at least about 1.2-fold, 1.25-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold. In some of any of the provided embodiments, the reduced transcription of Lp(a) leads to a reduction of Lp(a) and / or low-density lipoprotein (LDL).

[0096] In some aspects, provided herein is a method of reducing Lp(a), the method comprising introducing into a cell or population of cells any of the provided epigenetic -modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, any of the provided polynucleotides, any of the provided pluralities of polynucleotides, any of the provided vectors, any of the provided lipid nanoparticles, or a portion or a component of any of the foregoing.

[0097] In some aspects, provided herein is a method of reducing LDL, the method comprising introducing into a cell or population of cells any of the provided epigenetic -modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, any of the provided polynucleotides, any of the provided pluralities of polynucleotides, any of the provided vectors, any of the provided lipid nanoparticles, or a portion or a component of any of the foregoing.

[0098] In some of any of the provided embodiments, the cell or population of cells is a liver cell or comprises liver cells. In some of any of the provided embodiments, the cell or population of cells is in a subject and the method is carried out in vivo. In some of any of the provided embodiments, Lp(a) and / or LDL is reduced in the subject or a fluid, tissue, or organ thereof. In some of any of the provided embodiments, Lp(a) and / or LDL is reduced in the blood of a subject. In some of any of the provided embodiments, the subject is a human.

[0099] In some of any of the provided embodiments, the subject has or is suspected of having a disease, condition, or disorder, optionally wherein the disease, condition or disorder is a cardiovascular disease. In some of any of the provided embodiments, the subject has or is suspected of having one or more of: elevated levels of low-density lipoprotein in the blood, elevated levels of Lp(a) in the blood,20MF-366426018Attorney No. 224742004040 increased risk of cardiovascular disease, increased risk of early -onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in a low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia.

[0100] In some of any of the provided embodiments, the subject has is or is suspected of having elevated levels of Lp(a) in the blood. In some of any of the provided embodiments, the subject has is or is suspected of having familial hypercholesterolemia.

[0101] In some aspects, provided herein is a pharmaceutical composition comprising any of the provided epigenetic-modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, any of the provided polynucleotides, any of the provided pluralities of polynucleotides, any of the provided vectors, any of the provided lipid nanoparticles, or a portion or a component of any of the foregoing.

[0102] In some of any of the provided embodiments, the pharmaceutical composition is for use in reducing Lp(a) and / or low-density lipoprotein (LDL) in a subject. In some of any of the provided embodiments, the pharmaceutical composition is for use in treating a disease, condition, or disorder in a subject, optionally wherein the disease, condition or disorder is a cardiovascular disease. In some of any of the provided embodiments, the pharmaceutical composition is used in the manufacture of a medicament for reducing Lp(a) and / or low-density lipoprotein (LDL) in a subject. In some of any of the provided embodiments, the pharmaceutical composition is used in the manufacture of a medicament for treating a disease, condition, or disorder in a subject, optionally wherein the disease, condition or disorder is a cardiovascular disease.

[0103] In some of any of the provided embodiments, the subject has or is suspected of having a disease, condition, or disorder, optionally wherein the disease, condition or disorder is a cardiovascular disease. In some of any of the provided embodiments, the subject has or is suspected of having one or more of: elevated levels of low-density lipoprotein in the blood, elevated levels of Lp(a) in the blood, increased risk of cardiovascular disease, increased risk of early -onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in a low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia. In some of any of the provided embodiments, the subject has is or is suspected of having elevated levels of Lp(a) in the blood. In some of any of the provided embodiments, the subject has is or is suspected of having familial hypercholesterolemia.

[0104] In some of any of the provided embodiments, the pharmaceutical composition is to be administered to the subject in vivo. In some of any of the provided embodiments, the pharmaceutical composition is targeted to, or is to be administered to the liver of the subject. In some of any of the provided embodiments, following administration of the pharmaceutical composition, the expression of 21MF-366426018Attorney No. 224742004040 Lp(a) is reduced in cells of the subject. In some of any of the provided embodiments, following administration of the pharmaceutical composition, the expression of Lp(a) is reduced in liver cells of the subject.

[0105] In some of any of the provided embodiments, the pharmaceutical composition is for single dose infusion to the subject. In some of any of the provided embodiments, the pharmaceutical composition is for repeated dose administration, optionally a plurality of times at regular intervals. In some of any of the provided embodiments, the administration is a multiple dose administration comprising at least a first dose and a second dose. In some of any of the provided embodiments, the first dose and the second dose are the same. In some of any of the provided embodiments, the second dose is lower than the first dose, optionally wherein the second dose is 25% to 75% of the first dose (e.g., about 30%, about 40%, about 50%, about 60% or about 70%, or a percentage between any of the foregoing). In some of any of the provided embodiments, the second dose is higher than the first dose, optionally wherein the second dose is 150% to 500% of the first dose (e.g., about 200%, about 300%, about 400% or about 500%, or a percentage between any of the foregoing).

[0106] In some of any of the provided embodiments, the pharmaceutical composition comprises any of the provided the lipid nanoparticles.

[0107] In some aspects, provided herein is a method for treating a disease, condition, or disorder associated with elevated Lp(a) in a subject in need thereof, comprising administering to the subject any of the provided epigenetic -modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, any of the provided polynucleotides, any of the provided pluralities of polynucleotides, any of the provided vectors, any of the provided lipid nanoparticles, or a portion or a component of any of the foregoing.

[0108] In some aspects, provided herein is a method for treating a disease, condition, or disorder associated with elevated low-density lipoprotein (LDL) in a subject in need thereof, comprising administering to the subject any of the provided epigenetic -modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, any of the provided polynucleotides, any of the provided pluralities of polynucleotides, any of the provided vectors, any of the provided lipid nanoparticles, or a portion or a component of any of the foregoing.

[0109] In some of any of the provided embodiments, the disease, condition or disorder associated with elevated LDL is a cardiovascular disease. In some of any of the provided embodiments, the subject has or is suspected of having one or more of: elevated levels of low -density lipoprotein in the blood, elevated levels of Lp(a) in the blood, increased risk of cardiovascular disease, increased risk of early -onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in a low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia.22MF-366426018Attorney No. 224742004040

[0110] In some aspects, provided herein is a method for treating a familial hypercholesterolemia in a subject, comprising administering to the subject any of the provided epigenetic-modifying DNA-targeting systems, any of the provided the pluralities of gRNAs, any of the provided the Cas-gRNA combinations, any of the provided polynucleotides, any of the provided pluralities of polynucleotides, any of the provided vectors, any of the provided lipid nanoparticles, or a portion or a component of any of the foregoing.

[0111] In some of any of the provided embodiments, the administration is a single dose infusion to the subject. In some of any of the provided embodiments, the administration is repeated at least once, optionally a plurality of times at regular intervals. In some of any of the provided embodiments, the administration is a multiple dose administration comprising at least a first dose and a second dose. In some of any of the provided embodiments, the first dose and the second dose are the same. In some of any of the provided embodiments, the second dose is lower than the first dose, optionally wherein the second dose is 25% to 75% of the first dose (e.g., about 30%, about 40%, about 50%, about 60% or about 70%, or a percentage between any of the foregoing). In some of any of the provided embodiments, the second dose is higher than the first dose, optionally wherein the second dose is 150% to 500% of the first dose (e.g., about 200%, about 300%, about 400% or about 500%, or a percentage between any of the foregoing).

[0112] In some of any of the provided embodiments, any of the provided lipid nanoparticles is administered to the subject.Brief Description of the Drawings

[0113] FIG. 1 shows results from qRT-PCR to assess expression of Lp(a) in human hepatocellular carcinoma (Huh7) cells transfected with dSpCas9-KRAB-DNMT3A / L and a non-targeting gRNA (NT; negative control) or a gRNA targeting the promoter of human Lp(a). Results are shown for 7 days posttransfection. Dots represent expression levels for experimental replicates, bars represent mean of expression from experimental replicates. Expression is shown as fold change with respect to cells expressing the non-targeting gRNA.

[0114] FIG. 2 shows open chromatin peaks from ATACseq data of a genomic region upstream of Lp(a). The dashed box indicates the peak of open chromatin predicted to be an upstream enhancer for Lp(a).

[0115] FIG. 3A-3H shows results from qRT-PCR to assess expression of Lp(a) in human hepatocellular carcinoma (Huh7) cells transfected with dSpCas9-KRAB-DNMT3 A / L and a non-targeting gRNA (NT; negative control) or a gRNA targeting an enhancer of human Lp(a). Dots represent expression levels for experimental replicates, bars represent mean of expression from experimental 23MF-366426018Attorney No. 224742004040 replicates. Expression is shown as fold change with respect to cells expressing the non-targeting gRNA.FIG. 3A shows results from qRT-PCR expression to assess expression of Lp(a) mRNA in transfected Huh cells on day 3 post-transfection. FIG. 3B shows results from qRT-PCR expression to assess expression of Lp(a) mRNA in transfected Huh cells on day 10 post-transfection. FIG. 3C shows results from qRT-PCR expression to assess expression of Lp(a) mRNA in transfected Huh cells on day 21 posttransfection. FIG. 3D shows results from qRT-PCR expression to assess expression of Lp(a) mRNA in transfected Huh cells on day 28 post-transfection. FIG. 3E shows results from qRT-PCR expression to assess expression of Lp(a) mRNA in transfected Huh cells on day 38 post-transfection. FIG. 3F shows results from qRT-PCR expression to assess expression of Lp(a) mRNA in transfected Huh cells from day 3 to day 115 post-transfection. FIG. 3G shows the results from qRT-PCR expression of Lp(a) mRNA transfected Huh cells with two gRNAs showing 80% repression at about two months. FIG.3H shows repression of Lp(a) using a variety of gRNAs that target across the 5’ enhancer region.

[0116] FIG. 4A-4E show PCSK9 mRNA expression levels and PCSK9 CpG methylation in human hepatocellular carcinoma (Huh7) cells transfected with dSpCas9-KRAB-DNMT3 A / L and a non-targeting gRNA (NT; negative control) or a PCSK9-targeting gRNA. FIG. 4A shows results from qRT-PCR to assess expression of PCSK9 mRNA in transfected Huh cells on day 3, day 7, day 14, day 21, day 30, day 40, day 50, and day 62 post-transfection. FIG. 4B shows results from qRT-PCR to assess expression of PCSK9 mRNA in transfected Huh cells on day 120 post-transfection. Dots represent expression levels for experimental replicates, bars represent mean of expression from experimental replicates. Expression is shown as fold change with respect to cells expressing the non-targeting gRNA. FIG. 4C shows results from qRT-PCR to assess PCSK9 mRNA expression levels in Huh cells transfected with dSpCas9-KRAB-DNMT3A / L and a non-targeting gRNA (NT) or gRNA PCSK9-C from day 3 through day 180 post-transfection. Dots represent individual time points and error bars represent mean of expression from experimental replicates. FIG. 4D shows the % methylation of CpGs in the PCSK9 promoter in Huh cells transfected with dSpCas9-KRAB-DNMT3 A / L and a non-targeting gRNA (NT) or gRNA PCSK9-C. The methylation profiles are shown for cells expressing the non-targeting gRNA (NT) at day 103 posttransfection and for cells expressing PCSK9-C at day 21and day 103 post-transfection. The shaded bar indicates the location of the target site for gRNA PCSK9-C in the PCSK9 promoter. FIG. 4E shows the methylation profiles of CpG islands 1-22 at the PCSK9 promoter for Huh cells expressing the nontargeting gRNA (NT) at day 103 post-transfection and for cells expressing PCSK9-C at day 21 and day 103 post-transfection. The shaded bar indicates the location of the target site for gRNA PCSK9-C in the PCSK9 promoter.

[0117] FIG. 5 shows results from qRT-PCR to assess PCSK9 mRNA expression levels in primary human hepatocytes transfected with dSpCas9-KRAB-DNMT3 A / L and a non-targeting gRNA (NT) or gRNA PCSK9-C from day 3 through day 30 post-transfection. Dots represent individual time points and 24MF-366426018Attorney No. 224742004040 error bars represent mean of expression from experimental replicates.

[0118] FIG.6A-6B show PCSK9 mRNA and protein levels in Cynomolgus macaque primary hepatocytes transfected with dSpCas9-KRAB-DNMT3 A / L and a non-targeting gRNA (NT), a human PCSK9-targeting gRNA, or a cynomolgus-specific PCSK9-gRNA (cPCSK9-C). FIG. 6A shows results from qRT-PCR to assess expression of Cynomolgus PCSK9 (cPCSK9) mRNA levels in transfected hepatocytes at day 3 and day 14 post-transfection. mRNA expression is shown as fold change with respect to cells expressing the non-targeting gRNA. FIG. 6B shows PCSK9 protein levels as measured by ELISA in transfected hepatocytes at day 3 and day 14 post-transfection. Dots represent values for experimental replicates, bars represent mean values for experimental replicates.

[0119] FIG. 7 shows blood PCSK9 protein levels from three individual Cynomolgus macaques (NHP1, NHP2, and NHP3) intravenously infused with a high dose (3.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C. Protein levels are shown as % change from baseline at numerous time points spanning 49 days post-infusion for NHP1 and 28 days post-infusion for NHP2 and NHP3.

[0120] FIG. 8 shows average blood PCSK9 protein levels from Cynomolgus macaques intravenously infused with a high dose (3.0 mg / kg, n=3) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C as compared to a PBS control dose (n=2). Protein levels are shown as % change from baseline at numerous time points spanning 100 days post-infusion for the PBS treated group and 120 days post-infusion for high dose treated group. Dots represent individual time points and error bars represent mean of expression from experimental replicates.

[0121] FIG. 9 shows LDL-C measurements from three individual Cynomolgus macaques (NHP1, NHP2, and NHP3) intravenously infused with a high dose (3.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C. Blood LDL-C levels are shown as % of baseline at numerous time points spanning 49 days post-infusion for NHP1 and 28 days post-infusion for NHP2 and NHP3.

[0122] FIG. 10 shows average LDL-C levels from Cynomolgus macaques intravenously infused with a high dose (3.0 mg / kg, n=3) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C as compared to a PBS control dose (n=2). Blood LDL-C levels are shown as % change from baseline at numerous time points spanning 100 days post-infusion for the PBS treated group and 120 days post-infusion for high dose treated group. Dots represent individual time points and error bars represent mean of expression from experimental replicates.

[0123] FIG. 11A-11B show alanine transaminase (ALT) and asparate aminotransferase (AST) measurements from three individual Cynomolgus macaques (NHP1, NHP2, and NHP3) intravenously infused with a high dose (3.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C. FIG. 11A shows ALT levels for each NHP at various timepoints spanning 14 days pre-infusion through 28 days post-infusion. FIG. 11B shows AST levels for each NHP at various timepoints spanning 14 days preinfusion through 28 days post-infusion.25MF-366426018Attorney No. 224742004040

[0124] FIG. 12A-12B show average alanine transaminase (ALT) and asparate aminotransferase (AST) measurements from Cynomolgus macaques intravenously infused with a high dose (3.0 mg / kg, n=3) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C. FIG. 12A shows average ALT levels at various timepoints spanning 0-120 days post-infusion. FIG. 12B shows average AST levels at various timepoints spanning 0-120 days post-infusion.

[0125] FIG. 13 shows blood PCSK9 protein levels from three individual Cynomolgus macaques (NHP4, NHP5, and NHP6) intravenously infused with a low dose (1.0 mg / mL) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C and re-dosed at 1.0 mg / kg 21 days after the initial infusion. A dashed oval marks the re-dose time point. Protein levels are shown as % change from baseline at numerous time points spanning 28 days after the initial infusion.

[0126] FIG. 14A-14C show LDL-C, alanine transaminase (ALT), and asparate aminotransferase (AST) measurements from three individual Cynomolgus macaques (NHP4, NHP5, and NHP6) intravenously infused with a low dose (1.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C and re-dosed at 1.0 mg / kg 21 days after the initial infusion. A dashed oval marks the re-dose time point. FIG. 14A shows LDL-C levels as % of baseline at numerous time points spanning 28 days after the initial infusion. FIG. 14B shows ALT levels for each NHP at various timepoints spanning 14 days pre-infusion through 28 days post-infusion. FIG. 14C shows AST levels for each NHP at various timepoints spanning 14 days pre-infusion through 28 days post-infusion.

[0127] FIG. 15 shows PCSK9 mRNA levels measured from liver biopsies of Cynomolgus macaques intravenously infused with a PBS control, a high dose (3.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C, or a low dose (1.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C. Relative mRNA levels are shown from liver biopsies performed on day 7 postinfusion for each treatment group and biopsies performed on day 28 post-infusion for the low dose (1.0 mg / kg) and PBS treatment groups. mRNA levels are shown as fold change relative to the PBS controls. Dots represent values for experimental replicates, bars represent mean values for experimental replicates.

[0128] FIG. 16. shows PCSK9 mRNA levels measured from liver biopsies of three individual Cynomolgus macaques (NHP4, NHP5, and NHP6) following a third intravenous infusion of KRAB-DNMT3 A / L and gRNA cPCSK9-C at varying doses 126 days after the initial infusion compared to a PBS control (NHP7). NHP4 and NHP5 were redosed at 3.0 mg / kg and NHP6 was redosed at 1.0 mg / kg. Relative mRNA levels are shown from liver biopsies performed on day 140 post-infusion for NHP4, NHP6, and NHP7 and a biopsy performed on day 129 post-infusion for NHP5. mRNA levels are shown as fold change relative to the PBS controls.

[0129] FIG. 17 shows average blood PCSK9 protein levels from Cynomolgus macaques intravenously infused with dSpCas9-KRAB-DNMT3 A / L and gRNA cPCSK9-C at a high dose (3.0 mg / kg, n=3) and a low dose (1.0 mg / kg, n=3) as compared to a PBS control dose (n=2). 126 days after 26MF-366426018Attorney No. 224742004040 the initial infusion, NHP4 and NHP5 from the low dose group were redosed at 3.0 mg / kg and NHP6 was redosed at 1.0 mg / kg. Protein levels are shown as % change from baseline at numerous time points spanning 160 days post-infusion for the high dose treated group and 140 days post-infusion for the low dose and PBS treated groups. Dots represent individual time points and error bars represent mean of expression from experimental replicates.

[0130] FIG. 18 shows average LDL-C levels from Cynomolgus macaques intravenously infused with dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C at a high dose (3.0 mg / kg, n=3) and a low dose (1.0 mg / kg, n=3) as compared to a PBS control dose (n=2). 126 days after the initial infusion, NHP4 and NHP5 from the low dose group were redosed at 3.0 mg / kg and NHP6 was redosed at 1.0 mg / kg. Blood LDL-C levels are shown as % change from baseline at numerous time points spanning 160 days post-infusion for the high dose treated group and 140 days post-infusion for the low dose and PBS treated groups. Dots represent individual time points and error bars represent mean of expression from experimental replicates.

[0131] FIG. 19 shows the % methylation of CpGs in the PCSK9 promoter of DNA extracted from liver biopsies of Cynomolgus macaques intravenously infused with a PBS control (n=2)or a high dose (3.0 mg / kg, n=3) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C. Liver biopsies were performed at day 7 and day 85 post-infusion. The shaded bar indicates the location of the target site for gRNA cPCSK9-C in the cPCSK9 promoter.

[0132] FIG. 20 shows the methylation signature surrounding the CpGs in the PCSK9 promoter for Cynomolgus macaques intravenously infused with a PBS control (n=2) or a high dose (3.0 mg / kg, n=3) of dSpCas9-KRAB-DNMT3 A / L and gRNA cPCSK9-C at day 85 post-infusion. The cross-hatched bar indicates the location of the target site for gRNA cPCSK9-C in the cPCSK9 promoter.

[0133] FIG. 21 shows PCSK9 protein levels plotted against the average % CpG methylation across the 2.2 kB PCSK9 promoter region for individual Cynomolgus macaques intravenously infused with a PBS control (NHP7 and NHP8), a low dose (1.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA CPCSK9-C (NHP4, NHP5, and NHP6), or a high dose (3.0 mg / kg) of dSpCas9-KRAB-DNMT3A / L and gRNA cPCSK9-C (NHP1, NHP2, and NHP3). Measurements were taken on day 7 post-infusion.

[0134] FIG. 22A-22B show Lp(a) fold expression in primary human hepatocytes and primary Cynomolgus macaque hepatocytes infused with dSpCas9-KRAB-DNMT3 A / L and LPA-M gRNA as compared to a PBS control. FIG. 22A shows Lp(a) fold expression of primary human hepatocytes from different donors at 6 days (Yl) and 7 days (DI and D2) post-transfection compared to PBS controls. FIG. 22B shows Lp(a) expression of primary Cynomolgus macaque hepatocytes at 7 days posttransfection compared to PBS controls.

[0135] FIG. 23A-23D show the fold change of Lp(a) and PCSK9 in human hepatocellular carcinoma cell line (Huh7) or primary human hepatocytes transfected with mRNA of dSpCas9-KRAB- 27MF-366426018Attorney No. 224742004040 DNMT3 A / L combination with a gRNA targeting Lp(a), a gRNA targeting PCSK9, or both gRNAs with the transfection carried out with lipid nanoparticles that co-encapsulate both the mRNA and the gRNA(s) together. FIG. 23A shows fold expression of Lp(a) and PCSK9 in Huh7 cells transfected with mRNA of dSpCas9-KRAB-DNMT3 A / L combination with a gRNA targeting Lp(a), a gRNA targeting PCSK9, or both gRNAs at 3 days post-transfection. FIG. 23B shows fold expression of Lp(a) and PCSK9 in primary human hepatocytes transfected with mRNA of dSpCas9-KRAB-DNMT3 A / L combination with a gRNA targeting Lp(a), a gRNA targeting PCSK9, or both gRNAs at 7 days post-transfection. FIG. 23C shows fold expression of Lp(a) and PCSK9 in primary human hepatocytes transfected with mRNA of dSpCas9-KRAB-DNMT3A / L combination with a gRNA targeting Lp(a), a gRNA targeting PCSK9, or both gRNAs at 14 days post-transfection. FIG. 23D shows fold expression of Lp(a) and PCSK9 in primary human hepatocytes from a second donor transfected with mRNA of dSpCas9-KRAB-DNMT3 A / L combination with a gRNA targeting Lp(a), a gRNA targeting PCSK9, or both gRNAs at 14 days posttransfection.

[0136] FIG. 24A shows the fold change in PCSK9 mRNA expression in Huh7 cells relative to the untreated cells at Day 34 post-transfection following lipofection of the indicated fusion proteins from Table E8 in combination with an exemplary PCSK9-C gRNA.

[0137] FIG. 24B shows the fold change in PCSK9 mRNA expression in Hep3B cells relative to the untreated cells at Day 34 post-transfection following lipofection of the indicated fusion proteins from Table E8 in combination with an exemplary PCSK9-C gRNA.

[0138] FIG. 25 shows the number of differentially methylated loci (CpGs) from the custom off-target panel in Huh7 cells for each fusion protein at Day 7 and Day 45 post-transfection following transfection of either ApDNMT3L-dSpCas9 (As3L) or DNMT3 A-mDNMT3L-dSpCas9-KRAB (D3 A-m3L-K0Xl) positive control fusion proteins in combination with exemplary PCSK9-C gRNA.

[0139] FIG. 26A shows schematics of three tested dCas-9 effector fusion proteins.

[0140] FIG. 26B shows a dose response curve for fold change in PCSK9 mRNA relative to the lipid only controls for each fusion protein shown in FIG. 26A in combination with an exemplary PCSK9-C gRNA.

[0141] FIG. 27A shows the positions of the exemplary PCSK9-targeting gRNAs along the CpG island relative to the TSS and Exon 1 of PCSK9.

[0142] FIG. 27B shows the fold change in PCSK9 mRNA expression in Huh7 cells relative to the untreated cells at Day 4 post-transfection following transfection with the indicated fusion proteins in combination with an exemplary PCSK9-targeting gRNA from Table E10 or different negative controls.

[0143] FIG. 27C shows the fold change in PCSK9 mRNA expression in Huh7 cells relative to the untreated cells at Day 28 post-transfection following transfection with the indicated fusion proteins in combination with an exemplary PCSK9-targeting gRNA from Table E10 or different negative controls.28MF-366426018Attorney No. 224742004040

[0144] FIG. 27D shows the fold change in PCSK9 mRNA expression in Huh7 cells relative to the non-targeting gRNA (gNT) with the negative control (no ATG) fusion protein over time for each indicated fusion protein in combination with guides PCSK9-C (top), PCSK9-D (middle), and PCSK9-N (bottom)

[0145] FIG. 28 shows the fold change in Lp(a) mRNA expression in Huh7 cells relative to the nontargeting gRNA (gNT) with the negative control (no ATG) fusion protein at Day 28 post-transfection following transfection with the indicated fusion proteins in combination with an exemplary Lp(a)-targeting gRNA from Table Ell or different negative controls.

[0146] FIGS. 29A -29B depict an exemplary sequence alignment to depict identification of corresponding residues in a sequence compared to a reference sequence. The symbol “*” between two aligned amino acid indicates that the aligned amino acids are identical. The symbol indicates a gap in the alignment. Exemplary, non-limiting positions for amino acid substitution described herein are indicated with bold text. Based on the alignment of two similar sequences having identical residues in common, a skilled artisan can identify “corresponding” positions in a sequence by comparison to a reference sequence using conserved and identical amino acid residues as guides. Shown in the figure is an exemplary alignment of a reference mouse DNMT3L protein sequence set forth in SEQ ID NO: 281 (“mouse,” which contains the full-length mouse DNMT3L protein sequence with an ADD domain and a MTase-like domain but no initiating methionine residue) with a human DNMT3L protein sequence set forth in SEQ ID NO: 282 (“human,” which contains the full-length human DNMT3L protein sequence with an ADD domain and a MTase-like domain but no initiating methionine residue); aligning identical residues demonstrates, for example, that amino acid residue S60 in SEQ ID NO: 282 corresponds to residue S26 in SEQ ID NO: 282. It is within the level of a skilled artisan to carry out similar alignments between two similar protein sequences to identify corresponding residues, including based on the exemplification and description herein. Primary domains are annotated using italics with reference to the ADD domain and bold in reference to the MTase-like domain. A key is provided to correlate specific domains and regions of the DNMT3L protein involved in the recruitment of DNMT3A to the appropriate annotation. It is understood that to the extent that residues of a domain or region involved in DNMT3 A recruitment are present in a reference sequence that the corresponding domain or region involved in DNMT3A recruitment would similarly align between sequences. FIG. 29A shows the alignment of amino acid residues 1-240 of a mouse DNMT3L protein sequence (SEQ ID NO: 281) and amino acid resides 1-206 of a human DNMT3L protein sequence (SEQ ID NO: 282). FIG. 29B shows the alignment of amino acid residues 181-420 of a mouse DNMT3L protein sequence (SEQ ID NO: 281) and amino acid resides 147-385 of a human DNMT3L protein sequence (SEQ ID NO: 282).

[0147] FIGS. 30A-30D show repression of LP A mRNA in Huh7 cells following delivery of dSpCas9 fusion proteins and gRNAs. FIG. 30A shows fold change in LPA mRNA expression in Huh729MF-366426018Attorney No. 224742004040 cells at day 4 following transfection with D3 A-m3L-dSpCas9-KOXl and individual LPA-targeting gRNAs, as measured by qRT-PCR and normalized to untreated cells. FIG.30B shows fold change in LPA mRNA expression in Huh7 cells at day 74 following transfection with D3 A-m3L-dSpCas9-KOXl and individual LPA-targeting gRNAs, measured relative to untreated cells. FIG. 30C shows fold change in LPA mRNA expression in Huh7 cells at day 4 following transfection with As3L-dSpCas9 and individual LPA-targeting gRNAs, as measured by qRT-PCR relative to untreated cells. FIG.30D shows fold change in LPA mRNA expression in Huh7 cells at day 74 following transfection with As3L-dSpCas9 and individual LPA-targeting gRNAs, as measured by qRT-PCR relative to untreated cells.

[0148] FIG. 31 shows exemplary dose response curves for fold change in LPA mRNA expression in Huh7 cells following delivery of dSpCas9 fusion proteins and individual LPA-targeting gRNAs. Results are shown at day 28 post-transfection. The top panel shows the results using a D3 A-m3L-dSpCas9-KOX1 (3AL-KRAB) fusion protein and the bottom panel shows the results using a As3L-dSpCas9 (3L-only) fusion protein.

[0149] FIGS. 32A-32B show targeted LPA repression and methylation in primary human hepatocytes (PHH) following LNP delivery of mRNA encoding a either D3 A-m3L-dSpCas9-KOXl (3 AL-KRAB) fusion protein or a As3L-dSpCas9 (3L-only) fusion protein in combination with individual LPA-targeting gRNAs. FIG. 32A shows fold change in LPA mRNA expression in PHHs at 4 days following LNP transfection with a 130 ng “high dose” of mRNA. FIG. 32B shows % methylation of CpGs in the 5’ Lp(a) enhancer region in PHHs at 4 days following LNP transfection with either a 130 ng “high dose” of mRNA or a 50 ng “low does” of mRNA. The top panel shows the methylation pattern using the 3 AL-KRAB dCas effector and the bottom panel shows the methylation pattern using the 3L-only dCas effector. The bars indicates the locations of the target sites for gRNA LPA-U and gRNA LPA- AC in the Lp(a) 5’ enhancer.

[0150] FIG. 33A shows mean methylation % in the 5’ Lp(a) enhancer region in primary hepatocytes from Cynomolgus macaque (PCHs) following LNP delivery of mRNA encoding a either D3A-m3L-dSpCas9-KOXl (3 AL-KRAB) fusion protein or a As3L-dSpCas9 (3L-only) fusion protein in combination with individual LPA-targeting gRNAs. mRNAs were delivered at either a 130ng mRNA “high” dose or 50ng mRNA “low” dose. Negative controls included a untreated cells, use of a nontargeting gRNA (“NT”) instead of a Lp(a) targeting gRNA, a 3 AL-KRAB dCas effector lacking an ATG start codon (“No ATG”) and a catalytically dead 3 AL-KRAB dCas effector (“Cat dead”) effector with a C710A mutation in the catalytic domain of DNMT3A. Results are shown at Day 4 post-transfection.

[0151] FIG. 33B shows % methylation of CpGs in the 5’ Lp(a) enhancer region in PCHs at 4 days following LNP delivery of mRNA encoding a either D3 A-m3L-dSpCas9-KOXl (3 AL-KRAB) fusion protein or a As3L-dSpCas9 (3L-only) fusion protein in combination with an LPA-targeting gRNA. LNPs were delivered with either a 130 ng “high dose” of mRNA or a 50 ng “low does” of mRNA. The bar 30MF-366426018Attorney No. 224742004040 indicates the location of the target site for gRNA LPA-U in the Lp(a) 5’ enhancer.

[0152] FIG. 33C shows fold change in LPA mRNA expression in PCHs, as assessed by qRT-PCR, following lipofectamine-mediated transfection of a D3 A-m3L-dSpCas9-KOXl (3 AL-KRAB) fusion protein in combination with individual LPA-targeting gRNAs. Results are shown at Day 4 posttransfection.

[0153] FIG. 33D shows fold change in LPA mRNA expression in PCHs, as assessed by qRT-PCR, following lipofectamine-mediated transfection of a D3 A-m3L-dSpCas9-KOXl (3 AL-KRAB) fusion protein in combination with gRNA LPA-U. Results are shown at Day 4 post-transfection.

[0154] FIGS. 34A-34F show schematic representations and functional comparisons of DNMT3L dSpCas9 fusion proteins comprising domains from various DNMT3L orthologs. FIG. 34A shows schematic diagrams of a 3L-X80-dSpCas9 fusion protein, a 3 A-3L-X80-dSpCas9 fusion protein, and a 3A-ADD-3L-X-80-dSpCas9 fusion protein. The domains outlined dotted lines for substituted for corresponding domains from various DNMT3L orthologs. FIG. 34B shows LPA and PCSK9 repression activity associated with a 3L-X80-dSpCas9 (3L-dSpCas9) fusion proteins comprising 3L domains from the species indicated on the x-axis. Fold change in mRNA levels relative to untreated controls are shown for Huh7 cells 5 days after transfection with the dSpCas9 effector, an LPA-targeting gRNA and a PCSK9-targeting gRNA. FIG. 34C shows LPA and PCSK9 repression activity associated with a 3L-X80-dSpCas9 (3L-dSpCas9) fusion proteins comprising 3L domains from the species indicated on the x-axis. Fold change in mRNA levels relative to untreated controls are shown for Huh7 cells 13 days after transfection with the dSpCas9 effector, an LPA-targeting gRNA and a PCSK9-targeting gRNA. FIG.34D shows LPA and PCSK9 repression activity associated with a 3L-X80-dSpCas9 (3L-dSpCas9) fusion proteins comprising 3L domains from the species indicated on the x-axis. Fold change in mRNA levels relative to untreated controls are shown for Huh7 cells 21 days after transfection with the dSpCas9 effector, an LPA-targeting gRNA and a PCSK9-targeting gRNA. FIG. 34E shows LPA and PCSK9 repression activity associated with 3 A-3L-X80-dSpCas9 fusion protein and 3A-ADD-3L-X-80-dSpCas9 fusion proteins comprising 3L domains from the species indicated on the x-axis. Fold change in mRNA levels relative to untreated controls are shown for Huh7 cells 13 days after transfection with the dSpCas9 effector, an LPA-targeting gRNA and a PCSK9-targeting gRNA. FIG. 34E shows LPA and PCSK9 repression activity associated with 3 A-3L-X80-dSpCas9 fusion protein and 3A-ADD-3L-X-80-dSpCas9 fusion proteins comprising 3L domains from the species indicated on the x-axis. Fold change in mRNA levels relative to untreated controls are shown for Huh7 cells 24 days after transfection with the dSpCas9 effector, an LPA-targeting gRNA and a PCSK9-targeting gRNA

[0155] FIG. 35A shows dose response curves for fold change in PCSK9 expression in PHHs following LNP transfection with different dSpCas9 effector fusion proteins and individual PCSK9-targeting gRNAs. The results are shown in separate panels for each dSpCas9 effector fusion protein:31MF-366426018Attorney No. 224742004040 D3 A-m3L-Koxl (top panel), D3 A-Eq3L-Zim3 (middle panel) and As3L (bottom panel). FIG. 35B shows dose response curves for fold change in PCSK9 expression in PHHs for the following effector guide combinations: D3A-m3L-Koxl+ PCSK9-C; 3L only + PCSK9-C; and As3L only + PCSK9-D. FIG. 35C shows dose response curves for fold change in PCSK9 expression in PHHs for the following effector guide combinations: D3 A-m3L-Koxl+ PCSK9-C; D3 A-Eq3L-Zim3 + PCSK9-C; and D3 A-Eq3L-Zim3 + PCSK9-D.

[0156] FIG. 36 shows dose response curves for fold change in PCSK9 expression in PCHs following LNP transfection with different dSpCas9 effector fusion proteins and individual PCSK9-targeting gRNAs. The results are shown in separate panels for each dSpCas9 effector fusion protein: D3 A-m3L-Koxl (top panel), D3 A-Eq3L-Zim3 (middle panel) and As3L (bottom panel).

[0157] FIG. 37A shows a schematic of the 3' enhancer region, coding region, promoter region, and 5' enhancer region of the LPA locus. Open chromatin and CpG Islands associated with each region are depicted. FIGS. 37B-C shows repression of LPA mRNA in Huh7 cells following delivery of a D3A-m3L-dSpCas9-KOXl fusion protein and LPA-targeting gRNAs. FIG.37B. shows fold in change in LPA mRNA expression at day 4 post-transfection as measured by qRT-PCR and normalized to cells transfected with the non-targeting gRNA control (“NT”). FIG. 37C. shows fold in change in LPA mRNA expression at day 15 post-transfection as measured by qRT-PCR and normalized to cells transfected with the non-targeting gRNA control (“NT”).

[0158] FIG. 38A shows dose response curves for fold change in PCSK9 expression in PHHs following LNP transfection with a D3 A-m3L-Koxl dCas9 effector fusion and a PCSK9-targeting gRNA (PSCK9-C) at various mRNA-to-gRNA ratios.

[0159] FIG. 38B shows dose response curves for fold change in PCSK9 expression in PHHs following LNP transfection with different dCas9 effector fusions and a PCSK9-targeting gRNA (PSCK9-C or PCSK9-D) at a 2: 1 mRNA:gRNA ratio.

[0160] FIG. 39A shows dose response curves for fold change in PCSK9 expression in PHHs following LNP transfection with a D3 A-m3L-Koxl dCas9 effector fusion, a PCSK9-targeting gRNA (PSCK9-C), and a LPA-targeting gRNA (LPA-U) at various mRNA-to-gRNA-to-gRNA ratios.

[0161] FIG. 39B shows dose response curves for fold change in LPA expression in PHHs following LNP transfection with a D3A-m3L-Koxl dCas9 effector fusion, a PCSK9-targeting gRNA (PSCK9-C), and a LPA-targeting gRNA (LPA-U) at various mRNA-to-gRNA-to-gRNA ratios.

[0162] FIG. 39C shows dose response curves for fold change in PCSK9 expression in PHHs following LNP transfection with a As3L-only dCas9 effector fusion, a PCSK9 -targeting gRNA (PSCK9-C), and a LPA-targeting gRNA (LPA-U) at various mRNA-to-gRNA-to-gRNA ratios.

[0163] FIG. 39D shows dose response curves for fold change in LPA expression in PHHs following LNP transfection with a As3L-only dCas9 effector fusion, a PCSK9-targeting gRNA (PSCK9-C), and a 32MF-366426018Attorney No. 224742004040 LPA-targeting gRNA (LPA-U) at various mRNA-to-gRNA-to-gRNA ratios.

[0164] FIG. 39E shows dose response curves for fold change in PCSK9 expression in PHHs following LNP transfection with different dCas9 effector fusions, a PCSK9-targeting gRNA (PSCK9-C or PCSK9-D), and a LPA-targeting gRNA (LpA-U) at a 2:0.5:0.5 mRNA:gRNA:gRNA ratio.Detailed Description

[0165] Provided herein are epigenetic-modifying DNA-targeting systems for repressing transcription of Lipoprotein(a) (Lp(a)). In some embodiments, provided epigenetic-modifying DNA-targeting systems include fusion proteins comprising: (a) a DNA-binding domain for targeting to a target site of Lp(a), and (b) at least one transcriptional repressor domain. In some embodiments, the target site of Lp(a) is located in an enhancer of Lp(a). In some embodiments, the target site is located in a distal regulatory element approximately 15 kB upstream or approximately 140 kB downstream of the TSS of Lp(a). In some embodiments, the enhancer is a distal regulatory element located approximately 15 kB upstream or approximately 140 kB downstream of the transcriptional start site (TSS) of Lp(a). In some embodiments, the enhancer is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 or chr6: 160,519,674 and chr6: 160,520,973. In some embodiments, the target site of Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,78 or chr6: 160,519,674 and chr6: 160,520,9732. In some such embodiments, the DNA-binding domain is a CRISPR-Cas / guide RNA (gRNA) combination in which the epigenetic-modifying DNA-targeting system comprises (i) a fusion protein of a deactivated Cas protein and the at least one transcriptional repressor domain, and (ii) a gRNA that targets the fusion protein to the target site of LP(a).

[0166] Also provided herein are multiplexed epigenetic-modifying DNA-targeting systems with a plurality of targeting modules for repressing a plurality of genes such that the system is able to target both Lp(a) and proprotein convertase subtilisin / kexin type 9 (PCSK9). In such embodiments, the systems target Lp(a) at the target site located in a distal regulatory element approximately 15 kB upstream or approximately 140 kB downstream of the TSS of Lp(a), such as a target site of Lp(a) located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 or chr6: 160,519,674 and chr6: 160,520,973. In some embodiments, each module of the DNA-targeting system represses transcription of a different gene, for example one module represses transcription of Lp(a) and contains a fusion protein comprising: (a) a DNA-binding domain for targeting to a target site of Lp(a), and (b) at least one transcriptional repressor domain; and another module represses transcription of PCSK9 and contains a fusion protein comprising (a) a DNA-binding domain for targeting to a target site of PCSK9, and (b) at least one transcriptional repressor domain. In some such embodiments, each DNA-binding domain is a CRISPR-Cas / guide RNA (gRNA) combination in which the epigenetic-modifying DNA-targeting systemMF-366426018Attorney No. 224742004040 comprises (i) a fusion protein of a deactivated Cas protein and the at least one transcriptional repressor domain, (ii) a gRNA that targets the fusion protein to the target site of LP(a); and (iii) a gRNA that targets the fusion protein to the target site of PCSK9.

[0167] Among any of the provided embodiments, the epigenetic-modifying DNA-targeting system is provided to a subject (e.g., human subject) as a polynucleotide or a plurality of polynucleotides. In some embodiments, provided is a polynucleotide that encodes any of the provided epigenetic-modifying DNA-targeting systems, or a polynucleotide that encodes any of the fusion proteins of the DNA-targeting systems, such as a fusion protein of a deactivated Cas protein and at least one transcriptional repressor domain, that can combined with one or more gRNAs of the system. In some embodiments, provided is a plurality of polynucleotides in which the plurality of polynucleotides include (1) a polynucleotide, such as an mRNA, that encodes the fusion protein of a epigenetic-modifying DNA-targeting system and (2) any one or more gRNA of the system, in some embodiments, provided herein is a vector comprising any such polynucleotide or plurality of polynucleotides. In any of such embodiments, the one or more gRNAs may include a single gRNA targeting a distal regulatory element of Lp(a) as described. In any of such embodiments, the one or more gRNAs may include a gRNA targeting a distal regulatory element of Lp(a) and a gRNA targeting a regulatory element of PSCK9, such as any as described, for multiplexed repression of Lp(a) and PCSK9.

[0168] In some embodiments of the provided epigenetic-modifying DNA-targeting system, the DNA binding domain is a nuclease-inactive Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, such as a dead or deactivated Cas (dCas, e.g. dCas9), and the DNA-targeting system further includes at least one gRNA that can complex with the Cas. In such systems, the gRNA has a spacer sequence that is capable of hybridizing to the target site of Lp(a). In such systems, at least one gRNAs has a spacer sequence that is capable of hybridizing to the target site of Lp(a) and at least one gRNA has a spacer sequence that is capable of hybridizing to the target site of PCSK9. Also provided herein are related gRNAs, including Cas / gRNA combinations, polynucleotides, compositions and methods involving or related to the epigenetic-modifying DNA targeting systems.

[0169] Also provided herein are methods of using the epigenetic-modifying DNA-targeting systems for reducing levels of Lp(a) in a subject.

[0170] LPA is a gene that encodes Apolipoprotein(a) (also known as AK38, APO A, LP, Lipoprotein(a), Lp(a)). Apolipoprotein(a) is a constituent of lipoprotein(a). Lp(a) is an LDL-like particle identified as a risk factor for cardiovascular disease. Lp(a) comprises an LDL-like particle (e.g., containing apolipoprotein B- 100 (apoB-100)) associated with apolipoprotein(a) (apo(a)). For example, elevated Lp(a) increases the risk of atherosclerotic cardiovascular disease (ASCVD) which is a chronic inflammatory disease caused by build-up of plaque in the arteries. ASCVD is responsible for 1 in 4 deaths in the US, hundreds of millions globally, and is the underlying cause of ~50% of all deaths in 34MF-366426018Attorney No.224742004040 westernized society. In addition, 75% of acute heart attacks are due to sclerotic plaque rupture. Elevated Lp(a) levels also increase the risk of lower extremity arterial disease, aortic valve stenosis, and elevated low -density lipoprotein cholesterol (LDL-C).

[0171] Lp(a) levels are genetically determined and elevated Lp(a) affects approximately 20-30% of the general population. The LPA gene encodes apolipoprotein(a) (apo(a)), a protein component required for formation of the Lp(a) particle. Lp(a) is the primary carrier of oxidized phospholipids, which can trigger inflammation leading to plaque and blood clot formation. Elevated Lp(a) is more prevalent in individuals with Familial Hypercholesterolemia (FH). FH is a genetic disorder of cholesterol metabolism that is characterized by abnormally elevated blood serum levels of low-density lipoprotein (LDL) cholesterol, which can lead to early -onset cardiovascular disease and death. Unless stated otherwise, reference to LDL throughout this disclosure refers to LDL cholesterol (LDL-C) as this form is a standard measure of LDL and LDL-attributable cardiovascular disease risk. FH is a relatively common disorder, affecting approximately 1 in 250 individuals world-wide. FH can result from mutations in a number of genes, several of which play a role in LDL cholesterol metabolism (Konstantina Valanti, E. et al., Metabolism 116:154461 (2020); Defesche, J. C. et al. Nat. Rev. Dis. Primers 3:17093 (2017); Bouhairie, V. E. et al. Cardiol. Clin. 33(2): 169-179 (2015)). In addition to high levels of LDL, 30-50% of individuals with FH have elevated Lp(a), creating an added risk for cardiovascular disease.

[0172] Current therapies for lowering Lp(a) include lowering LDL-C using statins, a class of lipid lowering medications also known as HMG CoA reductase inhibitors, which reduce cholesterol synthesis. While statins are well-established for lowering LDL levels, Lp(a) levels are not effectively lowered by statins (de Boer, L. M. et al., Em. J. Prev. Cardio, 29(5): 779-792 (2021)). Statins can be used to stabilize but do not reverse atherosclerotic plaques. However, the need for repeated administration and side effects of statin treatments limit uptake and compliance. Lipoprotein apheresis, in which LDL and Lp(a) is removed from the blood of a patient in a clinical setting, is another treatment option for patients with elevated Lp(a), particularly those with FH. The cost and intensive nature of lipoprotein apheresis is disadvantageous, with lipoprotein apheresis typically requiring a 3 -hour treatment session every 1-2 weeks. Secondary analyses of clinical trials of PCSK9 monoclonal antibodies support a potential role for PCSK9 inhibitors to reduce Lp(a) levels. However, PCSK9 antibodies incur significant time commitment and cost, requiring injection once every 2 to 4 weeks. Gene editing approaches create DNA mutations and can have off-target effects. Therapies targeting LPA mRNA expression using antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) have been developed. These therapies suffer from issues with stability, durability and possible induction of off-target effects. (Waqas, A. M., et al. JACC, 81(16): 1633-1645 (2023)).

[0173] In summary, current therapies for lowering Lp(a) face a number of challenges. Statins and PCSK9 antibodies, which can be effective in lowering LDL-C, have limited efficacy in reducing Lp(a)35MF-366426018Attorney No. 224742004040 levels. There is a need for alternative therapies that effectively reduce Lp(a) and that exhibit improved treatment outcomes, such as greater or more sustained efficacy.

[0174] Provided embodiments herein relate to leveraging engineered DNA-binding systems to target genetic mechanisms contributing to regulation of Lp(a), including for treatment of cardiovascular disease and FH. Emerging technologies for targeted modulation of gene expression in vivo, present new opportunities for treating the disease. Sequence-specific DNA-binding systems found in nature, such as zinc -fingers, transcription-activator-like effectors, and CRISPR / Cas systems can now be engineered to target one or more genes for activation or repression in vivo (Adli, M. Nat. Commun. 9, 1911 (2018)). Hence, in some embodiments, the DNA-targeting systems comprise synthetic transcription factors that are able to modulate, such as reduce or repress, transcription of Lp(a) in a targeted manner, for example by reducing or repressing transcription of the LPA gene (encoding apo(a)), or, for the multiplexed systems, transcription of both Lp(a) and also PCSK9 in a targeted manner. In provided embodiments, reducing transcription of Lp(a) or Lp(a) and PCSK9 by the provided epigenetic -modifying DNA-targeting system thereby promotes an Lp(a)- and / or LDL-reducing phenotype. In some embodiments, reduced LPA transcription leads to reduced apo(a) expression and reduced formation and / or level of Lp(a) particles.

[0175] In aspects, the provided embodiments include introducing into a liver cell epigenetic modifications using effector domains that are repressors of transcription (i.e. transcriptional repressor domains), which can be directed to regions of Lp(a) (e.g. regulatory elements such as promoters or enhancers) for transcriptional repression and reduced expression of the Lp(a). In some embodiments, the targeted regions comprise regulatory elements of the LPA gene (encoding apo(a)). For instance, provided herein are epigenetic-modifying DNA binding systems combining a DNA-binding domain (e.g. a dCas and gRNA combination) and an effector domain, in which the effector domain is able to target a regulatory element of Lp(a) to precisely repress or reduce transcription of Lp(a) by epigenetic regulation. In some embodiments, such repression reduces LPA transcription and reduces apo(a) protein expression. The provided embodiments also relate to engineering the DNA-binding systems to target Lp(a) and PCSK9 to lower both Lp(a) and LDL. In some such embodiments, targeted repression of Lp(a) and PCSK9 in liver cells reduce Lp(a) expression. In some embodiments, targeted repression of LPA reduces apo(a) expression and thereby reduces Lp(a) particle formation and / or circulating Lp(a) levels. In some such embodiments, targeted repression of Lp(a) and PCSK9 promote a phenotype that reduces LDL in the blood, such as by increased expression of LDLR and / or increased LDL uptake. Transcriptional repression of Lp(a) and PCSK9, leading to reduced gene expression, reduces levels of Lp(a) and / or increases the capacity of the cell to clear LDL from the blood.

[0176] The ability to epigenetically control the capacity of liver cells to produce Lp(a) and / or clear LDL provides an advantageous approach for treating cardiovascular disease and FH, eliminating the need 36MF-366426018Attorney No. 224742004040 for other therapies or combinations of therapies which can be ineffective, toxic, expensive, or inconvenient. Moreover, the epigenetic modification of the cell does not modify DNA at the sequence level, thereby avoiding safety concerns with gene editing approaches. In addition, the ability to target both Lp(a) and PCSK9 for repression allows for reduction of Lp(a) and LDL via more than one mechanism with a single treatment, thereby avoiding the need for complicated schedules and toxicity associated with some combination therapies. Epigenetic repression of genes may provide a long-lasting solution to reducing Lp(a) and LDL, with the potential for administration schedules that are less intensive than current therapies.

[0177] The approach provided herein, therefore, offers substantial clinical solutions to treating cardiovascular disease, such as atherosclerotic cardiovascular disease (ASCVD), and FH by reducing Lp(a) and / or circulating LDL, while circumventing the problems associated with current therapies. In particular, provided embodiments relate to methods for targeted repression of Lp(a) by provided DNA-targeting systems. Results herein demonstrate that targeting a region approximately 15 kB upstream of Lp(a) with a DNA-targeting repressor system results in a surprisingly sustained level of Lp(a) transcriptional repression of up to 85% more than a month after a single transient transfection of a human hepatocellular carcinoma cell line. Results demonstrate that the targeted genomic region, spanning GRCh38 (hg38) genomic coordinates chr6: 160, 678,873-160,679,782, functions as an enhancer of Lp(a). A previous study tested this region for enhancer activity using a luciferase-based assay and observed no activity (Wade, D. P. et al., J. Biol. Chem., Vol. 272, No. 48:30387-30399 (1997)). The provided DNA-targeting systems for targeted repression of Lp(a) offers the potential to durably reduce Lp(a) levels for the prevention or treatment of cardiovascular disease.

[0178] Provided embodiments also relate to methods for targeted multiplexed repression of Lp(a) and PCSK9 by provided DNA-targeting systems. Results herein also demonstrate a surprisingly high sustained level of repression of PCSK9 in serum of about 80% more than 22 weeks after a single transient dose to an animal model provided with a DNA-targeting repressor system specific to the PCSK9 promoter. Results demonstrate that silencing PCSK9 results in reduction of LDL-cholesterol and thus is a therapeutic target for the prevention or treatment of cardiovascular disease and FH. While currently approved PCSK9 inhibitors require repeat administration to maintain efficacy, the provided DNA-targeting systems for targeted repression of PCSK9 offers the potential to durably silence PCSK9 without altering the genetic sequence. Multiplexed repression of Lp(a) and PCSK9 by provided DNA-targeting systems could thus eliminate the need for combination therapies and provide a single treatment for reducing both Lp(a) and LDL, which are independent risk factors of cardiovascular disease. Since individuals with FH have both elevated LDL and a higher prevalence of elevated Lp(a), targeted epigenetic repression of both Lp(a) and PCSK9 provides a meaningful clinical solution for the treatment of FH and cardiovascular disease.37MF-366426018Attorney No. 224742004040

[0179] Also provided herein are methods of using the epigenetic-modifying DNA-targeting system for modulating transcription or a phenotype of liver cells. Also provided herein are methods of using the epigenetic-modifying DNA-targeting systems for reducing Lp(a) and / or LDL in a subject. In some embodiments, the method can be used in therapies for reducing Lp(a), such as for treatment or prevention of cardiovascular disease. In some embodiments, the methods can be used in therapies for reducing LDL, such as for treatment of FH.

[0180] In some embodiments, the provided methods reduce LDL in a subject. In some embodiments, the methods can be used in therapies for reducing Lp(a), such as for treatment of cardiovascular disease, such as atherosclerotic cardiovascular disease (ASCVD). In some embodiments, the methods can be used in therapies for reducing LDL, such as for treatment of Familial Hypercholesterolemia (FH). In some embodiments, the methods can be used for therapies for reducing Lp(a) and LDL, such as for reducing the risk of cardiovascular disease.

[0181] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0182] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. LPA DNA-TARGETING SYSTEMS

[0183] Provided herein are epigenetic-modifying DNA-targeting systems for repression of Lp(a). The terms “LP(a)” and “LPA” with reference to a gene are used interchangeably herein to refer to the gene encoding apolipoprotein(a) (apo(a)) and associated genomic loci and regulatory elements. The term “Lp(a)” or “lipoprotein(a)” is also known to refer to the circulating lipoprotein(a) particle. Context clarifies whether a reference is to the gene / locus versus the particle. In some embodiments, Lp(a) refers to the lipoprotein(a) particle and / or to expression of the LPA gene (encoding apolipoprotein(a) (apo(a))). In some embodiments, “LP(a)” refers to the LPA gene. In some embodiments, provided DNA-targeting systems are capable of specifically targeting a target site in an enhancer of Lp(a). In provided embodiments, the enhancer is an enhancer that is distal to the transcription start site (TSS) of LP(a), either upstream or downstream of the TSS. In some embodiments, the enhancer is at or about 10 kB-150 kB or more upstream or downstream of the TSS of LP(a). In provided embodiments, the DNA-targeting systems include a DNA-binding domain that binds to a target site in a distal regulatory element located approximately 15 kB upstream of the TSS of Lp(a). In provided embodiments, the DNA-targeting 38MF-366426018Attorney No. 224742004040 systems include a DNA-binding domain that binds to a target site in a distal regulatory element located approximately 140 kB downstream of the TSS of Lp(a). In provided embodiments, the DNA-targeting systems include a DNA-binding domain that binds in proximal regulatory element located within 500 bp of the TSS of Lp(a). In provided embodiment, the DNA-targeting systems include a DNA-binding domain that binds the promoter of Lp(a).

[0184] In provided embodiments, the DNA-targeting systems are capable of targeting sites in more than one Lp(a) regulatory element. In some embodiments, the DNA-targeting systems target a target site in a distal regulatory element located approximately 15 kB upstream of the TSS of Lp(a) and target site in a distal regulatory element located approximately 140 kB downstream of the TSS of Lp(a). In some embodiments, the DNA-targeting systems target a target site in a distal regulatory element located approximately 15 kB upstream of the TSS of Lp(a) and target site in a proximal regulatory element located within 500 bp of the TSS of Lp(a). In some embodiments, the DNA-targeting systems target a target site in a distal regulatory element located approximately located approximately 140 kB downstream of the TSS of Lp(a) and target site in a proximal regulatory element located within 500 bp of the TSS of Lp(a).

[0185] In some embodiments, the DNA-targeting systems additionally include at least one effector domain that is able to epigenetically modify one or more DNA bases of the distal regulatory element, in which the epigenetic modification results in a reduction in transcription of Lp(a) (e.g. inhibits transcription or reduces transcription of Lp(a) compared to the absence of the DNA-targeting system). In some embodiments, the at least one effector domain is able to epigenetically modify one or more DNA bases of the proximal regulatory element. In some embodiments, reduced transcription of Lp(a) comprises reduced transcription of the LPA gene, resulting in reduced apo(a) expression and reduced formation and / or level of Lp(a) particles. Hence, the terms DNA-targeting system and epigenetic-modifying DNA-targeting system may be used herein interchangeably. In some embodiments, the DNA-targeting systems include a fusion protein comprising (a) at least one DNA-binding domain capable of being targeted to the target site of Lp(a); and (b) at least one effector domain capable of reducing transcription of Lp(a). For instance, the at least one effector domain is a transcription repressor domain.

[0186] In aspects of the provided embodiments, a DNA-targeting system provided herein targets a distal regulatory element located approximately 15 kB, a distal regulatory element located approximately 140 kB downstream upstream of the TSS of Lp(a), or a proximal regulatory element located within 500 bp of the TSS of Lp(a) to reduce transcription of Lp(a) in a liver cell, such as a hepatocyte, in which the reduced transcription modulates one or more activities or functions of liver cells, such as a phenotype of the liver cell. In some embodiments, reduced transcription of Lp(a) results in a reduced expression of Lp(a), i.e. reduced gene expression, in the liver cell. In some embodiments, reduced transcription comprises reduced transcription of the LPA gene, resulting in reduced apo(a) mRNA and / or apo(a)39MF-366426018Attorney No. 224742004040 protein in the liver cell. In some embodiments, the reduced transcription of Lp(a), such as reduced gene expression, promotes a phenotype that leads to reduction of Lp(a), such as in a subject. In some embodiments, reduction of Lp(a) comprises reduced apo(a) expression and reduced formation and / or reduced circulating levels of Lp(a) particles. In some embodiments, the reduced transcription of Lp(a) promotes a phenotype that leads to reduction of LDL, such as in a subject (i.e. an enhanced LDL-reducing phenotype).

[0187] In some aspects, the cell is a liver cell, such as a hepatocyte. In some aspects, the cell is a hepatocyte. For instance, provided herein is a DNA-targeting system that targets a distal regulatory element located approximately 15 kB upstream or approximately 140 kB downstream of the TSS of Lp(a) to reduce transcription of Lp(a) in a hepatocyte, in which the reduced transcription modulates one or more activities or functions of the hepatocyte, such as a phenotype of the hepatocyte. In some embodiments, reduced transcription of Lp(a) results in a reduction in expression of Lp(a), i.e. reduced gene expression, in the hepatocyte. In some embodiments, reduced expression comprises reduced expression of apo(a) encoded by the LPA gene. In some aspects, the cell is from a human subject. In some aspects the cell is a cell in a subject (i.e. a cell in vivo).

[0188] In some embodiments, the DNA-binding domain comprises or is derived from a CRISPR associated (Cas) protein, zinc finger protein (ZFP), transcription activator-like effectors (TALE), meganuclease, homing endonuclease, I-Scel enzyme, or variants thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive (e.g. nuclease-inactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA-binding domain comprises a deactivated Cas9 (dCas9) protein or variant thereof that is a catalytically inactivated so that it is inactive for nuclease activity and is not able to cleave the DNA.

[0189] In some embodiments, the DNA-binding domain comprises or is derived from a Cas protein or variant thereof, such as a nuclease-inactive Cas or dCas (e.g. dCas9), and the DNA-targeting system comprises one or more guide RNAs (gRNAs), such as a combination of gRNAs (e.g. two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence that is capable of targeting and / or hybridizing to the target site. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some aspects, the gRNA directs or recruits the Cas protein or variant thereof to the target site.

[0190] In some embodiments, the effector domain comprises a transcription repressor domain, and / or is capable of reducing transcription of the gene. In some embodiments, the effector domain directly or indirectly leads to reduced transcription of the gene. In some embodiments, the effector domain induces, catalyzes or leads to transcription repression. In some embodiments, the effector domain induces transcription repression. In some aspects, the effector domain is selected from a KRAB domain, ERF repressor domain, MXI1 domain, SID4X domain, MAD-SID domain, a DNMT family protein 40MF-366426018Attorney No. 224742004040 domain (e.g. DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g. DNMT3A / L, which comprises a fusion of DNMT3A and DNMT3L domains), LSD1, EZH2, a SunTag domain, a partially or fully functional fragment or domain of any of the foregoing, or a combination of any of the foregoing. In some embodiments, the effector domain is a KRAB domain. In some embodiments, the effector domain is DNMT3 A / L. In some embodiments, the effector domain comprises KRAB and DNMT3 A / L.

[0191] In some embodiments, the fusion protein of the DNA-targeting system comprises a dCas9-KRAB fusion protein. In some embodiments, the fusion protein of the DNA-targeting system comprises a DNMT3 A / L-dCas9-KRAB fusion protein. For purposes herein, unless a particular SEQ ID NO is identified or a particular order specified, the term DNMT3 A / L-dCas9-KRAB may refer to any orientation of the fusion protein as described above. In some embodiments, the fusion protein of the DNA-targeting system comprises in N- to C- terminal order a DNMT3 A / L-dCas9-KRAB- fusion protein. In some embodiments, the fusion protein of the DNA-targeting system comprises in N- to C-terminal order a KRAB-dCas9- DNMT3A / L- fusion protein.

[0192] Exemplary components and features of the DNA-targeting systems are provided below in the following subsections.A. Lp(a) Target Sites

[0193] In some aspects, provided herein are target sites for repression of Lp(a). In provided embodiments, reference to a target site or complement thereof (interchangeably, “complementary sequence”) refers to the region of DNA to which the spacer sequence hybridizes within and can be designated by a sequence that is complementary to the spacer (including the reverse complement when accounting for the 5’ to 3 ’ directionality of a sequence) or the sequence of the other strand of the paired DNA in the region. For instance, for purposes herein, the target site is typically designated by the same sequence as the spacer sequence (except that the target site sequence is DNA instead of RNA), which is the sequence of the other strand of the paired DNA that is in the region that the spacer hybridizes to. It is understood that reference to a target site or complement thereof (or variations thereof, such as “complementary”) can be a complement of the target site sequence, including the reverse complement (e.g., taking into account 5’ to 3’ directionality of a sequence).

[0194] In some embodiments, the target site is targeted using any of the provided DNA-targeting systems. In some embodiments, the target site is located within an enhancer of Lp(a). In some embodiments, the enhancer is an enhancer that is distal to the transcription start site (TSS) of Lp(a), either upstream or downstream of the TSS. In some embodiments, the enhancer is at or about 10 kB-150 kB or more upstream or downstream of the TSS of LP(a).41MF-366426018Attorney No. 224742004040

[0195] In some embodiments, the target site is located in a distal regulatory element approximately 15 kB upstream of the TSS of Lp(a). In some embodiments, the target site is located within an enhancer. In some embodiments, the enhancer is a distal regulatory element located approximately 15 kB upstream of the transcriptional start site (TSS) of Lp(a). In some embodiments, the target site is located between 14.5 and 15.5 kB upstream of the TSS of Lp(a), wherein the TSS of Lp(a) is located at hg38 genomic coordinate chr6: 160,664,275. In some embodiments, the target site is located between 14.5 and 15.5 kB upstream of hg38 genomic coordinate chr6: 160,664,275. In some embodiments, the enhancer is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782.

[0196] In some embodiments, the target site for Lp(a) is located between the human genome assembly GR38 (hg38) genomic coordinates chr6: 160, 678,800 and chr6: 160,679,650. In some embodiments, the target site for Lp(a) is located between the human genome assembly GR38 (hg38) genomic coordinates chr6: 160, 678,280 and chr6: 160,679,602. In some embodiments, the target site for Lp(a) is located between the human genome assembly GR38 (hg38) genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782. In some embodiments, the target site forLp(a) is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160, 679,000 and club:160,679,350. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700. In some embodiments, the target site forLp(a) is located between the hg38 genomic coordinates chr6: 160, 679,000-160,679,200. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 200-160,679,400. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 400-160,679,600, In some embodiments, the target site forLp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 500-160,679,700.

[0197] In some embodiments, the target site is within or overlaps the coordinates club: 160,679,292-160,679,311. In some embodiments, the target site is or includes the coordinates club: 160,679,292-160,679,311. In some embodiments, the target site is within or overlaps the coordinates club:160,679,619-160,679,638. In some embodiments, the target site is or includes the coordinates club: 160,679,619-160,679,638. In some embodiments, the target site is within or overlaps the coordinates club: 160,679,225,160,679,244. In some embodiments, the target site is or includes the coordinates club: 160,679,225,160,679,244. In some embodiments, the target site is within or overlaps the coordinates club: 160,679,477-160,679,496. In some embodiments, the target site is or includes the coordinates club: 160,679,477-160,679,496. In some embodiments, the target site is within or overlaps the coordinates club: 160,679,066-160,679,085. In some embodiments, the target site is or includes the coordinates club: 160,679,066-160,679,085. In some embodiments, the target site is within or overlaps the coordinates club: 160,678,956-160,678,975. In some embodiments, the target site is or includes the coordinates club:42MF-366426018Attorney No. 224742004040 160,678,956-160,678,975. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,679,313-160,679,332. In some embodiments, the target site is or includes the coordinates chr6: 160,679,313-160,679,332. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,679,046-160,679,065. In some embodiments, the target site is or includes the coordinates chr6: 160,679,046-160,679,065. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,679,124-160,679,143. In some embodiments, the target site is or includes the coordinates chr6: 160,679,124-160,679,143. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,679,280-160,679,299. In some embodiments, the target site is or includes the coordinates chr6: 160,679,280-160,679,299. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,679,345-160,679,364. In some embodiments, the target site is or includes the coordinates chr6: 160,679,345-160,679,364. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,679,583-160,679,602. In some embodiments, the target site is or includes the coordinates chr6: 160,679,583-160,679,602. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,679,865-160,679,884. In some embodiments, the target site is or includes the coordinates chr6: 160,679,865-160,679,884.

[0198] In some embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35, 256-258, 418-424, 528-533, 546, and 604 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS: 31-35, 256-258, 418-424, 528-533, 546, and 604 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOS: 31-35,256-258, 418-424, 528-533, 546, and 604.

[0199] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 31 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO:31, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing.. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 31, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the target site is the sequence set forth in SEQ ID NO:31.

[0200] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 32 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO:32, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some43MF-366426018Attorney No. 224742004040 embodiments, the target site comprises the sequence set forth in SEQ ID NO: 32, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 32. In some embodiments, the target site is the sequence set forth in SEQ ID NO:32.

[0201] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 33 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO:33, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing.. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 33, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 33. In some embodiments, the target site is the sequence set forth in SEQ ID NO:33.

[0202] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 34 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO:34, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing.. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 34, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the target site is the sequence set forth in SEQ ID NO:34.

[0203] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 35 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO:35, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing.. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 35, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 35. In some embodiments, the target site is the sequence set forth in SEQ ID NO:35.

[0204] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:256 or SEQ ID NO: 418 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 256 or SEQ ID NO: 418, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing, of any of the foregoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 256 or SEQ ID NO: 418, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 256 or SEQ ID NO: 418. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 256 or SEQ ID NO: 418.

[0205] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:257 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set 44MF-366426018Attorney No. 224742004040 forth in SEQ ID NO:257, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 257, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 257. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 257.

[0206] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:258 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO:258, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 258, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 258. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 258.

[0207] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:419 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO:419, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 419, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 419. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 419.

[0208] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 420 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 420, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 420, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 420. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 420.

[0209] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:421 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 421, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 421, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 421. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 421.

[0210] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 422 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set 45MF-366426018Attorney No. 224742004040 forth in SEQ ID NO: 422, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 422, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 422. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 422.

[0211] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:423 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 423, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 423, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 423. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 423.

[0212] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 424 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 424, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 424, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 424. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 424.

[0213] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 528 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 528, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 528, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 528. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 528.

[0214] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 529 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 529, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 529, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 529. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 529.

[0215] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 530 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set 46MF-366426018Attorney No. 224742004040 forth in SEQ ID NO: 530, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 530, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 530. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 530.

[0216] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 531 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 531, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 531, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 531. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 531.

[0217] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 532 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 532, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 532, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 532. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 532.

[0218] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 533 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 533, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 533, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 533. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 533.

[0219] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 546 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 546, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 546, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 546. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 546.

[0220] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 604 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set 47MF-366426018Attorney No. 224742004040 forth in SEQ ID NO: 604, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 604, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 604. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 604.

[0221] In some embodiments, the target site is located approximately 140 kB downstream of the transcriptional start site (TSS) of Lp(a) that has the genomic coordinates chr6: 160,664,275. In some embodiments, the enhancer is a distal regulatory element located approximately 10 kB downstream of the coding sequence of Lp(a) which ends at genomic coordinate 160,531,482. and 9kB away from lipoproteinA like2 (LPAL2) which is located at club: 160,466,555-160,511,124 In some embodiments, the upstream enhancer also is an enhancer that is approximately 9 kB upstream of the TSS of lipoprotein(a) like 2 pseudogene (LPAL2) which starts at genomic coordinate 160,511,124. LPAL2 gene also is located on chromosome 6 in the same region that contains the LP(a) gene. LPAL2 is considered a pseudogene because it is not known to produce functional proteins. The LP(a) gene is located on chromosome 6 at 6q25.3-q26. Specifically, the genomic coordinates forLPA are club: 160,531,482-160,664,275 in the GRCh38 reference genome. The LPAL2 gene, which is a pseudogene related to LP(a), has genomic coordinates club: 160,466,555-160,511, 124. In some embodiments, the enhancer is located between the hg38 genomic coordinates club: 160,519,674 and 160,520,973.

[0222] In some embodiments, the target site for Lp(a) is located between the human genome assembly GR38 (hg38) genomic coordinates club: club: 160,519,674 and 160,520,973. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club:160,519,674 and club: 160,520,320. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160,520,320 and club: 160,520,973. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160,519,674 and club:160,519,974. In some embodiments, the target site forLp(a) is located between the hg38 genomic coordinates club: 160,519,974 and 160,520,174. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160,520,174 and 160,520,374. In some embodiments, the target site forLp(a) is located between the hg38 genomic coordinates club: 160,520,374 and 160,520,574, In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160,520,574 and 160,520,774. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates club: 160,520,774 and 160,520,973.

[0223] In some embodiments, the target site is within or overlaps the coordinates club: 160,519,674- 160,519,974. In some embodiments, the target site is or includes the coordinates club: 160,519,674- 160,519,974. In some embodiments, the target site is within or overlaps the coordinates club:160,519,974-160,520,174. In some embodiments, the target site is or includes the coordinates club:48MF-366426018Attorney No. 224742004040 160,519,974-160,520,174. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,520,174-160,520,374. In some embodiments, the target site is or includes the coordinates chr6: 160,520,174-160,520,374. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,520,374-160,520,574. In some embodiments, the target site is or includes the coordinates chr6: 160,520,374-160,520,574. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,520,574-160,520,774. In some embodiments, the target site is or includes the coordinates chr6: 160,520,574-160,520,774. In some embodiments, the target site is within or overlaps the coordinates chr6: 160,520,774-160,520,973. In some embodiments, the target site is or includes the coordinates chr6: 160,520,774-160,520,973.

[0224] In some embodiments, the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 512-521 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 512-521 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NO: 512-521.

[0225] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 512 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 512, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 512, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 512. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 512.

[0226] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 513 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 513, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 513, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 513. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 513.

[0227] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 514 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 514, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some 49MF-366426018Attorney No. 224742004040 embodiments, the target site comprises the sequence set forth in SEQ ID NO: 514, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 514. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 514.

[0228] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 515 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 515, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 515, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 515. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 515.

[0229] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 516 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 516, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 516, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 516. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 516.

[0230] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 517 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 517, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 517, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 517. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 517.

[0231] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 517 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 518, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 518, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 518. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 518.

[0232] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 519 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 519, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some 50MF-366426018Attorney No. 224742004040 embodiments, the target site comprises the sequence set forth in SEQ ID NO: 519, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 519. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 519.

[0233] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 520 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 520, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 520, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 520. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 520.

[0234] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 521 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 521, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 521, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 521. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 521.

[0235] In some embodiments, the target site for Lp(a) is located within 500bp of the TSS located at hg38 genomic coordinate chr6: 160,664,275. In some embodiments, the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160,663,920-160,664,656. In some embodiments, the target site for Lp(a) is located is located within 250 bp the hg38 genomic coordinate chr6: 160,664,275.

[0236] In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOS: 1-10 and 522-524, a contiguous portion thereof of at least 14 nucleotides of any one of SEQ ID NOS: 1-10 and 522-524, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS: 1-10 and 522-524 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOS: 1-10 and 522-524.

[0237] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 4 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 4, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 4, or a complementary 51MF-366426018Attorney No. 224742004040 sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 4.

[0238] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 7 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 7, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 7, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 7.

[0239] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 522 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 522 or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 522, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 522. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 522.

[0240] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 523 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 523 or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 523, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 523. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 523.

[0241] In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 524 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 524 or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 524, or a complementary sequence thereof. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 524. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 524.

[0242] In some embodiments, the DNA-targeting systems target a first Lp(a) target site and a second Lp(a) target site. In some embodiments, the first target site is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and the second target site is within 500 bp of the hg38 genomic coordinate chr6: 160,664,275. In some embodiments, the first target site is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and the second target 52MF-366426018Attorney No. 224742004040 site is located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973. In some embodiments, the first target site is located between the hg38 genomic coordinates 160,519,674 and chr6: 160,520,973 and the second target site is within 500 bp of the hg38 genomic coordinate chr6: 160,664,275.B. CRISPR-Based DNA-Targeting Systems

[0243] Provided herein are epigenetic -targeting DNA-targeting systems based on CRISPR / Cas systems, i.e. CRISPR / Cas-based DNA-targeting systems, that are able to bind to a target site of Lp(a). In some embodiments, the CRISPR / Cas DNA-binding domain is nuclease inactive, such as includes a dCas (e.g. dCas9) so that the system binds to the target site without mediating nucleic acid cleavage at the target site. The CRISPR / Cas-based DNA-targeting systems may be used to modulate expression of Lp(a) in a cell, such as a hepatocyte. In some embodiments, the target site of Lp(a) may include any as described herein, including any described above in Section I. A. In some embodiments, the CRISPR / Cas-based DNA-targeting system can include any known Cas enzyme, and generally a nuclease-inactive or dCas. In some embodiments, the CRISPR / Cas-based DNA-targeting system includes a fusion protein of a nuclease-inactive Cas protein or a variant thereof and an effector domain that reduces transcription of a gene (e.g. a transcriptional repressor), and at least one gRNA.

[0244] The CRISPR system (also known as CRISPR / Cas system, or CRISPR-Cas system) refers to a conserved microbial nuclease system, found in the genomes of bacteria and archaea, that provides a form of acquired immunity against invading phages and plasmids. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), refers to loci containing multiple repeating DNA elements that are separated by non-repeating DNA sequences called spacers. Spacers are short sequences of foreign DNA that are incorporated into the genome between CRISPR repeats, serving as a 'memory' of past exposures. Spacers encode the DNA-targeting portion of RNA molecules that confer specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, which can act as RNA-guided nucleases for mediating the cleavage, as well as non-protein coding DNA elements that encode RNA molecules capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0245] In Type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The trans-activating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex.

[0246] Naturally occurring CRISPR / Cas systems, such as those with Cas9, have been engineered to 53MF-366426018Attorney No. 224742004040 allow efficient programming of Cas / RNA RNPs to target desired sequences in cells of interest, both for gene-editing and modulation of gene expression. The tracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, commonly referred to as a guide RNA (gRNA), for example as described in WO 2013 / 176772, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), or Cong, L. et al. Science 339(6121):819-23 (2013). The spacer sequence of the gRNA can be chosen by a user to target the Cas / gRNA RNP complex to a desired locus, e.g. a desired target site in the target gene.

[0247] Cas proteins have also been engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. An exemplary deactivated Cas9 (dCas9) derived from S. pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013 / 176772, WO 2014 / 093661, Jinek, M. et al. Science 337(6096):816-21 (2012), and Qi, L. et al. Cell 152(5): 1173-83 (2013).Exemplary dCas variants derived from the Cas 12 system (i.e. Cpfl) are described, for example in WO 2017 / 189308 and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0248] dCas-fusion proteins with transcriptional and / or epigenetic regulators have been used as a versatile platform for ectopically regulating gene expression in target cells. These include fusion of a Cas with an effector domain, such as a transcriptional activator or transcriptional repressor. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide composed of four tandem copies of VP16, a 16 amino acid transactivation domain of the Herpes simplex virus) can result in robust induction of gene expression. Alternatively, fusing dCas9 with a transcriptional repressor such as KRAB (Krüppel associated box) can result in robust repression of gene expression. A variety of dCas-fusion proteins with transcriptional and epigenetic regulators can be engineered for regulation of gene expression, for example as described in WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2021 / 247570, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973–976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833–838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977–979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nuñez, J.K. et al. Cell 184(9):2503-2519 (2021).

[0249] In some aspects, provided is a DNA-targeting system comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or variant thereof, and an effector 54MF-366426018Attorney No. 224742004040 domain for reducing transcription or inducing transcriptional repression (i.e. a transcriptional repressor) when targeted to the target gene in the cell (e.g. hepatocyte). In such embodiments, the DNA-targeting system also includes one or more gRNAs, provided in combination or as a complex with the dCas protein or variant thereof, for targeting of the DNA-targeting system to the target site of Lp(a). In some embodiments, the fusion protein is guided to a specific target site sequence of the target gene by the guide RNA, wherein the effector domain mediates targeted epigenetic modification to reduce or repress transcription of Lp(a). Any of a variety of effector domains that reduce or repress transcription can be used as described further below.1. CRISPR-based DNA-binding domains

[0250] In some aspects, the DNA-binding domain comprises a CRISPR-associated (Cas) protein or variant thereof, or is derived from a Cas protein or variant thereof. In particular embodiments here, the Cas protein is nuclease-inactive (i.e. is a dCas protein).

[0251] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e. multiple Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e. single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Type V CRISPR system.

[0252] CRISPR / Cas systems may be multi-protein systems or single effector protein systems. Multiprotein, or Class 1, CRISPR systems include Type I, Type III, and Type IV systems. In some aspects, Class 2 systems include a single effector molecule and include Type II, Type V, and Type VI. In some embodiments, the DNA targeting system comprises components of CRISPR / Cas systems, such as a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Class 1 CRISPR system (i.e., multiple Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is from a Class 2 CRISPR system (i.e., single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system.

[0253] Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S. B. et al. Exp. Mol. Med. 51, 1–11 (2019), Zhang, F. Q. Rev. Biophys. 52, E6 (2019), and Makarova K. S. et al. Methods Mol. Biol. 1311:47-75 (2015).

[0254] Type I CRISPR / Cas systems employ a large multi-subunit ribonucleoprotein (RNP) complex called Cascade that recognizes double-stranded DNA (dsDNA) targets. After target recognition and verification, Cascade recruits the signature protein Cas3, a fused helicase-nuclease, to degrade DNA.

[0255] In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described 55MF-366426018Attorney No. 224742004040 in WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013). In Type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The transactivating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex. Cas9 mediates cleavage of target DNA if a correct protospacer-adjacent motif (PAM) is also present at the 3' end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer-adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage.

[0256] Different Type II systems have differing PAM requirements. The S. pyogenes CRISPR system may have the PAM sequence for this Cas9 (SpCas9) as 5'-NRG-3', where R is either A or G, and characterized the specificity of this system in human cells. A unique capability of the CRISPR / Cas9 system is the straightforward ability to simultaneously target multiple distinct genomic loci by coexpressing a single Cas9 protein with two or more sgRNAs. For example, the Streptococcus pyogenes Type II system typically prefers to use an “NGG” (SEQ ID NO: 157) sequence, where “N” can be any nucleotide, but also accepts other PAM sequences, such as “NAG” in engineered systems (Hsu et al., Nature Biotechnology (2013) doi: 10.1038 / nbt.2647). Similarly, the Cas9 derived from Neisseria meningitidis (NmCas9) normally has a native PAM of NNNNGATT (SEQ ID NO: 159), but has activity across a variety of PAMs, including a highly degenerate NNNNGNNN (SEQ ID NO: 242) PAM (Esvelt et al. Nature Methods (2013) doi: 10.1038 / nmeth.2681). In another example, the Cas9 derived from Campylobacter jejuni typically uses 5'-NNNNACAC-3' (SEQ ID NO: 243) or 5'-NNNNRYAC-3' (SEQ ID NO: 160) PAM sequences, where “N” can be any nucleotide, “R” can be either guanine (G) or adenine (A), and “Y” can be either cytosine (C) or thymine (T). In some aspects, the PAM sequences for spacer targeting depends on the type, ortholog, variant or species of the Cas protein.

[0257] In Type III systems, the RNP complex is multimeric with a helicoid structure similar to Cascade. In contrast to Type I CRISPR / Cas systems, the Type III RNP complex recognizes complementary RNA sequences instead of dsDNA. RNA recognition stimulates a nonspecific DNA cleavage activity of the exemplary Type III Cas 10 nuclease that is part of the RNP complex, such that DNA cleavage is achieved co-transcriptionally.

[0258] In some embodiments, the Cas protein is from a Type V CRISPR system. Exemplary Cas proteins of a Type V CRISPR system include Casl2a (also known as Cpfl), Casl2b (also known as C2cl), Casl2e (also known as CasX), Casl2k (also known as C2c5), Casl4a, and Casl4b. In some 56MF-366426018Attorney No. 224742004040 embodiments, the Cas protein is from a Casl2 protein (i.e., Cpfl) or variant thereof, for example as described in WO 2017 / 189308, WO2019 / 232069 and Zetsche et al. Cell. 163 (3):759-71 (2015).

[0259] Exemplary Type V systems include those based on a Cas 12 effector, and the C-terminus with only one RuvC endonuclease domain is the defining characteristic of the Type V systems. The RuvC nuclease domain cleaves dsDNA adjacent to protospacer adjacent motif (PAM) sequences and singlestranded DNA (ssDNA) nonspecifically. The Type V systems can be further divided into subtypes, each characterized by different signature proteins, PAM sequences, and properties. Non-limiting exemplary Cas proteins derived from Type V CRISPR systems include Casl2a (Cpfl), UnlCasl2fl, Casl2j (CasPhi, such as CasPhi-2), Casl2k, and CasMini. For example, Type V-A includes, for example, Cas 12a, which uses “TTTV” (SEQ ID NO: 164) PAM sequence, where “V” is adenine (A), cytosine (C), or guanine (G). Type V-F is includes, for example, Casl2f, which can use “TTTR” (SEQ ID NO: 239), where “R” is G or A, or “TTTN” (SEQ ID NO: 240), where “N” is any nucleotide. Type V-K is includes, for example, Cas 12k, which uses “GGTT” (SEQ ID NO: 241) PAM sequence.

[0260] In some embodiments, the Cas protein is derived from a Cas 12 protein (i.e. Cpfl) or variant thereof, for example as described in WO 2017 / 189308 and Zetsche, B. et al. Cell. 163 (3):759-71 (2015). In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described in WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121): 819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013). Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S. B. et al. Exp. Mol. Med. 51, 1–11 (2019), Zhang, F. Q. Rev. Biophys. 52, E6 (2019), and Makarova K. S. et al. Methods Mol. Biol. 1311:47-75 (2015).

[0261] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes.

[0262] Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in Acaryochloris marina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium 57MF-366426018Attorney No. 224742004040 vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS 10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. LochMaree; Clostridium botulinum Ba4 str. 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua', Lyngbya sp. PCC 8106; Marinobacter sp. ELB17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis', Nitrosococcus halophilus Nc4;Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinae spiralis ATCC 25486; Streptococcus thermophilus', Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

[0263] In some aspects, the Cas protein is a variant that lacks nuclease activity (i.e. is a dCas protein). In some embodiments, the Cas protein is mutated so that nuclease activity is reduced or eliminated. Such Cas proteins are referred to as deactivated Cas or dead Cas (dCas) or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9, or iCas9) protein.

[0264] In some embodiments, the DNA-targeting systems or fusion proteins comprise a Cas protein or variant thereof, such as a Cas protein or variant thereof set forth in any one of SEQ ID NOs: 125-128 and 216-237, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 125-128 and 216-237. In some embodiments, the Cas protein or variant thereof of any of the DNA-targeting systems or fusion proteins provided herein comprise a sequence set forth in any one of SEQ ID NOs: 125-128 and 216-237. In some aspects, the Cas protein or variant thereof lacks an initial methionine residue. In some aspects, the Cas protein or variant thereof comprises an initial methionine residue.

[0265] In some embodiments, the Cas9 protein or a variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a58MF-366426018Attorney No. 224742004040 Staphylococcus aureus dCas9 protein (dSaCas9) that comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 126 or SEQ ID NO: 216, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 126, which lacks an initial methionine residue. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 216, which includes an initial methionine residue.

[0266] In some embodiments, the Cas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein that comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 127. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 128 or SEQ ID NO: 217, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 128, which lacks an initial methionine residue. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 217, which includes an initial methionine residue.

[0267] In some embodiments, the Cas9 protein or variant thereof is a Campylobacter jejuni Cas9 (CjCas9) protein or a variant thereof. In some embodiments, the variant Cas9 comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 218 or 219. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 224, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 225, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 225, which lacks an initial methionine residue. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 224, which includes an initial methionine residue.

[0268] In some embodiments, the Cas protein or a variant thereof is a Casl2a protein or a variant thereof. In some embodiments, the variant Cas protein is a variant Casl2a protein that lacks nuclease activity or that is a deactivated Casl2a (dCasl2a) protein. In some embodiments, the Casl2a protein or variant thereof is a Acidaminococcus sp. Cas 12a (AsCasl2a) protein or a variant thereof. In some embodiments, the variant Casl2a is a Acidaminococcus sp. dCasl2a (dAsCasl2a) protein that comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 222 or 223. In some 59MF-366426018Attorney No. 224742004040 embodiments, the variant Casl2a protein comprises the sequence set forth in SEQ ID NO: 220, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Casl2a protein comprises the sequence set forth in SEQ ID NO: 221, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Casl2a protein comprises the sequence set forth in SEQ ID NO: 221, which lacks an initial methionine residue. In some embodiments, the variant Casl2a protein comprises the sequence set forth in SEQ ID NO: 220, which includes an initial methionine residue.

[0269] In some embodiments, the Cas protein or a variant thereof is a CasPhi-2 protein or a variant thereof. In some embodiments, the variant Cas protein is a variant CasPhi-2 protein that lacks nuclease activity or that is a deactivated CasPhi-2 (dCasPhi-2) protein. In some embodiments, the variant CasPhi-2 comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 226 or 227. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 228, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 229, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 229, which lacks an initial methionine residue. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 228, which includes an initial methionine residue.

[0270] In some embodiments, the Cas protein or a variant thereof is a UnlCasl2fl protein or a variant thereof. In some embodiments, the variant Cas protein is a variant UnlCasl2f 1 protein that lacks nuclease activity or that is a deactivated UnlCasl2fl (dUnlCasl2fl) protein. In some embodiments, the variant UnlCasl2fl comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 230 or 231. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO: 232, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO: 233, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO: 233, which lacks an initial methionine residue. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO: 232, which includes an initial methionine residue.

[0271] In some embodiments, the Cas protein or a variant thereof is a Cas 12k protein or a variant thereof. In some embodiments, the Casl2k protein comprises the sequence set forth in SEQ ID NO: 234, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%60MF-366426018Attorney No. 224742004040 sequence identity thereto. In some embodiments, the Casl2k protein comprises the sequence set forth in SEQ ID NO: 235, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the Casl2k protein comprises the sequence set forth in SEQ ID NO: 235, which lacks an initial methionine residue. In some embodiments, the Casl2k protein comprises the sequence set forth in SEQ ID NO: 234, which includes an initial methionine residue.

[0272] In some embodiments, the Cas protein or a variant thereof is a CasMini protein or a variant thereof, such as an engineered Cas protein or variant based on a Casl2f (also known as Casl4), including those described in Xu et al., Mol. Cell 81(20):4333-4345 (2021) or set forth in SEQ ID NO: 238. In some embodiments, the variant Cas protein is a variant CasMini protein that lacks nuclease activity or that is a deactivated CasMini (dCasMini) protein. In some embodiments, the variant CasMini comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 238. In some embodiments, the variant CasMini protein comprises the sequence set forth in SEQ ID NO: 238, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CasMini protein comprises the sequence set forth in SEQ ID NO: 238. In some embodiments, the variant CasMini protein comprises the sequence set forth in SEQ ID NO: 236 or 237, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CasMini protein comprises the sequence set forth in SEQ ID NO: 237, which lacks an initial methionine residue. In some embodiments, the CasMini protein comprises the sequence set forth in SEQ ID NO: 236, which includes an initial methionine residue.2. Guide RN As

[0273] In some embodiments, the Cas protein (e.g. dCas9) is provided in combination or as a complex with one or more guide RNA (gRNA). In some aspects, the gRNA is a nucleic acid that promotes the specific targeting or homing of the gRNA / Cas RNP complex to the target site of the target gene, such as any described above. In some embodiments, a target site of a gRNA may be referred to as a protospacer. In some embodiments the gRNA hybridizes to the sequence complementary to the sequence defined as the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid.

[0274] Provided herein are gRNAs, such as gRNAs that target or bind to a target site of a target gene (i.e., Lp(a)). In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e., a spacer sequence or a guide sequence) that is capable of hybridizing to the target site, or that is complementary to the target site, such as any target site described in Section I. A or further below. In some embodiments,61MF-366426018Attorney No. 224742004040 the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein.

[0275] In some embodiments, the gRNAs provided herein are chimeric gRNAs. In general, gRNAs can be unimolecular (i.e. composed of a single RNA molecule), or modular (comprising more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, wherein sequences from the separate modular RNA molecules are comprised in a single gRNA molecule, sometimes referred to as a chimeric gRNA, synthetic gRNA, or single gRNA. In some embodiments, the chimeric gRNA is a fusion of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence, for example as described in WO 2013 / 176772, or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0276] In some aspects, the spacer sequence of a gRNA is a polynucleotide sequence comprising at least a portion that has sufficient complementarity with the target gene or DNA regulatory element thereof (e.g. any described in Section I. A) to hybridize with a target site in the target gene and direct sequence-specific binding of a CRISPR complex to the sequence of the target site. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. In some embodiments, the gRNA comprises a spacer sequence that is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary), to the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid.

[0277] In some embodiments, the gRNA spacer sequence is between about 14 nucleotides (nt) and about 26 nt, or between 16 nt and 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt or 22 nt in length. In some embodiments, the gRNA spacer sequence is 19 nt in length.

[0278] A target site of a gRNA may be referred to as a protospacer. In some aspects, the gRNA spacer is designed to target a protospacer with a specific protospacer-adjacent motif (PAM), i.e. a sequence immediately adjacent to the protospacer that contributes to and / or is required for Cas binding specificity. Different CRISPR / Cas systems have different PAM requirements for targeting. For example, in some embodiments, S. pyogenes Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO:86), where N is any nucleotide. S. aureus Cas9 uses the PAM 5’- NNGRRT-3’ (SEQ ID NO:158), where N is any nucleotide, and R is G or A. N. meningitidis Cas9 uses the PAM 5'-NNNNGATT -3 ’ (SEQ ID NO: 159), where N is any nucleotide. C. jejuni Cas9 uses the PAM 5'-NNNNRYAC-3' (SEQ ID NO: 160), where N is any nucleotide, R is G or A, and Y is C or T. S', thermophilus uses the PAM 5’-NNAGAAW-3’(SEQ ID NO: 161), where N is any nucleotide and W is A or T. F. Novicida Cas9 uses the PAM 5’-NGG-3 ’(SEQ 62MF-366426018Attorney No. 224742004040 ID NO:162), where N is any nucleotide. T. denticola Cas9 uses the PAM 5’-NAAAAC-3’(SEQ ID NO: 163), where N is any nucleotide. Casl2a (also known as Cpfl) from various species, uses the PAM 5’-TTTV-3’(SEQ ID NO: 164). Cas proteins may use or be engineered to use different PAMs from those listed above. For example, variant SpCas9 proteins may use a PAM selected from: 5’-NGG-3’ (SEQ ID NO:86), 5’-NGAN-3’ (SEQ ID NO:165), 5’-NGNG-3’(SEQ ID NO:166), 5’-NGAG-3’(SEQ ID NO: 167), or 5’-NGCG-3’(SEQ ID NO: 168). In some embodiments, the PAM sequence for complexing with S'. pyogenes Cas9 or variant thereof is set forth in SEQ ID NO: 157. In some embodiments, the PAM sequence for complexing with S'. aureus Cas9 or variant thereof is set forth in SEQ ID NO: 158. Methods for designing or identifying gRNA spacer sequences and / or protospacer sequences in a particular region, are known. gRNA spacer sequences and / or protospacer sequences can be determined based on the type of Cas protein used and the associated PAM sequence.

[0279] A gRNA spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0280] In some embodiments, the gRNA (including the guide sequence) will comprise the base uracil (U), whereas DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the guide sequence with the target sequence contributes to specificity of the interaction of the gRNA molecule / Cas molecule complex with a target nucleic acid. It is understood that in a guide sequence and target sequence pair, the uracil bases in the guide sequence will pair with the adenine bases in the target sequence. A gRNA spacer sequence herein may be defined by the DNA sequence encoding the gRNA spacer, and / or the RNA sequence of the spacer.

[0281] In some embodiments, one, more than one, or all of the nucleotides of a gRNA can have a modification, e.g., to render the gRNA less susceptible to degradation and / or improve bio-compatibility. By way of non-limiting example, the backbone of the gRNA can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the gRNA can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0282] Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., International PCT Pub. Nos. WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, WO 2015 / 089427, WO 2016 / 049258, WO 2016 / 123578, WO 2021 / 076744, WO 2014 / 191128, WO 2015 / 161276, WO 2017 / 193107, and WO 2017 / 093969.

[0283] In some aspects, the gRNA comprises scaffold sequences. In some aspects, the scaffold sequence (in some cases including a crRNA sequence and / or a tracrRNA sequence) will be different depending on the Cas protein. In some aspects, different CRISPR / Cas systems have different gRNA 63MF-366426018Attorney No. 224742004040 scaffold sequences for associating with Cas protein. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO: 85, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:85. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:85. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:259, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 259. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO: 259.

[0284] In some embodiments, an exemplary scaffold sequence comprises a 3 ’ polyU sequence. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:450 or SEQ ID NO: 451, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 450 or SEQ ID NO: 451. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO: 450 or SEQ ID NO: 451.

[0285] In some aspects, the gRNA can target the DNA-targeting system to direct the activities of an associated polypeptide (e.g., fusion protein, DNA-targeting system, effector domain, etc.) to a specific target site within a target nucleic acid (e.g., an enhancer of Lp(a)).

[0286] In some embodiments, a gRNA provided herein targets a target site located within an enhancer of Lp(a).

[0287] In some embodiments, the gRNA targets a target site located in a distal regulatory element approximately 15 kB upstream of the TSS of Lp(a). In some embodiments, the gRNA targets a target site located between 14.5 and 15.5 kB upstream of the TSS of Lp(a), wherein the TSS of Lp(a) is located at hg38 genomic coordinate chr6: 160,664,275. In some embodiments, the gRNA targets a target site that is located between the human genome assembly GR38 (hg38) genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782, which is an enhancer of Lp(a). In some embodiments, the gRNA targets a target site that is located between the human genome assembly GR38 (hg38) genomic coordinates chr6: 160, 678,800 and chr6: 160,679,650. In some embodiments, the gRNA targets a target site that is located between the human genome assembly GR38 (hg38) genomic coordinates chr6: 160, 678,280 and chr6: 160,679,602. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160, 679,000 and chr6:160,679,350. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160, 679,000- 64MF-366426018Attorney No. 224742004040 160,679,200. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160, 679, 200-160,679,400. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160, 679, 400-160,679,600, In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160, 679, 500-160,679,700.

[0288] In some embodiments, the gRNA targets a target site located in a distal regulatory element approximately 15 kB upstream of the TSS of Lp(a). In some embodiments, the gRNA targets a target site that comprises a sequence selected from any one of SEQ ID NOS: 31-35, 256-258, 418-424, 528-533, 546, and 604, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS:31-35, 256-258, 418-424, 528-533, 546, and 604 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOS: 31-35, 256-258, 418-424, 528-533, 546, and 604. In some embodiments, the gRNA targets a target site in an enhancer of Lp(a).

[0289] In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:31 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:31. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:32 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:32. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:33 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:33. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:34 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:34. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:35 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:35. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 256 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 256. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 257 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 257. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 258 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set 65MF-366426018Attorney No. 224742004040 forth in SEQ ID NO: 258. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 418 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 418. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 419 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 419. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 420 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 420. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 421 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 421. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 422 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 422. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 423 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 423. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 424 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 424.

[0290] In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 528 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 528. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 529 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 529. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 530 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 530. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 531 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 531. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 532 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 532. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 533 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 533. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 546 or a contiguous portion thereof of at least 14 nucleotides.66MF-366426018Attorney No. 224742004040 In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 546. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 604 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 604.

[0291] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOS: 36-40,253-255, 417, 425-430, 534-539, 547, and 605 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOS: 36-40,253-255, 417, 425-430, 534-539, 547, and 605 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 36–40, 253–255, 417, 425–430, 534–539, 547, and 605.

[0292] In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:36 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:36. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:37 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:37. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:38 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:38. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:39 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:39. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:40 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:40. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 253 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 253. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 254 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 254. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 255 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 255. In some embodiments, the spacer sequence of the gRNA includes 67MF-366426018Attorney No. 224742004040 or is set forth in SEQ ID NO: 417 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 417. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 425 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 425. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 426 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 426. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 427 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 427. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 428 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 428. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 429 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 429. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 430 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 430.

[0293] In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 534 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 534. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 535 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 535. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 536 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 536. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 537 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 537. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 538 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 538. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 539 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 1968MF-366426018Attorney No. 224742004040 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 539. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 547 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 547. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 605 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 605.

[0294] In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOS: 41–45, 247–252, 439–449, 485–511, 540–545, 547, and 606–607 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 41–45, 247–252, 439–449, 485–511, 540–545, 547, and 606–607. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 41–45, 247–252, 439–449, 485–511, 540–545, 547, and 606–607.

[0295] In some embodiments, the gRNA targets a target site located in a distal regulatory element approximately 140 kB downstream of the transcriptional start site (TSS) of Lp(a). In some embodiments, the gRNA targets a target site that is located between the human genome assembly GR38 (hg38) genomic coordinates chr6: 160,519,674 and 160,520,973, which is an enhancer of Lp(a). In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160,519,674 and club: 160,520,320. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates club: 160,520,320 and club: 160,520,973. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates club: 160,519,674 and club: 160,519,974. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates club: 1160,519,974 and 160,520,174. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates club: 160,520,174 and 160,520,374. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates club: 160,520,374 and 160,520,574. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates club: 160,520,574 and 160,520,774. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates club: 160,520,774 and 160,520,973.

[0296] In some embodiments, the gRNA targets a target site for Lp(a) that comprises a sequence selected from any one of SEQ ID NOS: 512-521, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS: 512-521 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set 69MF-366426018Attorney No. 224742004040 forth in any one of SEQ ID NOS: 512-521.

[0297] In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:512 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:512. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:513 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:513. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:514 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 514. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 515 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:515. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:516 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:516. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 517 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:517. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:518 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:518. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO: 519 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:519. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:520 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:520. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:521 or a contiguous portion thereof of at least 14 nucleotides. In some embodiments, the gRNA targets a target site that includes or is set forth in SEQ ID NO:521.

[0298] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOS: 577-586 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOS: 577-586 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 577-586.

[0299] In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:577 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 1970MF-366426018Attorney No. 224742004040 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:577. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:578 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:578. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:579 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:579. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:580 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:580. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:581 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 581. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:582 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 582. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:583 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 583. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:584 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 584. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:585 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 585. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:586 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 586.

[0300] In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOS: 590–599 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 590–599. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 590–599.

[0301] In some embodiments, the gRNA targets a target site located in a proximal regulatory within 500 bp of the TSS of Lp(a). In some embodiments, the gRNA targets a target site located within 500bp of the TSS located at hg38 genomic coordinate chr6: 160,664,275. In some embodiments, the gRNA targets a target site that is located between the hg38 genomic coordinates chr6: 160,663,920-160,664,656. In 71MF-366426018Attorney No. 224742004040 some embodiments, the gRNA targets a target site that is located is located within 250 bp the hg38 genomic coordinate chr6: 160,664,275.

[0302] In some embodiments, the gRNA targets a target site that comprises a sequence selected from any one of SEQ ID NOS: 1-10 and 522-524, a contiguous portion thereof of at least 14 nucleotides of any one of SEQ ID NOS: 1-10 and 522-524, or a complementary sequence of any of the foregoing. In some embodiments, the gRNA targets a target site that is a contiguous portion of any one of SEQ ID NOS: 1-10 and 522-524 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the gRNA targets a target site that is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the gRNA targets a target site that is the sequence set forth in any one of SEQ ID NOS: 1-10 and 522-524.

[0303] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 4 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 4, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 4, or a complementary sequence thereof. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the gRNA targets a target site that is the sequence set forth in SEQ ID NO: 4.

[0304] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 7 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 7, or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 7, or a complementary sequence thereof. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, the gRNA targets a target site that is the sequence set forth in SEQ ID NO: 7.

[0305] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 522 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 522 or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 522, or a complementary sequence thereof. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 522. In some embodiments, the gRNA 72MF-366426018Attorney No. 224742004040 targets a target site that is the sequence set forth in SEQ ID NO: 522.

[0306] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 523 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 523 or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 523, or a complementary sequence thereof. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 523. In some embodiments, the gRNA targets a target site that is the sequence set forth in SEQ ID NO: 523.

[0307] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 524 or a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to the sequence set forth in SEQ ID NO: 524 or is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the forgoing. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 524, or a complementary sequence thereof. In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 524. In some embodiments, the gRNA targets a target site that is the sequence set forth in SEQ ID NO: 524.

[0308] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOS: 11–20 and 587–589 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOS: 11–20 and 587–589 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOS: 11–20 and 587–589.

[0309] In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 14 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 14. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO: 17 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 17. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:587 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:587. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:588 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the 73MF-366426018Attorney No. 224742004040 gRNA is set forth in SEQ ID NO:588. In some embodiments, the spacer sequence of the gRNA includes or is set forth in SEQ ID NO:589 or a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides). In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:589.

[0310] In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOS: 600-602, 607 and 608 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 600-602, 607 and 608. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 600-602, 607 and 608.

[0311] In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:85, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:85. In some embodiments, the scaffold sequence is set forth in SEQ ID NO: 85.

[0312] In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:259, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:259. In some embodiments, the scaffold sequence is set forth in SEQ ID NO: 259.

[0313] In some embodiments, the scaffold sequence comprises a 3 ’ polyU sequence. In some embodiments, the scaffold sequence comprises a sequence set forth in SEQ ID NO:450 or SEQ ID NO: 451, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 450 or SEQ ID NO: 451. In some embodiments, the scaffold comprises a sequence set forth in SEQ ID NO: 450 or SEQ ID NO: 451.

[0314] In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOS: 41–45, 247–252, 439–449, 485–511, 540–545, 547, and 606–607 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 41–45, 247–252, 439–449, 485–511, 540–545, 547, and 606–607. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 41–45, 247–252, 439–449, 485–511, 540–545, 547, and 606–607.

[0315] In some embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 85. In some embodiments, the gRNA, including a spacer sequence and a scaffold sequence, comprises the sequence selected from any one of SEQ ID NOS: 41-45, 247-252, and 439-449 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 41-45,247-252, and 439-449. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 41-45,247-252, and 439-449.74MF-366426018Attorney No. 224742004040

[0316] In some embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 259. In some embodiments, the gRNA, including a spacer sequence and a scaffold sequence, comprises the sequence selected from any one of SEQ ID NO: 250, 252 and 439-444 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 250, 252 and 439-444. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 250, 252 and 439-444.

[0317] In some embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 450.. In some embodiments, the gRNA, including a spacer sequence and a scaffold sequence, comprises the sequence selected from any one of SEQ ID NOS: 485-494 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 485-494. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 485-494.

[0318] In some embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 451. In some embodiments, the gRNA, including a spacer sequence and a scaffold sequence, comprises the sequence selected from any one of SEQ ID NOS: 495-511, 540-545, 548, 590-602, and 607-708 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOS: 495-511, 540-545, 548, 590-602, and 607-708. In some embodiments, the gRNA is set forth in any one of SEQ ID NOS: 495-511, 540-545, 548, 590-602, and 607-708.

[0319] In some embodiments, the gRNA, including a spacer sequence and a scaffold sequence, comprises the sequence set forth in SEQ ID NO: 508 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 508. In some embodiments, the gRNA is set forth in SEQ ID NO: 508.

[0320] In some embodiments, the gRNA, including a spacer sequence and a scaffold sequence, comprises the sequence set forth in SEQ ID NO: 544 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 544. In some embodiments, the gRNA is set forth in SEQ ID NO: 544.

[0321] In some embodiments, any of the provided gRNA sequences is complexed with or is provided in combination with a Cas9. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSpCas9, such as a dSpCas9 set forth in SEQ ID NO: 128, or a variant and / or fusion thereof.

[0322] In some embodiments, a provided epigenetic -modifying DNA-targeting system for epigenetic modification of Lp(a) includes any of the aforementioned gRNAs complexed with a Cas protein, such as a Cas9 protein. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSpCas9, such as a dSpCas9 set forth in SEQ ID NO: 128, or a variant and / or fusion thereof.75MF-366426018Attorney No. 224742004040

[0323] In some embodiments, the the DNA-targeting system comprises an mRNA encoding the fusion protein and one gRNA targeting a target site of Lp(a). In some embodiments the ratio of the mRNA to the one gRNA targeting a target site of Lp(a) is between 5:1 and 1:5. In some embodiments, the ratio of mRNA:gRNA is 4: 1. In some embodiments, the ratio of mRNA:gRNA is 3: 1. In some embodiments, the ratio of mRNA:gRNA is 2: 1. In some embodiments, the ratio of mRNA:gRNA is 1:1. In some embodiments, the ratio of mRNA:gRNA is 1:2. In some embodiments, the amount of the mRNA is twice the amount of the gRNA.

[0324] In some embodiments, a provided epigenetic -modifying DNA-targeting system for epigenetic modification of Lp(a) includes more than one gRNA that targets a site for Lp(a). In some embodiments, the DNA-targeting system comprises a first gRNA that targets a first Lp(a) target site and second gRNA that targets a second Lp(a) target site. In some embodiments, the first target site located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and the second target site is within 500 bp of the hg38 genomic coordinate chr6: 160,664,275. In some embodiments, the first target site is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and the second target site is located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973. In some embodiments, the first target site is located between the hg38 genomic coordinates 160,519,674 and club: 160,520,973 and the second target site is within 500 bp of the hg38 genomic coordinate chr6: 160,664,275.C. Other DNA-binding domains

[0325] In some of any of the provided embodiments, the DNA-binding domain comprises a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing.

[0326] In some embodiments, a ZFP, a zinc finger DNA binding protein, or zinc finger DNA binding domain, is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial, or engineered, ZFPs, comprising ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3, and 6) on a zinc finger recognition helix.76MF-366426018Attorney No.224742004040 Thus, for example, the ZFP or ZFP -containing molecule is non-naturally occurring, e.g., is engineered to bind to a target site of choice.

[0327] In some embodiments, zinc fingers are custom-designed (i.e. designed by the user), or obtained from a commercial source. Various methods for designing zinc finger proteins are available. For example, methods for designing zinc finger proteins to bind to a target DNA sequence of interest are described, for example in Liu, Q. et al., PNAS, 94(11):5525-30 (1997); Wright, D. A. et al., Nat. Protoc., 1(3): 1637-52 (2006); Gersbach, C. A. etal., Acc. Chem. Res., 47(8):2309-18 (2014); Bhakta M. S. etal., Methods Mol. Biol., 649:3-30 (2010); and Gaj et al., Trends Biotechnol, 31(7):397-405 (2013). In addition, various web-based tools for designing zinc finger proteins to bind to a DNA target sequence of interest are publicly available. See, for example, the Zinc Finger Tools design web site from Scripps available on the world wide web at scripps.edu / barbas / zfdesign / zfdesignhome.php. Various commercial services for designing zinc finger proteins to bind to a DNA target sequence of interest are also available. See, for example, the commercially available services or kits offered by Creative Biolabs (world wide web at creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly Kit available from Addgene (world wide web at addgene.org / kits / zfc-modular-assembly / ), or the CompoZr Custom ZFN Service from Sigma Aldrich (world wide web at sigmaaldrich.com / life-science / zinc-finger-nuclease-technology / custom-zfn.html).

[0328] Transcription activator-like effectors (TALEs), are proteins naturally found in Xanthomonas bacteria. TALEs comprise a plurality of repeated amino acid sequences, each repeat having binding specificity for one base in a target sequence. Each repeat comprises a pair of variable residues in position 12 and 13 (repeat variable diresidue; RVD) that determine the nucleotide specificity of the repeat. In some embodiments, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A. In some embodiments, RVDs can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. Binding domains with similar modular base-per-base nucleic acid binding properties can also be derived from different bacterial species. These alternative modular proteins may exhibit more sequence variability than TALE repeats.

[0329] In some embodiments, a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains, each comprising a repeat variable diresidue (RVD), are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at 77MF-366426018Attorney No. 224742004040 least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat units. See, e.g., U. S. Pat. Nos. 8,586,526 and 9,458,205.

[0330] In some embodiments, a TALE is a fusion protein comprising a nucleic acid binding domain derived from a TALE and an effector domain.

[0331] Zinc finger and TALE DNA-binding domains can be engineered to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein, by engineering of the amino acids in a TALE repeat involved in DNA binding (the repeat variable diresidue or RVD region), or by systematic ordering of modular DNA-binding domains, such as TALE repeats or ZFP domains. Therefore, engineered zinc finger proteins or TALE proteins are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. A designed protein is a protein not occurring in nature whose design / composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U. S. Pat. Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496.D. Effector Domains

[0332] In some aspects, the DNA-targeting systems provided herein further include one or more effector domains. In some embodiments, provided herein is a DNA-targeting system comprising a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in a gene or regulatory DNA element thereof, such as any described above, and (b) at least one effector domain. In some aspects, the effector domain is capable of reducing transcription of the gene or combination of genes. In some aspects, the effector domain comprises a transcriptional repressor domain. In some aspects, the effector domain is a single transcriptional repressor domain. In some embodiments, provided herein is a DNA-targeting system comprising a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in a gene or regulatory DNA element thereof, such as any described above, and (b) at least one transcriptional repressor domain. In some embodiments, the term “effector” domain may be used interchangeably with “transcriptional repressor” domain.

[0333] In some aspects, the effector domain induces, catalyzes, or leads to repressed and / or reduced transcription of a gene when ectopically recruited to the gene or DNA regulatory element thereof. In some embodiments, the effector domain induces, catalyzes or leads to transcription repression, transcription co-repression, histone modification, histone acetylation, histone deacetylation, nucleosome 78MF-366426018Attorney No.224742004040 remodeling, chromatin remodeling, heterochromatin formation, proteolysis, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, splicing, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain induces, catalyzes, or leads to transcription repression or transcription co-repression. In some embodiments, the effector domain induces transcription repression. In some embodiments, the effector domain has one of the aforementioned activities itself (i.e. acts directly). In some embodiments, the effector domain recruits and / or interacts with a protein or polypeptide domain that has one of the aforementioned activities (i.e. acts indirectly).

[0334] In some embodiments, the at least one transcriptional repressor domain is capable of methylating the target site and / or a region surrounding the target site of the gene or combination of genes (e.g. Lp(a)). In certain embodiments, methylation comprises CpG methylation mediated directly or indirectly by a DNA methyltransferase or a DNA methyltransferase-recruiting domain. In some embodiments, such methylation results in durable or heritable repression of transcription of the gene or combination of genes, such as Lp(a) and / or PCSK9.

[0335] Gene expression of endogenous mammalian genes, such as human genes, can be achieved by targeting a fusion protein comprising a DNA-binding domain, such as a dCas9, and an effector domain, such as a transcription repression domain, to mammalian genes or regulatory DNA elements thereof (e.g. a promoter or enhancer) via one or more gRNAs. Any of a variety of effector domains for transcriptional repression (e.g. transcription repression domains) are known and can be used in accord with the provided embodiments. Transcription repression domains, as well as transcriptional repression of target genes using Cas fusion proteins with the transcription repression domains, are described, for example, in WO 2014 / 197748, WO 2017 / 180915, WO 2021 / 226077, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, Adli, M. Nat. Commun. 9, 1911 (2018), and Gilbert, L. A. et al. Cell 154(2):442-451 (2013).

[0336] In some embodiments, the effector domain may comprise a KRAB domain, ERF repressor domain, MXI1 domain, SID4X domain, MAD-SID domain, a DNMT family protein domain (e.g.DNMT3 A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g.DNMT3A / L, which comprises a fusion of DNMT3A and DNMT3L domains), LSD1, EZH2, a SunTag domain, a partially or fully functional fragment or domain of any of the foregoing, or a combination of any of the foregoing. For example, the fusion protein may be dCas9-KRAB, or DNMT3 A / L-dCas9-KRAB. In some embodiments, the fusion protein may be dCas9-KRAB. In some embodiments, the fusion protein may be DNMT3A / L-dCas9-KRAB. In some embodiments, the fusion protein may be KRAB-dCas9- DNMT3A / L.

[0337] In some embodiments, the effector domain comprises a transcriptional repressor domain described in WO 2021 / 226077.79MF-366426018Attorney No. 224742004040

[0338] In some embodiments, the effector domain comprises a KRAB domain, or a variant thereof. The KRAB -containing zinc finger proteins make up the largest family of transcriptional repressors in mammals. The Krüppel associated box (KRAB) domain is a transcriptional repressor domain present in many zinc finger protein-based transcription factors. The KRAB domain comprises charged amino acids and can be divided into sub-domains A and B. The KRAB domain recruits corepressors KAP1 (KRAB-associated protein-1), epigenetic readers such as heterochromatin protein 1 (HP1), and other chromatin modulators to induce transcriptional repression through heterochromatin formation. KRAB-mediated gene repression is associated with loss of histone H3 -acetylation and an increase in H3 lysine 9 trimethylation (H3K9me3) at the repressed gene promoters. KRAB domains, including in dCas fusion proteins, have been described, for example, in WO 2017 / 180915, WO 2014 / 197748, US 2019 / 0127713, WO 2013 / 176772, Urrutia R. et al. Genome Biol. 4, 231 (2003), Groner A. C. et al. PLoS Genet. 6, e 1000869 (2010). In some embodiments, the effector domain comprises at least one KRAB domain or a variant thereof. In some embodiments, the KRAB domain is from KOX1, ZIM3, or ZNF324. In some embodiments, the effector domain comprises a ZNF10 KRAB domain. In some embodiments, an exemplary KRAB domain is set forth in SEQ ID NO: 130. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 130, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130. In some embodiments, an exemplary KRAB domain is set forth in SEQ ID NO: 169. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 169, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 169. In some embodiments, an exemplary KRAB domain is set forth in SEQ ID NO: 276. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 276, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:276. In some embodiments, an exemplary KRAB domain is set forth in SEQ ID NO: 527. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 527, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:527.

[0339] In some embodiments, the effector domain comprises at least one ERF repressor domain, or a variant thereof. ERF (ETS2 repressor factor) is a strong transcriptional repressor that comprises a conserved ets-DNA-binding domain, and represses transcription via a distinct domain at the carboxylterminus of the protein. ERF repressor domains, including in dCas fusion proteins, have been described, for example, in W02017180915, WO2014197748, WO2013176772, Mavrothalassitis, G., Ghysdael, J. Proteins of the ETS family with transcriptional repressor activity. Oncogene 19, 6524–6532 (2000). In some embodiments, the effector domain comprises at least one ERF repressor domain or a variant 80MF-366426018Attorney No. 224742004040 thereof. An exemplary ERF repressor domain is set forth in SEQ ID NO: 139. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 129, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0340] In some embodiments, the effector domain comprises at least one MXI1 domain, or a variant thereof. The MXI1 domain functions by antagonizing the myc transcriptional activity by competing for binding to myc-associated factor x (MAX). MXI1 domains, including in dCas fusion proteins, have been described, for example, in W02017180915, WO2014197748, US20190127713. In some embodiments, the effector domain comprises at least one MXI1 domain or a variant thereof. An exemplary MXI1 domain is set forth in SEQ ID NO: 140. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 140, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0341] In some embodiments, the effector domain comprises at least one SID4X domain, or a variant thereof. The mSin3 interacting domain (SID) is present on different transcription repressor proteins. It interacts with the paired amphipathic alpha-helix 2 (PAH2) domain of mSin3, a transcriptional repressor domain that is attached to transcription repressor proteins such as the mSin3 A corepressor. A dCas9 molecule can be fused to four concatenated mSin3 interaction domains (SID4X). SID domains, including in dCas fusion proteins, have been described, for example, in W02017180915, WO2014197748, WO2014093655. In some embodiments, the effector domain comprises at least one SID domain or a variant thereof. An exemplary SID domain is set forth in SEQ ID NO: 141. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 141, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0342] In some embodiments, the effector domain comprises at least one MAD domain, or a variant thereof. The MAD family proteins, Madl, Mxil, Mad3, and Mad4, belong to the basic helix-loop-helix-zipper class and contain a conserved N terminal region (termed Sin3 interaction domain (SID)) necessary for repressional activity. MAD-SID domains, including in dCas fusion proteins, have been described, for example, in W02017180915, WO2014197748, WO2013176772. In some embodiments, the effector domain comprises at least one MAD-SID domain or a variant thereof. An exemplary MAD-SID domain is set forth in SEQ ID NO: 142. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 142, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0343] In some embodiments, the effector domain comprises at least one DNMT3 domain, or a variant thereof. In some embodiments, the at least one DNMT3 domain, or a variant thereof, is from a DNMT3 or is a portion or a functionally active variant thereof with DNA methyltransferase activity. The 81MF-366426018Attorney No.224742004040 DNMT3 A and DNMT3B are two DNA methyltransferases that catalyze de novo methylation, which depending on the site may be associated with transcriptional repression. DNMTs, such as DNMT3s, mediate transfer of a methyl group from the universal methyl donor, S-adenosyl-L-methionine (SAM), to the 5-position of cytosine residues. In some aspects, these DNMT3 DNA methyltransferases induce de novo methylation of a cytosine base to methylated 5-methylcytosine. DNMT3, including in dCas fusion proteins, have been described, for example, in US20190127713, Liu, X. S. et al. Cell 167, 233–247.e17 (2016), Lei, Y. et al. Nat. Commun. 8, 16026 (2017). DNMT3 proteins, such as DNMT3A and DNMT3B, contain an N-terminal part that is naturally involved in regulatory activity and targeting, and a C-terminal catalytic domain termed the MTase C5-type domain. In some embodiments, an effector domain in embodiments provided herein includes a catalytically active portion of a DNMT3 A or a DNMT3B that contains a catalytically active C-terminal domain. In particular, isolated catalytic domains of DNMT3a and DNMT3b are catalytically active (see e.g. Gowher and Jeltsch (2002) J. Biol. Chem., 277:20409).

[0344] In some embodiments, the effector domain comprises at least one DNMT3 domain or a variant thereof. In some embodiments, the DNMT3 domain may be an effector domain of DNMT3A or DNMT3B that is catalytically active. In some embodiments, the effector domain may be the full-length of DNMT3 A or DNMT3B or a catalytically active portion thereof. In some embodiments, the effector domain is a catalytically active portion that is less than the full-length sequence of DNMT3A or DNMT3B. In some embodiments, a catalytically active portion is a contiguous sequence of amino acids that confers DNA methyltransferase activity, such as by mediating methylation of a cytosine base to methylated 5-methylcytosine. In some embodiments, the contiguous sequence of amino acids is a contiguous C-terminal portion of a DNMT3 protein, such as DNMT3 A, or DNMT3B, that is from 280 amino acids to 330 amino acids in length. In some embodiments, the contiguous portion is 280 amino acids, 290 amino acids, 300 amino acids, 310 amino acids, 320 amino acids, or 330 amino acids in length, or is a length of any value between any of the foregoing. In some embodiments, a catalytically active portion of a DNMT, such as a DNMT3, includes a SAM-dependent MTase C5-type domain. In some embodiments, the DNMT3 domain, such as a domain of DNMT3A or DNMT3B, is of human origin.

[0345] An exemplary DNMT3 A domain is set forth in SEQ ID NO: 132 or 170. An exemplary DNMT3B domain is set forth in SEQ ID NO: 143. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 132, SEQ ID NO: 170 or SEQ ID NO: 143, or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0346] In some embodiments, the DNMT3 A domain is set forth in SEQ ID NO: 132, or is a catalytically active portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%,82MF-366426018Attorney No.224742004040 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 132 or the catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3 A domain is set forth in SEQ ID NO: 132. In some embodiments, the DNMT3 A domain is encoded by the nucleotide sequence set forth in SEQ ID NO: 132.

[0347] In some embodiments, the DNMT3 A domain is set forth in SEQ ID NO: 170, or is a catalytically active portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 170 or the catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3 A domain is set forth in SEQ ID NO: 170.

[0348] In some embodiments, the effector domain is from DNMT3B or a catalytically active portion or variant thereof that exhibits DNA methyltransferase activity. An exemplary DNMT3B domain is set forth in SEQ ID NO: 143, or is a catalytically active portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 143 or the catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the catalytically active portion is a contiguous portion of amino acids of SEQ ID NO: 143 that includes the SAM-dependent MTase C5-type domain (e.g. corresponding to amino acids 575-853 of SEQ ID NO: 143). In some embodiments, the contiguous sequence of amino acids of SEQ ID NO: 143 includes at least 250 amino acids, 275 amino acids, 300 amino acids or 325 amino acids, or any value between any of the foregoing. In some embodiments, the contiguous sequence of amino acids is a contiguous portion of SEQ ID NO:224 that includes amino acids 575-853 and is from 280 amino acids to 330 amino acids in length. In some embodiments, the contiguous portion is 280 amino acids, 290 amino acids, 300 amino acids, 310 amino acids, 320 amino acids, or 330 amino acids in length, or is a length of any value between any of the foregoing.

[0349] Any of a variety of assays are known to assess or monitor methyltransferase (MTase) ativity. In some embodiments, exemplary assays to assess DNA methyltransferase activity include, but are not limited to, radio DNA MTase assays, colorimetric DNA MTase activity assays, fluorescent DNA MTase activity assays, chemiluminescent / bioluminescent DNA MTase activity assays, electrochemical DNA MTase activity assays, and electrogenerated chemiluminescence (ECL) DNA MTase activity assays. Exemplary assays are described in Poh et al. Theranostics, 2016, 6:369-391; Li et al., Methods Appl. Fluoresc., 2017, 5:012002; Deng et al., Anal Chem., 2014, 86:2117-23; and Ma et al. J Mater Chem B., 2020, 8:3488-3501.

[0350] In some embodiments, the effector domain comprises a catalytically inactive DNA methyltransferase domain or portion thereof. In some embodiments, the effector domain comprises a DNMT3L protein or portion thereof. In some embodiments, the effector domain includes at least one DNMT3L domain, or a variant thereof. In some embodiments, the effector domain is a DNMT3L 83MF-366426018Attorney No.224742004040 domain. In some embodiments, the DNMT3L domain comprises a DNMT3L ADD domain, a DNMT3L MTase-like domain, or a DNMT3L ADD domain and a DNMT3L MTase-like domain. In some embodiments, the effector domain is a DNMT3L MTase-like domain.

[0351] In some embodiments, an effector domain provided herein is a single transcriptional repressor domain. In some embodiments, the single transcriptional repressor domain is a DNMT3L domain or a functional portion thereof. In some embodiments, the DNMT3L domain or functional portion thereof is the only transcriptional repressor domain. In some embodiments, the effector domain consists of a DNMT3L domain or a functional portion thereof as the sole transcriptional repressor domain. In some embodiments, the single transcriptional repressor domain is exclusively a DNMT3L domain. In some embodiments, the effector domain does not include any other transcriptional repressor domains, such as DNMT3A, KRAB, SID, or other repressor motifs. In some embodiments, the effector domain is free of other transcriptional repressor domains, including but not limited to DNMT3 A, KRAB domains, SID domains, or other repressor motifs. In some embodiments, transcriptional repression is mediated exclusively by the DNMT3L domain or functional portion thereof. In some embodiments, the single transcriptional repressor domain is a DNMT3L MTase-like domain.

[0352] The DNMT3L domain or a variant thereof may be a DNMT3L or a portion of DNMT3L, or a variant of DNMT3L or the portion thereof. DNMT3L (DNA (cytosine-5)-methyltransferase 3-like) is a catalytically inactive regulatory factor of DNA methyltransferases that can either promote or inhibit DNA methylation depending on the context. DNMT3L is essential for the function of DNMT3A and DNMT3B; DNMT3L interacts with DNMT3 A and DNMT3B and significantly enhances their catalytic activity. For instance, DNMT3L interacts with the catalytic domain of DNMT3A to form a heterodimer, demonstrating that DNMT3L has dual functions of binding an unmethylated histone tail and activating DNA methyltransferase. Without wishing to be bound by theory, it is also believed that a DNMT3L protein or portion thereof is sufficient to recruit and bind to domains with DNA methyltransferase activity, such as DNMT3 A or DNMT3B, without being fused to the domains with DNA methyltransferase activity.

[0353] DNMT3L is characterized by a regulatory ATRX-DNMT3-DNMT3L (ADD) domain and an MTase-like domain. An exemplary full-length amino acid sequence of a wild-type (also called “unmodified”) DNMT3L polypeptide derived from a mouse comprising both an ADD domain and an MTase-like domain is set forth in SEQ ID NO: 171. With reference to the exemplary wild-type DNMT3L polypeptide set forth in SEQ ID NO: 171, the ADD domain is the contiguous sequence set forth as amino acid residues 68-207 or amino acid residues 75-207. With reference to the exemplary wild-type DNMT3L set forth in SEQ ID NO: 171, the MTase-like domain is the contiguous sequence set forth as amino acid residues 208-421. It is within the level of a skilled artisan to identify domains in an DNMT3L protein, including portion thereof, e.g., an MTase-like domain, such as by alignment of a 84MF-366426018Attorney No. 224742004040 reference sequence (e.g. SEQ ID NO: 171) with other DNMT3L sequences, e.g., human DNMT3L protein sequence set forth in SEQ ID NO: 134. An exemplary alignment identifying domains is exemplified in FIGS. 29A-29B, which shows residues in a human DNMT3L protein sequence without an initial methionine, set forth in SEQ ID NO: 282 (“human”), that correspond to the numbering of positions in a mouse DNMT3L protein sequence without an initial methionine, set forth in SEQ ID NO: 281 (“mouse”).

[0354] A DNMT3L ADD domain is characterized by controlling DNA methyltransferase activity by preventing methylation of regions marked by tri-methylation of the histone tail H3K4 (H3K4me3). H3K4me3 is a chromatin mark associated with the promoters of actively transcribed genes, including transcribed CpG island-containing genes with hypomethylated promoters that may otherwise be prime targets for de novo methylation by domains and / or proteins with methyltransferase activity, such as DNMT3 A or DNMT3B. The ADD domain sterically interferes with the DNMT3 A-DNMT3L binding interface on the DNMT3L. When the H3K4 is tri-methylated, the ADD domain cannot bind to the histone tail, and so the DNMT3 A or DNMT3B protein cannot methylate. However, if a gene is no longer actively transcribed and the H3K4 tail is not methylated, the ADD domain can bind H3K4meO, and swing out of the inhibitory confirmation enabling DNA methylation. Further, the ADD domain has also been demonstrated to interact with the common suite of heterochromatin proteins, analogously to a KRAB domain.

[0355] A DNMT3L MTase-like domain is characterized by associating with proteins with methyltransferase activity (e.g., DNMT3A or DNMT3B) to both stimulate the DNA methylation activity and also to enhance the recruitment of proteins such as DNMT3A or DNMT3B to genomic sites to be methylated. Without wishing to be bound by theory, it is believed that the DNMT3L MTase-like domain alone is sufficient to recruit domains and / or proteins with methyltransferase activity, such as DNMT3 A or DNMT3B. Specifically, regions of the DNMT3L MTase-like domain are known to be involved in the binding or recruitment of DNMT3 A through their role as part of the DNMT3 A-DNMT3L binding interface on the DNMT3L protein. Exemplary regions of the DNMT3L MTase-like domain involved in the DNMT3 A-DNMT3L binding interface are known in the literature, see, e.g., Jia et al., Nature, 2007 and Jurkowska et al., Nucleic Acids Res., 2008. With reference to the exemplary wild-type human DNMT3L set forth in SEQ ID NO: 134, the region of the DNMT3L MTase-like domain involved in the DNMT3 A-DNMT3L binding interface includes the contiguous sequence set forth as amino acid residues 226-234, 258-274 and / or 292-303. Without wishing to be bound by theory, it is believed that a pair of phenyalanine residues (F) play a role in forming the DMNT3 A-DNMT3L interface. An exemplary pair of phenyalanine residues include F297 and F337 with reference to the exemplary wild-type mouse DNMT3L set forth in SEQ ID NO: 171. It is within the level of a skilled artisan to identify regions involved in the DNMT3 A-DNMT3L binding interface in an DNMT3L protein, e.g., a pair of85MF-366426018Attorney No. 224742004040 phenyalanine residues, such as by alignment of a reference sequence (e.g. SEQ ID NO: 171 or SEQ ID NO: 134) with other DNMT3L sequences.

[0356] In some embodiments, the effector domain is 50, 100, 250, 300, 350, 400, 450, 50, 550, or 600 amino acids in length, or within a range defined by any of the foregoing. In some embodiments, the effector domain is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, or 421 amino acids in length, or within a range defined by any of the foregoing. In some embodiments, the effector domain is 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids in length, or within a range defined by any of the foregoing. In some embodiments, the effector domain is at least 10, 15, 20, 22, 25, 30, 35, 37, 40, 42, 45, 47, 49, 50, 55, 57, 60, 61, 62, 65, 70, 72, 75, 76, or 80 amino acids in length. In some embodiments, the effector domain is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 amino acids in length, or within a range defined by any of the foregoing. In some embodiments, the effector domain is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 amino acids in length, or within a range defined by any of the foregoing. In some embodiments, the effector domain is 22, 37, 42, 47, 49, 57, 61, 62, 70, 72, 76, or 80 amino acids in length, or within a range defined by any of the foregoing.

[0357] In some embodiments, the effector domain is less than 800, less than 700, less than 600, less than 550, less than 540, less than 530, less than 520, less than 510, less than 500, less than 490, less than 480, less than 470, less than 460, less than 450, less than 440, less than 430, less than 420, less than 410, less than 400, less than 390, less than 380, less than 370, less than 360, less than 350, less than 340, less than 330, less than 320, less than 310, less than 300, less than 290, less than 280, less than 270, less than 260, less than 250, less than 200, less than 100, less than 50, less than 40, less than 30, less than 25, less than 20, or less than 15 amino acids in length. In some embodiments, the effector domain is less than 800 amino acids in length. In some embodiments, the effector domain is less than 600 amino acids in length. In some embodiments, the effector domain is less than 520 amino acids in length. In some embodiments, the effector domain is less than 510 amino acids in length. In some embodiments, the effector domain is less than 400 amino acids in length. In some embodiments, the effector domain is less than 300 amino acids in length. In some embodiments, the effector domain is less than 250 amino acids in length.

[0358] In some embodiments, the effector domain is between 10 and 800, between 10 and 700, between 10 and 600, between 10 and 550, between 10 and 510, between 10 and 500, between 10 and 400, between 10 and 300, between 10 and 250, between 10 and 200, between 10 and 100, between 10 and 50, 15 and 800, between 15 and 700, between 15 and 600, between 15 and 550, between 15 and 510, between 15 and 500, between 15 and 400, between 15 and 300, between 15 and 250, between 15 and 200, between 15 and 100, between 15 and 50, 40 and 800, between 40 and 700, between 40 and 600, between 40 and 550, between 40 and 510, between 40 and 500, between 40 and 400, between 40 and 86MF-366426018Attorney No. 224742004040 300, between 40 and 250, between 40 and 200, between 40 and 100, between 40 and 50, 100 and 800, between 100 and 700, between 100 and 600, between 100 and 550, between 100 and 510, between 100 and 500, between 100 and 400, between 100 and 300, between 100 and 250, between 100 and 200, between 250 and 800, between 250 and 700, between 250 and 600, between 250 and 550, between 250 and 510, between 250 and 500, between 250 and 400, between 250 and 300, between 300 and 800, between 300 and 700, between 300 and 600, between 300 and 550, between 300 and 510, between 300 and 500, between 300 and 400, between 350 and 800, between 350 and 700, between 350 and 600, between 350 and 550, between 350 and 510, between 350 and 500, between 350 and 400, between 400 and 800, between 400 and 700, between 400 and 600, between 400 and 550, between 400 and 510, between 400 and 500, between 500 and 800, between 500 and 700, between 500 and 600, between 500 and 550, between 500 and 510, between 510 and 800, between 510 and 700, between 510 and 600, or between 510 and 550 amino acids in length. In some embodiments, the effector domain is between 250 and 510 amino acids in length. In some embodiments, the effector domain is between 250 and 400 amino acids in length.

[0359] In some embodiments, the effector domain comprises a DNMT3L protein or portion thereof. In some embodiments, the DNMT3L protein or portion thereof is selected from one of the following species: Acomys russatus, Ailuropoda melanoleuca, Apodemus sylvaticus, Arvicanthis niloticus, Bos indicusm, Callithrix jacchus, Camelus bactrianus, Capricomis sumatraensis, Carlito syrichta, Castor canadensis, Cavia porcellus, Chinchilla lanigera, Choloepus didactylus, Chrysochloris asiatica, Cricetulus griseus, Cynocephalus volans, Dasypus novemcinctus, Desmodus rotundus, Diceros bicomis minor, Dipodomys spectabilis, Echinops telfairi, Elephantulus edwardii, Enhydra lutris kenyoni, Eptesicus fuscus, Equus caballus, Erinaceus europaeus, Eschrichtius robustus, Eubalaena glacialis, Eulemur rufifrons, Fukomys damarensis, Globicephala melas, Heterocephalus glaber, Hippopotamus amphibius kiboko, Hipposideros armiger, Homo sapiens, Hyaena hyaena, Jaculus jaculus, Lipotes vexillifer, Loxodonta Africana, Macaca fascicularis, Manis pentadactyla, Marmota monax, Mastomys coucha, Meriones unguiculatus, Mesoplodon densirostris, Microtus ochrogaster, Miniopterus natalensis, Molossus molossus, Monodelphis domestica, Monodon monoceros, Muntiacus muntjak, Mus musculus, Mustela nigripes, Myodes glareolus, Myotis lucifugus, Myotis yumanensis, Nannospalax galili, Neofelis nebulosa, Neogale vison, Neotoma lepida, Notamacropus eugenii, Nyctereutes procyonoides, Nycticebus coucang, Ochotona curzoniae, Ochotona princeps, Octodon degus, Odobenus rosmarus divergens, Onychomys torridus, Orcinus orca, Orycteropus afer afer, Ovis aries, Perognathus longimembris pacificus, Phacochoerus africanus, Phoca vitulina, Phocoena Phocoena, Phocoena sinus, Phyllostomus hastatus, Physeter macrocephalus, Pipistrellus kuhlii, Propithecus coquereli, Pteronotus mesoamericanus, Pteropus vampyrus, Rattus norvegicus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Saccopteryx leptura, Sagmatias obliquidens, Saimiri boliviensis boliviensis, Sarcophilus harrisii, Sigmodon hispidus,87MF-366426018Attorney No. 224742004040 Smutsia gigantea, Sturnira hondurensis, Sus scrofa, Trichechus manatus latirostris, Tupaia chinensis, Urocitellus parryii, Ursus arctos, Vicugna pacos, Vombatus ursinus, and Vulpes vulpes.

[0360] Table 1 lists the SEQ ID NOs for full-length DNMT3L protein sequences from the species listed above as well the corresponding start and stop amino acid positions for the ADD and MTase-like domains for each sequence. For example, the protein sequence for the full-length DNMT3L protein from Apodemus sylvaticus (Wood mouse) is set forth in SEQ ID NO: 291. With reference to SEQ ID NO: 291, the DNMT3L ADD domain from Apodemus sylvaticus starts at amino acid position 75 and ends at amino acid position 207 while the DNMT3L MTase domain from Apodemus sylvaticus starts at amino acid position 208 and ends at amino acid position 420.Table 1. DMNT3L orthologsDNMT3L ADD ADD Mtase Mtase SEQ ID Scientific Common Name Position Position Position Position NO. Name (Start) (End) (Start) (End)Golden spiny310 Acomys 76 208 209 419 russatus mouse311 AiluropodaGiant panda 1 123 124 336 melanoleuca291 ApodemusWood mouse 75 207 208 420 sylvaticus312 ArvicanthisNile grass rat 79 211 212 425 niloticus313 Bos indicusm Zebu 221 353 354 566 Callithrix Common314 74 206 207 418 jacchus marmoset315 Camelus Bactrian camel 41 173 174 402 bactrianus316 Capricornis Sumatran serow 82 214 215 427 sumatraensis317 Carlito syrichta Philippine tarsier 81 213 216 426 Castor North American318 41 173 174 388 canadensis beaver319 Domestic guineaCavia porcellus 30 162 163 376Pig320 Chinchilla Long-tailed 83 215 216 429 lanigera chinchilla321 Choloepus Linnaeus's 258 390 391 603 didactylus two-toed sloth322 ChrysochlorisCape golden mole 85 217 220 399 asiaticaCricetuhis323 Chinese hamster 75 207 208 423 griseus324 Cynocephalus Philippine flying 216 348 349 561 volans lemur325 Dasypus Nine-banded 72 204 205 417novemcinctus armadillo88MF-366426018Attorney No. 224742004040Common vampire326 Desmodus 80 212 213 425 rotundus batSouthwestern327 Diceros bicomis 79 211 212 424 minor black rhinoceros328 Dipodomys Banner-tailed 1 122 123 384 spectabilis, kangaroo rat329 Echinops telfairi Lesser hedgehog 67 199 200 412tenrec330 Elephantulus Cape elephant 83 215 216 494 edwardii shrew331 Enhydra lutris Northern sea otter 38 170 170 384 kenyoni332 Eptesicus fuscus Big brown bat 75 207 208 420333 Equus caballus Domestic horse 41 173 174 386334 Erinaceus European 64 191 192 398 europaeus hedgehog335 EschrichtiusGray whale 102 259 260 504 robustus336 Eubalaena North Atlantic 222 354 355 567 glacialis right whaleEulemur337 Red-fronted lemur 141 273 274 486 rufifrons338 Fukomys Damaraland 27 159 160 370 damarensis mole-rat339 Globicephala Long-finned pilot 71 203 204 416 melas whale340 Heterocephalusglaber Naked mole-rat 61 193 194 407 Hippopotamus East African341 amphibius 41 173 174 386hippopotamuskiboko342 Hipposideros Great roundleaf 41 173 174 386 armiger bat134 Homo sapiens Human 41 173 174 386343 Hyaena hyaena Striped hyena 100 232 233 445Lesser Egyptian344 Jaculus jaculus 68 200 203 416jerboa345 LipotesBaiji 252 384 385 597 vexilliferAfrican bush346 Loxodonta 35 167 168 380 Africana elephant347 Macaca Crab eating 41 173 174 387 fascicularis macaque348 ManisChinese pangolin 115 247 248 460 pentadactyla349 Marmota monax Groundhog 115 247 248 459Southern350 Mastomysmultimammate 76 208 209 422 conchamouse89MF-366426018Attorney No. 224742004040351 Meriones Mongolian gerbil 76 208 209 424 unguiculatusMesoplodon352 Blainville's 229 361 362 574 densirostris beaked whale353 Microtusochrogaster Prairie vole 101 233 234 492 354 Miniopterus Natal 50 182 183 396 natalensis long-fingered bat355 Molossus Velvety 80 212 213 425 molossus free-tailed bat356 Monodelphis Gray short-tailed 74 207 209 413 domestica opossumMonodon357 Narwhal 229 361 362 574 monocerosMuntiacus358 muntjak Indian muntjac 62 194 195 407171 Mus musculus House mouse 75 207 208 421 Mustela Black-footed359 135 267 267 492 nigripes ferret360 Myodes Bank vole 75 207 208 423 glareolus361 MyotisLittle brown bat 41 173 174 386 lucifugus362 MyotisYuma myotis 111 243 244 456 yumanensisNannospalax363 Mount Carmel 78 210 211 426 galili blind mole-ratNeofelis364 Clouded leopard 65 197 198 412 nebulosa365 Neogale vison American mink 56 188 189 414366 Neotoma lepida Desert woodrat 54 186 187 414 Notamacropus367 1 132 135 343 eugenii Tammar wallaby368 Nyctereutes Raccoon dog 86 218 219 431 procyonoides369 Nycticebuscoucang Sunda slow loris 76 208 209 423 370 Ochotona Plateau pika 85 217 218 431 curzoniae371 Ochotona American pika 63 195 196 409 princeps372 Octodon degus Degu 84 216 217 431 Odobenus373 rosmarus Pacific walrus 69 201 202 414 divergensSouthern374 Onychomys grasshopper 76 208 209 422 torridusmouse375 Orcinus orca Killer whale 56 213 214 42690MF-366426018Attorney No. 224742004040376 Orycteropus Aardvark 77 209 210 423afer afer377 Ovis aries Domestic sheep 101 233 234 776 Perognathus378 Pacific pocketlongimembris 62 194 195 411 mousepacificus379 PhacochoerusCommon warthog 27 159 160 410 africanus380 Phoca vitulina Harbor seal 97 229 230 442381 PhocoenaHarbor porpoise 194 326 327 539 phocoena382 Phocoena sinus Vaquita 187 319 320 532383 Phyllostomus Greater 117 249 250 462 hastatus spear-nosed batPhyseter384 Sperm whale 41 173 174 386 macrocephalus385 Pipistrelluskuhlii Kuhl's pipistrelle 67 199 200 413 386 PropithecusCoquerel's sifaka 94 226 229 440 coquereliMesoamerican387 Pteronotus 145 277 278 490 mesoamericanus mustached bat388 Pteropus Large flying fox 104 236 237 447 vampyrus389 Rattus Brown rat 76 208 209 422 norvegicus390 Rhinolophus Greater horseshoe 72 204 205 410 ferrumequinum bat391 RousettusEgyptian fruit bat 69 201 202 412 aegyptiacusSaccopteryx Lesser sac -winged392 59 191 193 404 leptura batPacific393 Sagmatiaswhite-sided 1 147 153 365 obliquidens dolphinSaimiriBolivian squirrel394 boliviensis 41 173 174 385monkeyboliviensis395 Sarcophilus Tasmanian devil 74 207 209 452 harrisiiSigmodon396 Hispid cotton rat 87 219 232 468 hispidus397 Smutsia Giant pangolin 34 172 173 386 giganteaHonduran398 Stumira yellow-shouldered 41 173 174 386 hondurensisbat399 Sus scrofa Wild boar 72 204 205 41491MF-366426018Attorney No. 224742004040 Trichechus400 manatus Florida manatee 90 222 223 435 latirostris401 Tupaia Chinese tree 187 319 320 532 chinensis shrew402 Urocitellus Arctic ground 68 200 201 414 parry ii squirrel403 Ursus arctos Brown bear 331 463 464 676404 Vicugna pacos Alpaca 41 173 174 402405 Vombatus Common wombat 100 233 235 444ursinus406 Vulpes vulpes Red fox 91 249 250 462

[0361] In some embodiments, the effector domain comprises a DNMT3L protein or portion thereof. In some embodiments, the DNMT3L protein comprises any one of the sequences set forth in SEQ ID NOs: 134, 171, and 310-406, as shown in Table 1. In some embodiments, the effector domain comprises the DNMT3L ADD domain of any one of SEQ ID NOs: 134, 171, and 310-406, corresponding to the sequence encompassed by the ADD domain start and stop amino acid positions set forth in Table 1. In some embodiments, the effector domain comprises the DNMT3L MTase-like domain of any one of SEQ ID NOs: 134, 171, and 310-406, corresponding to the sequence encompassed by MTase domain start and stop amino acid positions set forth in Table 1. In some embodiments, the effector domain comprises the DNMT3L ADD domain and the amino acid sequence of the DNMT3L MTase-like domain of any one of SEQ ID NOs: 134, 171, and 310-406, corresponding to the sequence encompassed by the ADD domain start and stop amino acid positions and the sequence encompassed by MTase-like domain start and stop amino acid positions set forth in Table 1.

[0362] In some embodiments, the DNMT3L protein or portion thereof is selected from one of the following species: Apodemus sylvaticus, Mus musculus, Homo sapiens, Equus caballus, Arvicanthis niloticus, Chinchilla lanigera, Desmodus rotundus, Echinops telfairi, Eptesicus fuscus, Fukomys damarensis, Globicephala melas, Jaculus jaculus, Mastomys coucha, Neofelis nebulosa, Neotoma lepida Ochotona princeps, Onychomys torridus, Perognathus longimembris pacificus, Phoca vitulina, Pteronotus mesoamericanus, and Saccopteryx leptura. In some embodiments, the DNMT3L protein or portion thereof is selected from one of the following species: Homo sapiens, Mus musculus, Apodemus sylvaticus, Rattus norvegicus, Bos taurus, Papio Anubis, Cebus imitator, Macaca nemestrina, Pongo abelii, Lexodonta Africana, Pan troglodytes, and Chlorocebus sabaeus. In some embodiments, the DNMT3L protein or portion thereof is selected from one of the following species: Apodemus sylvaticus, Mus musculus, Homo sapiens, Equus caballus, Neotoma lepida, Ochotona princeps, and Onychomys torridus. In some embodiments, the DNMT3L protein of portion thereof is selected from one of the following species: Homo sapiens, Mus musculus, and Apodemus sylvaticus. In some embodiments, the 92MF-366426018Attorney No.224742004040 DNMT3L protein of portion thereof is from Homo sapiens. In some embodiments, the DNMT3L protein of portion thereof is from Mus musculus. In some embodiments, the DNMT3L protein or portion thereof is from Apodemus sylvaticus. In some embodiments, the DNMT3L protein or portion thereof is from Equus caballus. In some embodiments, the DNMT3L protein or portion thereof is from Neotoma lepida. In some embodiments, the DNMT3L protein or portion thereof is from Ochotona princeps. In some embodiments, the DNMT3L protein or portion thereof is from Onychomys torridus.

[0363] In some embodiments, the effector domain comprises a DNMT3L protein or portion thereof. In some embodiments, the effector domain is a contiguous portion of a DNTM3L protein that comprises at least 10 amino acids from a reference DNMT3L MTase-like domain. In some embodiments, the effector domain comprises less than a full-length DNMT3L MTase-like domain. In some embodiments, the effector domain comprises a DNMT3L MTase-like domain. In some embodiments, the effector domain is a DNMT3L MTase-like domain. In some embodiments, the effector domain comprises a contiguous portion of the DNMT3L protein that is greater than a full-length DNMT3L MTase-like domain, for example the effector domain may comprise a DNMT3L MTase-like domain and further comprise a DNMT3L ADD domain or portion thereof. In some embodiments, the effector domain comprises a DNMT3L protein. In some embodiments, the effector domain is a DNMT3L protein.

[0364] In some embodiments, the effector domain comprises a DNMT3L protein or portion thereof. In some embodiments, the DNMT3L protein or portion thereof comprises a portion of the DNMT3L protein that is a contiguous portion that is less than a full-length DNMT3L MTase-like domain and comprises at least 10 amino acids from a reference DNMT3L MTase-like domain. In some embodiments, the contiguous portion is involved in a DNMT3 A-DNMT3L interface. Without wishing to be bound by theory, it is believed that a contiguous portion of a DNMT3L protein that is involved in a DNMT3 A-DNMT3L interface is able to recruit domains with DNA methyltransferase activity (e.g., DNMT3A) to a target site, such as any target site described in Section I. A. or Section II. A. Provided herein are portions of the DNMT3L protein that are a contiguous portion that comprise at least 10 amino acids from a reference DNMT3L MTase-like domain, such as any reference DNMT3L MTase-like domain described herein, and are further involved in a DNMT3 A-DNMT3L interface.

[0365] In some embodiments, the contiguous portion comprises the sequence set forth as amino residues 258-274 or 293-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134. In some embodiments, the contiguous portion comprises the sequence set forth as amino residues 258-274 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134. In some embodiments, the contiguous portion comprises the sequence set forth as amino residues 293-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134. In some embodiments, the contiguous portion comprises the sequence set forth as amino residues 258-30393MF-366426018Attorney No. 224742004040 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134. In some embodiments, the contiguous portion comprises the sequence set forth as amino residues 226-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134.

[0366] In some embodiments, the reference DNMT3L MTase-like domain is a DNMT3L MTase-like domain selected from one of the following species: Acomys russatus, Ailuropoda melanoleuca, Apodemus sylvaticus, Arvicanthis niloticus, Bos indicusm, Callithrixjacchus, Camelus bactrianus, Capricomis sumatraensis, Carlito syrichta, Castor canadensis, Cavia porcellus, Chinchilla lanigera, Choloepus didactylus, Chrysochloris asiatica, Cricetuhis griseus, Cynocephalus volans, Dasypus novemcinctus, Desmodus rotundus, Diceros bicomis minor, Dipodomys spectabilis, Echinops telfairi, Elephantulus edwardii, Enhydra lutris kenyoni, Eptesicus fuscus, Equus caballus, Erinaceus europaeus, Eschrichtius robustus, Eubalaena glacialis, Eulemur rufifrons, Fukomys damarensis, Globicephala melas, Heterocephalus glaber, Hippopotamus amphibius kiboko, Hipposideros armiger, Homo sapiens, Hyaena hyaena, Jaculus jaculus, Lipotes vexillifer, Loxodonta Africana, Macaca fascicularis, Manis pentadactyla, Marmota monax, Mastomys coucha, Meriones unguiculatus, Mesoplodon densirostris, Microtus ochrogaster, Miniopterus natalensis, Molossus molossus, Monodelphis domestica, Monodon monoceros, Muntiacus muntjak, Mus musculus, Mustela nigripes, Myodes glareolus, Myotis lucifugus, Myotis yumanensis, Nannospalax galili, Neofelis nebulosa, Neogale vison, Neotoma lepida, Notamacropus eugenii, Nyctereutes procyonoides, Nycticebus coucang, Ochotona curzoniae, Ochotona princeps, Octodon degus, Odobenus rosmarus divergens, Onychomys torridus, Orcinus orca, Orycteropus afer afer, Ovis aries, Perognathus longimembris pacificus, Phacochoerus africanus, Phoca vitulina, Phocoena Phocoena, Phocoena sinus, Phyllostomus hastatus, Physeter macrocephalus, Pipistrellus kuhlii, Propithecus coquereli, Pteronotus mesoamericanus, Pteropus vampyrus, Rattus norvegicus, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Saccopteryx leptura, Sagmatias obliquidens, Saimiri boliviensis boliviensis, Sarcophilus harrisii, Sigmodon hispidus, Smutsia gigantea, Sturnira hondurensis, Sus scrofa, Trichechus manatus latirostris, Tupaia chinensis, Urocitellus parryii, Ursus arctos, Vicugna pacos, Vombatus ursinus, and Vulpes vulpes.

[0367] In some embodiments, the reference DNMT3L MTase-like domain is a DNMT3L MTase-like domain selected from one of the following species: Apodemus sylvaticus, Mus musculus, Homo sapiens, Equus caballus, Arvicanthis niloticus, Chinchilla lanigera, Desmodus rotundus, Echinops telfairi, Eptesicus fuscus, Fukomys damarensis, Globicephala melas, Jaculus jaculus, Mastomys coucha, Neofelis nebulosa, Neotoma lepida Ochotona princeps, Onychomys torridus, Perognathus longimembris pacificus, Phoca vitulina, Pteronotus mesoamericanus, and Saccopteryx leptura. In some embodiments, the reference DNMT3L MTase-like domain is a DNMT3L MTase-like domain selected from one of the following species: Homo sapiens, Mus musculus, Apodemus sylvaticus, Rattus norvegicus, Bos taurus,94MF-366426018Attorney No. 224742004040 Papio Anubis, Cebus imitator, Macaca nemestrina, Pongo abelii, Lexodonta Africana, Pan troglodytes, and Chl...

Claims

Attorney No. 224742004040CLAIMS1. A plurality of polynucleotides encoding an epigenetic-modifying DNA-targeting system comprising:a) a polynucleotide encoding a fusion protein comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional repressor domain; andb) at least one gRNA that targets a target site of Lp(a) located between the hg38 genomic coordinates club: 160, 678,873 and chr6: 160,679,782 or between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973.

2. The plurality of polynucleotides encoding an epigenetic -modifying DNA-targeting system of claim 1, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782.

3. The plurality of polynucleotides of claim 1 or claim 2, further comprising:c) at least one gRNA that targets a target site of PCSK9 located within 500bp of the hg38 genomic coordinate chr1:55,039,548.

4. The plurality of polynucleotides of any one of claims 1-3, wherein the at least one gRNA that a targets a target site of Lp(a) is a first gRNA that targets a first Lp(a) target site and a second gRNA that targets a second Lp(a) target site.

5. The plurality of polynucleotides of claim 4, wherein the first and second Lp(a) target sites are:a) a first target site located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and a second target site within 500 bp of the hg38 genomic coordinate chr6: 160,664,275; or b) a first target site located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and a second target site located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973; orc) a first target site located between the hg38 genomic coordinates 160,519,674 and chr6: 160,520,973 and a second target site within 500 bp of the hg38 genomic coordinate chr6:160,664,275.340MF-366426018Attorney No. 224742004040 6. The plurality of polynucleotides of any one of claims 1-5, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 678,800 and chr6: 160,679,650.

7. The plurality of polynucleotides of any of claims 1-6, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 678,280 and chr6: 160,679,602.

8. The plurality of polynucleotides of any of claims 1-7, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782.

9. The plurality of polynucleotides of any of claims 1-8, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650.

10. The plurality of polynucleotides of any of claims 1-9, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679,000 and chr6: 160,679,350.

11. The plurality of polynucleotides of any of claims 1-9, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700.

12. The plurality of polynucleotides of any of claims 1-9, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679, 000-160,679,200; chr6: 160, 679, 200-160,679,400; chr6:160, 679, 400-160,679,600; or chr6:160, 679, 500- 160,679,700.

13. The plurality of polynucleotides of any of claims 1-10 and 12, wherein at least one gRNA targets a target site for Lp(a) that comprises the hg38 genomic coordinates chr6: 160, 679,280-160, 679, 299.

14. The plurality of polynucleotides of any of claims 1-9, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 31-35, 256-258, 418- 341MF-366426018Attorney No. 224742004040 424, 528-533, 546, and 604 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

15. The plurality of polynucleotides of any of claims 1-9 and 14, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 31-35,256-258, 418-424, 528-533, 546, and 604.

16. The plurality of polynucleotides of any of claims 1-9 and 14-15, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in SEQ ID NO: 421, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

17. The plurality of polynucleotides of any of claims 1-9 and 14-16, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in SEQ ID NO: 421.

18. The plurality of polynucleotides of any of claims 1-17, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973.

19. The plurality of polynucleotides of any of claims 1-18, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 512-521, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

20. The plurality of polynucleotides of any of claims 1-19, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 512-521.

21. The plurality of polynucleotides of any of claims 1-20, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160,663,920-160,664,656.

22. The plurality of polynucleotides of any of claims 1-21, wherein at least one gRNA targets a target site for Lp(a) that is located is located within 250 bp the hg38 genomic coordinate chr6: 160,664,275.

23. The plurality of polynucleotides of any of claims 1-22, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 1-10 and 522- 342MF-366426018Attorney No. 224742004040 524, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

24. The plurality of polynucleotides of any of claims 1-23, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 1-10 and 522-524.

25. The plurality of polynucleotides of any of claims 1-24, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 4, 7 and 522-524, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

26. The plurality of polynucleotides of any of claims 1-25, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 4, 7 and 522-524.

27. The plurality of polynucleotides of any of claims 3-26, wherein at least one gRNA targets a target site of PCSK9 that is located within 110 bp of the hg38 genomic coordinatechrl: 55,039,548.

28. The plurality of polynucleotides of any of claims 3-27, wherein at least one gRNA targets a target site forPCSK9 that is within the coordinates chr1: 55,039,438-55,039,658.

29. The plurality of polynucleotides of any of claims 3-28, wherein at least one gRNA targets a target site of PCSK9 that is located within 80 bp of the hg38 genomic coordinatechrl: 55,039,548.

30. The plurality of polynucleotides of any of claims 3-29, wherein at least one gRNA targets a target site forPCSK9 that is within the coordinates chr1: 55,039,470-55,039,597.

31. The plurality of polynucleotides of any of claims 3-30, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in any one of SEQ ID NOs: 46-58,89-100, 409-412, 568, 569, and 574, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.343MF-366426018Attorney No. 224742004040 32. The plurality of polynucleotides of any of claims 3-31, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in any one of SEQ ID NOs: 46-58,89-100, 409-412, 568, 569, and 574.

33. The plurality of polynucleotides of any of claims 3-32, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in any one of SEQ ID NOs: 48, 49, 52 and 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

34. The plurality of polynucleotides of any of claims 3-33, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in any one of SEQ ID NOs: 48, 49, 52 and 89.

35. The plurality of polynucleotides of any of claims 3-34, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in SEQ ID NO: 48, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

36. The plurality of polynucleotides of any of claims 3-35, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in SEQ ID NO: 48.

37. The plurality of polynucleotides of any of claims 3-36, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in SEQ ID NO: 49, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

38. The plurality of polynucleotides of any of claims 3-37, wherein at least one gRNA targets a target site for PCSK9 that has the sequence set forth in SEQ ID NO: 49.

39. The plurality of polynucleotides of any of claims 1-38, wherein the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein.

40. The plurality of polynucleotides of any of claims 1-39, wherein the dCas protein lacks nuclease activity.

41. The plurality of polynucleotides of claim 39 or claim 40, wherein the dCas protein is a dCas9 protein.344MF-366426018Attorney No. 22474200404042. The plurality of polynucleotides of claim 40 or claim 41, wherein the dCas protein is a dCas12 protein.

43. The plurality of polynucleotides of claim 41, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

44. The plurality of polynucleotides of claim 43, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125.

45. The plurality of polynucleotides of claim 43 or claim 44, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 126, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

46. The plurality of polynucleotides of any of claims 43-45, wherein the dSaCas9 is set forth in SEQ ID NO: 126.

47. The plurality of polynucleotides of claim 41, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

48. The plurality of polynucleotides of claim 47, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 127.

49. The plurality of polynucleotides of claim 47 or claim 48, wherein the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

50. The plurality of polynucleotides of any of claims 47-49, wherein the dSpCas9 is set forth in SEQ ID NO: 128.

51. The plurality of polynucleotides of any of claims 1-50, wherein each of the at least one gRNA comprises a gRNA spacer sequence that is complementary to the target site of the respective gene.345MF-366426018Attorney No. 224742004040 52. The plurality of polynucleotides of any of claims 1-51, wherein each of the at least one gRNA independently comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

53. The plurality of polynucleotides of any of claims 1-52, wherein each of the at least one gRNA independently comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

54. The plurality of polynucleotides of any of claims 1-9 and 14-53, wherein at least one gRNA that targets a target site of Lp(a) comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36–40, 253–255, 417, 425–430, 534–539, 547, and 605 or a contiguous portion thereof of at least 14 nt.

55. The plurality of polynucleotides of any of claims 1-9 and 14-54, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOs: 36-40,253-255, 417, 425-430, 534-539, 547, and 605.

56. The plurality of polynucleotides of any of claims 1-9 and 14-55, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in SEQ ID NO: 42757. The plurality of polynucleotides of any of claims 1-9 and 14-56, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 577-586 or a contiguous portion thereof of at least 14 nt.

58. The plurality of polynucleotides of any of claims 1-9 and 14-57, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOs: 577-586.

59. The plurality of polynucleotides of any of claims 1-9 and 14-58, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 11-20 and 587-589 or a contiguous portion thereof of at least 14 nt.

60. The plurality of polynucleotides of any of claims 1-9 and 14-59, wherein at least one gRNA that target a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOs: 11-20 and 587-589.346MF-366426018Attorney No. 22474200404061. The plurality of polynucleotides of any of claims 1-9 and 14-60, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 14, 17, and 587-589 or a contiguous portion thereof of at least 14 nt.

62. The plurality of polynucleotides of any of claims 1-9 and 14-61, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOs: 14, 17, and 587-589.

63. The plurality of polynucleotides of any of claims 3-62, wherein at least one gRNA that targets a target site of PCSK9 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 59-71,101-112, 413-416, 570, 571, and 575, or a contiguous portion thereof of at least 14 nt; optionally wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:61 or a contiguous portion of at least 14 nt.

64. The plurality of polynucleotides of any of claims 3-63, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 59-71,101-112, 413-416, 570, 571, and 575,; optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:61.

65. The plurality of polynucleotides of any of claims 3-64, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 61, 62, 65 and 101.

66. The plurality of polynucleotides of any of claims 3-65, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in SEQ ID NO: 61.

67. The plurality of polynucleotides of any of claims 3-66, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in SEQ ID NO: 62.

68. The plurality of polynucleotides of any of claims 47-67, wherein each of the at least one gRNA further comprises a scaffold sequence set forth in SEQ ID NO:85, 259, 450 or 451.347MF-366426018Attorney No. 224742004040 69. The plurality of polynucleotides of any of claims 47-68, wherein at least one gRNA that targets a target site of Lp(a) comprises the sequence set forth in any one of SEQ ID NOS:41-45, 247-252, 439-449, 485-511, 540-545, 547, and 606-607.

70. The plurality of polynucleotides of any of claims 47-69, wherein at least one gRNA that targets a target site of Lp(a) is set forth in any one of SEQ ID NOS:41-45,247-252, 439-449, 485-511, 540-545, 547, and 606-60771. The plurality of polynucleotides of any of claims 47-70, wherein at least one gRNA that targets a target site of Lp(a) is set forth in SEQ ID NO: 508.

72. The plurality of polynucleotides of any of claims 47-71, wherein at least one gRNA that targets a target site of Lp(a) comprises the sequence set forth in any one of SEQ ID NOs: 590-599.

73. The plurality of polynucleotides of any of claims 47-72, wherein at least one gRNA that targets a target site of Lp(a) is set forth in any one of SEQ ID NOs: 590-599.

74. The plurality of polynucleotides of any of claims 47-73, wherein at least one gRNA that targets a target site of Lp(a) comprises the sequence set forth in any one of SEQ ID NOs: 600-602, 607 and 608.

75. The plurality of polynucleotides of any of claims 47-74, wherein at least one gRNA that targets a target site of Lp(a) is set forth in any one of SEQ ID NOs: 600-602, 607 and 608.

76. The plurality of polynucleotides of any of claims 47-75, wherein at least one gRNA that targets a target site of PCSK9 comprises the sequence set forth in any one of SEQ ID NOS:72-84,113-124, 431-438, 452, 454-484, 572, 573, 576, and 603; optionally wherein gRNA comprises the sequence set forth in SEQ ID NO: 74 or 431.

77. The plurality of polynucleotides of any of claims 47-76, wherein at least one gRNA that targets a target site of PCSK9 is set forth in any one of SEQ ID NOS: 72-84,113-124, 431-438, 452, 454-484, 572, 573, 576, and 603 optionally wherein the gRNA is set forth in SEQ ID NO:74 or 431.348MF-366426018Attorney No. 224742004040 78. The plurality of polynucleotides of any of claims 47-77, wherein at least one gRNA that targets a target site of PCSK9 is set forth in SEQ ID NO: 477.

79. The plurality of polynucleotides of any of claims 47-77, wherein at least one gRNA that targets a target site of PCSK9 is set forth in SEQ ID NO: 478.

80. The plurality of polynucleotides of any of claims 1-79, wherein each of the at least one gRNA comprises modified nucleotides for increased stability.

81. The plurality of polynucleotides of any of claims 1-80, wherein the at least one transcriptional repressor domain is capable of reducing transcription of the Lp(a) gene in a cell.

82. The plurality of polynucleotides of any of claims 3-81, wherein the at least one transcriptional repressor domain is capable of reducing transcription of the PCSK9 gene in a cell.

83. The plurality of polynucleotides of any of claims 1-82, wherein the at least one transcriptional repressor domain is capable of methylating or inducing DNA methylation at the target site and / or a region surrounding the target site in the Lp(a) gene and / or the PCSK9 gene.

84. The plurality of polynucleotides of any of claims 1-83, wherein the at least one transcriptional repressor domain comprises a DNA methyltransferase (DNMT).

85. The plurality of polynucleotides of any of claims 1-84, wherein the at least one transcriptional repressor domain comprises a DNMT3L domain or a variant or a functional portion thereof that exhibits transcriptional repressor activity.

86. The plurality of polynucleotides of any of claims 1-85, wherein the at least one transcriptional repressor domain is a single transcriptional repressor domain.

87. The plurality of polynucleotides of claim 86, wherein the single transcriptional repressor domain comprises a catalytically inactive DNA methyltransferase domain or a functional portion thereof.

88. The plurality of polynucleotides of claim 87, wherein the catalytically inactive DNA methyltransferase domain or a functional portion thereof is capable of recruiting domains with DNA methyltransferase activity.349MF-366426018Attorney No. 22474200404089. The plurality of polynucleotides of any one of claims 86-88, wherein the length of the single transcriptional repressor domain is less than 750 amino acids, 600 amino acids, 510 amino acids, 400 amino acids, or 300 amino in length.

90. The plurality of polynucleotides of any of claims 86-89, wherein the single transcriptional repressor domain comprises a DNMT3L domain or functional portion thereof.

91. The plurality of polynucleotides of any of claims 86-90, wherein the single transcriptional repressor domain is a DNMT3L domain or a functional portion thereof.

92. The plurality of polynucleotides of claim 91, wherein the DNMT3L domain or a functional portion thereof is the only transcriptional repressor domain present.

93. The plurality of polynucleotides of any of claims 86-92, wherein the single transcriptional repressor domain consists of a DNMT3L domain or functional portion thereof94. The plurality of polynucleotides of any of claims 85-93, wherein the plurality of polynucleotides are devoid of any nucleic acid sequence encoding additional transcriptional repressor domains other than the DNMT3L domain or functional portion thereof, optionally wherein the plurality of polynucleotides are devoid of a nucleic acid encoding a DNMT3 A domain, a KRAB domain, aSID domain, or other heterochromatin-inducing domain.

95. The plurality of polynucleotides of any of claims 85-94, wherein the DNMT3L domain or functional portion thereof is selected from one of the following species: Apodemus sylvaticus, Mus musculus, Homo sapiens, Equus caballus, Arvicanthis niloticus, Chinchilla lanigera, Desmodus rotundus, Echinops telfairi, Eptesicus fuscus, Fukomys damarensis, Globicephala melas, Jaculus jaculus, Mastomys coucha, Neofelis nebulosa, Neotoma lepida Ochotona princeps, Onychomys torridus, Perognathus longimembris pacificus, Phoca vitulina, Pteronotus mesoamericanus, and Saccopteryx leptura.

96. The plurality of polynucleotides of any of claims 85-95, wherein the DNMT3L domain or functional portion thereof is selected from one of the following species: Apodemus sylvaticus, Mus musculus, Homo sapiens, Equus caballus, Neotoma lepida, Ochotona princeps, and Onychomys torridus.350MF-366426018Attorney No. 224742004040 97. The plurality of polynucleotides of any of claims 85-96, wherein the DNMT3L domain or functional portion thereof is selected from one of the following species: Homo sapiens, Mus musculus, and Apodemus sylvaticus.

98. The plurality of polynucleotides of any of claims 85-97, wherein the functional portion of the DNMT3L domain is a contiguous portion that is less than a full-length DNMT3L MTase-like domain and comprises at least 10 amino acids from a reference DNMT3L MTase-like domain, wherein the contiguous portion of at least 10 amino acids is involved in a DNMT3 A-DNMT3L interface.

99. The plurality of polynucleotides of claim 98, wherein the contiguous portion is or comprises:a) the sequence set forth in WYX1FQFHRX2LQYAX3PX4X5 (SEQ ID NO: 293), wherein Xi is L or M, X2is L or I, X3is L or R, X4is K or R, and X5is P or Q;b) the sequence set forth in X1DX2X3X4X5X6RFLX7 (SEQ ID NO: 294), wherein Xi is E or D, X2is L or Q, X3is D, E, or M, X4is V or T, X5is A or T, X6is S, T, or V, and X7is E or Q;c) the sequence set forth in WYXiFQFHRX2LQYAX3PX4X5X6SX7X8PFFWX9FXioDNLXiiLXi2Xi3Xi4DXi5Xi6Xi7Xi8Xi9RFLX2o (SEQ ID NO: 302), wherein Xi is L or M, X2is L or I, X3is L or R, X4is K or R, X5is P or Q, X6is G or E, X7is P, Q, or absent, X8is R or Q, X9is M or I, Xw is V or M, Xu is V or L, X12 is N or T, Xnis K or E, X14 is E or D, X15 is L or Q, Xie is D, E, or M, X17 is V or T, Xis is A or T, X19 is S, T, or V, and X20 is E or Q; ord) the sequence set forth in XiX2VRX3DVEX4WGPFDLX5YGX6TX7PLGX8X9CDRXioPXiiWYXi2FQFHRXi3LQYAXi4PXi5Xi6Xi7SXi8Xi9PFFWX2oFX2iDNLX22LX23X24X25DX26X27X28X29X3oRFLX3i (SEQ ID NO: 306), wherein Xi is D or N, X2is T or V, X3is K or R, X4is E or K, X5is V or L, X6is A or S, X7is P or Q, X8is H or S, X9is T or S, Xw is P or C, Xu is S or G, X12 is L or M, Xnis L or I, XMis L or R, Xu is K or R, Xi6is P or Q, Xi7is G or E, Xis is P, Q, or absent, X19 is R or Q, X20 is M or I, X21 is V or M, X22 is V or L, X23 is N or T, X24 is K or E, X25 is E or D, X2e is L or Q, X27is D, E, or M, X28is V or T, X29 is A or T, X30 is S, T, or V, and X31 is E or Q.

100. The plurality of polynucleotides of claim 98 or claim 99, wherein the contiguous portion is or comprises:e) the sequence set forth as amino acid residues 258-274 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134;351MF-366426018Attorney No. 224742004040 f) the sequence set forth as amino acid residues 292-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134;g) the sequence set forth as amino acid residues 258-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134; orh) the sequence set forth as amino acid residues 226-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134.

101. The plurality of polynucleotides of any one of claims 98-100, wherein the contiguous portion is or comprises:e) the sequence set forth in any one of SEQ ID NOs: 296-298;f) the sequence set forth in any one of SEQ ID NOs: 299-301;g) the sequence set forth in any one of SEQ ID NOs: 303-305; orh) the sequence set forth in any one of SEQ ID NOs: 307-309.

102. The plurality of polynucleotides of any one of claims 98-101, wherein the contiguous portion comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or at least 75 amino acids.

103. The plurality of polynucleotides of any one of claims 98-102, wherein the reference DNMT3L MTase-like domain is or comprises the sequence set forth in any one of SEQ ID NOs: 272-274, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

104. The plurality of polynucleotides of any one of claims 85-103, wherein the DNMT3L domain or functional portion thereof is a DNMT3L MTase-like domain or a portion of a DNMT3L MTase-like domain.

105. The plurality of polynucleotides of any one of claims 85-104, wherein the DNMT3L domain or functional portion thereof is or comprises the sequence set forth in any one of SEQ ID NOs: 272-274 and 549-566, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.352MF-366426018Attorney No. 224742004040106. The plurality of polynucleotides of any one of claims 85-105, wherein the DNMT3L domain or functional portion thereof is or comprises the sequence set forth in any one of SEQ ID NOs: 272-274 and 549-566.

107. The plurality of polynucleotides of any one of claims 85-106, wherein the DNMT3L domain or functional portion thereof is or comprises the sequence set forth in SEQ ID NO: 274.

108. The plurality of polynucleotides of any one of claims 98-107, wherein the at least one transcriptional repressor domain further comprises a DNMT3L ADD domain.

109. The plurality of polynucleotides of claim 108, wherein the at least one transcriptional repressor domain comprises, from N-terminus to C-terminus:a) the DNMT3L ADD domain and the DNMT3L MTase-like domain; orb) the DNMT3L MTase-like domain and the DNMT3L ADD domain.

110. The plurality of polynucleotides of claim 108 or claim 109, wherein the DNMT3L ADD domain is or comprises the sequence set forth in any one of SEQ ID NOs: 283, and 285-287.

111. The plurality of polynucleotides of any one of claims 1-102 and 108-110, wherein the at least one transcriptional repressor domain comprises the sequence set forth in any one of SEQ ID NOs: 284, 288-290, and 567, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

112. The plurality of polynucleotides of any one of claims 1-102 and 108-111, wherein the at least one transcriptional repressor domain comprises:a) the sequence set forth in any one of SEQ ID NOs: 134, 171, and 291 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing; orb) the sequence set forth in any one of SEQ ID NOs: 281, 282, and 292 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

113. The plurality of polynucleotides of any of claims 1-112, wherein the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 134, a portion thereof,353MF-366426018Attorney No. 224742004040 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 134.

114. The plurality of polynucleotides of any of claims 1-86 and 95-113, wherein the at least one transcriptional repressor domain comprises a histone methyltransferase, or a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase.

115. The plurality of polynucleotides of any of claims 1-86 and 95-114, wherein the at least one transcriptional repressor domain comprises a DNA methyltransferase and a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase.

116. The plurality of polynucleotides of any of claims 1-86 and 95-115, wherein the at least one transcriptional repressor domain comprises a DNA methyltransferase and a histone methyltransferase.

117. The plurality of polynucleotides of any of claims 1-86 and 95-116, wherein one or more of the at least one transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3 A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3 A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxil repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing.

118. The plurality of polynucleotides of any of claims 1-86 and 95-117, wherein the at least one transcriptional repressor domain comprises a KRAB domain or a variant or portion thereof that exhibits transcriptional repressor activity.

119. The plurality of polynucleotides of any of claims 1-86 and 95-118, wherein the at least one transcriptional repressor domain comprises a KRAB domain, a DNMT3 A domain, a DNMT3L domain, or a combination of any of the foregoing.

120. The plurality of polynucleotides of any of claims 1-86 and 95-119, wherein the at least one transcriptional repressor domain is or comprises the sequence set forth in SEQ ID NO: 130 or SEQ354MF-366426018Attorney No. 224742004040 ID NO: 527, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

121. The plurality of polynucleotides of any of claims 1-86 and 95-120, wherein the at least one transcriptional repressor domain comprises a DNMT3 A domain or a variant or portion thereof that exhibits transcriptional repressor activity.

122. The plurality of polynucleotides of any of claims 1-86, 95-118 and 121, wherein the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 132, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 132.

123. The plurality of polynucleotides of any of claims 1-86 and 95-122, wherein the at least one transcriptional repressor domain is a DNMT3 A-DNMT3L fusion protein domain or a variant thereof that exhibits transcriptional repressor activity.

124. The plurality of polynucleotides of any of claims 1-86, 95-123, wherein the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 136 or SEQ ID NO: 138, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

125. The plurality of polynucleotides of any of claims 1-86 and 95-117, wherein at least one transcriptional repressor domain comprises the sequence set forth in any one of SEQ ID NOS:130, 132, 134, 136, 138-146, and 527, or a domain thereof, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

126. The plurality of polynucleotides of any of claims 1-125, wherein the at least one transcriptional repressor domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain.

127. The plurality of polynucleotides of any of claims 1-126, wherein the fusion protein further comprises one or more nuclear localization signals (NLS).355MF-366426018Attorney No. 224742004040 128. The plurality of polynucleotides of claim 127, wherein the fusion protein further comprises one or more linkers connecting two or more of: the DNA-binding domain, the at least one transcriptional repressor domain, and the one or more nuclear localization signals.

129. The plurality of polynucleotides of claim 128, wherein the one or more linkers comprise the sequence set forth in SEQ ID NO: 407 or SEQ ID NO: 190, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

130. The plurality of polynucleotides of claim 128 or claim 129, wherein the one or more linkers comprise the sequence set forth in SEQ ID NO: 190.

131. The plurality of polynucleotides of any of claims 1-113 and 126-130 wherein the fusion protein is devoid of any domains with DNA methyltransferase activity, domains capable of recruiting heterochromatin inducing factors, and H3K4meO peptides.

132. The plurality of polynucleotides of any of claims 41-113 and 126-131, wherein the fusion protein consists essentially of the dCas9 protein and the DNMT3L domain or functional portion thereof.

133. The plurality of polynucleotides of any of claims 85-113 and 126-132, wherein the DNMT3L domain or functional portion thereof is the sole transcriptional repressor domain of the fusion protein.

134. The plurality of polynucleotides of any of claims 85-113 and 126-133, wherein the fusion protein is devoid of any other transcriptional repressor domains, optionally wherein the fusion protein is devoid of a DNMT3A domain, a KRAB domain, a SID domain, or other repressor motifs.

135. The plurality of polynucleotides of any of claims 41-113 and 126-134, wherein the fusion protein comprises from N-terminus to C-terminus:c) the dCas9 protein, the linker, and the DNMT3L domain or functional portion thereof; or d) the DNMT3L domain or functional portions thereof, the linker, and the dCas9 protein.

136. The plurality of polynucleotides of any of claims 1-113 and 126-135, wherein the fusion protein comprises the sequence set forth in any one of SEQ ID NOS: 260-262, or an amino acid sequence356MF-366426018Attorney No. 224742004040 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

137. The plurality of polynucleotides of any of claims 1-113 and 126-135, wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 262.

138. The plurality of polynucleotides of any of claims 1-120 and 126-130, wherein the fusion protein comprises the sequence set forth in any one of SEQ ID NOS: 88, 148, 150, 152, and 279 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, optionally wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 150.

139. The plurality of polynucleotides of any of claims 1-138, wherein the polynucleotide encoding the fusion protein is mRNA.

140. The plurality of polynucleotides of claim 139, wherein the DNA-targeting system comprises an mRNA encoding the fusion protein and one gRNA targeting a target site of Lp(a).

141. The plurality of polynucleotides of claim 140, wherein the ratio of the mRNA to the one gRNA targeting a target site of Lp(a) is between 5:1 and 1:5.

142. The plurality of polynucleotides of claim 140 or claim 141, wherein the ratio of mRNA:gRNA is 2:1.

143. The plurality of polynucleotides of claim 139, wherein the DNA-targeting system comprises an mRNA encoding the fusion protein, one gRNA targeting a target site of Lp(a), and one gRNA targeting a target site of PSCK9.

144. The plurality of polynucleotides of claim 143, wherein the ratio of the mRNA to the one gRNA targeting a target site of Lp(a) to the one gRNA targeting a target site of PCSK9 is between 2:1.5:0.1 to 2:0.1:1.5.

145. The plurality of polynucleotides of claim 143 or claim 144, wherein the amount of the gRNA targeting a target site of Lp(a) is equal to the amount of the gRNA targeting a target site of PCKS9.357MF-366426018Attorney No. 224742004040146. The plurality of polynucleotides of any of claims 143-145, wherein the ratio of mRNA:gRNA:gRNA is 2:0.5:0.5.

147. The plurality of polynucleotides of any of claims 139-146, wherein the amount of mRNA is twice the amount of gRNA(s).

148. The plurality of polynucleotides of any of claims 139-147, wherein the amount of mRNA is at least 75 ng.

149. The plurality of polynucleotides of any one of claims 139-148, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 156, or a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

150. The plurality of polynucleotides of any one of claims 139-149, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 153.

151. An epigenetic -modifying DNA-targeting system, comprising a fusion protein comprising:(a) a DNA-binding domain for targeting to a target site of Lp(a), wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 or between the hg38 genomic coordinates club: 160,519,674 and 160,520,973, and(b) at least one transcriptional repressor domain.

152. The epigenetic-modifying DNA-targeting system of claim 151, wherein the target site forLp(a) is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782.

153. The epigenetic-modifying DNA-targeting system of claim 151 or claim 152, wherein the DNA-targeting system does not introduce a genetic disruption or a DNA break.

154. The epigenetic-modifying DNA-targeting system of claim 152 or claim 153, wherein the DNA-binding domain is selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof,358MF-366426018Attorney No. 224742004040 optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing.

155. The epigenetic-modifying DNA-targeting system of any of claims 151-154, wherein the DNA-binding domain is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and the system further comprises at least one gRNA for targeting the DNA-binding domain to a target site of Lp(a).

156. An epigenetic -modifying DNA-targeting system, comprising:(a) a fusion protein comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof and at least one transcriptional repressor domain; and(b) at least one gRNA that targets a target site of Lp(a), wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782, or between the hg38 genomic coordinates club: 160,519,674 and 160,520,973.

157. The epigenetic-modifying DNA-targeting system of claim 156, wherein the at least one gRNA that targets a site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782.

158. The epigenetic-modifying DNA-targeting system of claim 156 or claim 157, wherein the at least one gRNA that a targets a target site of Lp(a) is a first gRNA that targets a first Lp(a) target site and a second gRNA that targets a second Lp(a) target site.

159. The epigenetic-modifying DNA-targeting system of claim 158, wherein the first and second Lp(a) target sites are:a) a first target site located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and a second target site within 500 bp of the hg38 genomic coordinate chr6: 160,664,275; or b) a first target site located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and a second target site located between the hg38 genomic coordinates chr6: 160,519,674 and chr6:160,520,973; orc) a first target site located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973 and a second target site within 500 bp of the hg38 genomic coordinate chr6:160,664,275.359MF-366426018Attorney No. 224742004040 160. The epigenetic-modifying DNA-targeting system of any of claims 155-159, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 678,800 and chr6: 160,679,650.

161. The epigenetic-modifying DNA-targeting system of any of claims 155-160, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 678,280 and chr6: 160,679,602.

162. The epigenetic-modifying DNA-targeting system of any of claims 155-161, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782.

163. The epigenetic-modifying DNA-targeting system of any of claims 155-162, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650.

164. The epigenetic-modifying DNA-targeting system of any of claims 155-162, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679,000 and chr6: 160,679,350.

165. The epigenetic-modifying DNA-targeting system of any of claims 155-162, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700.

166. The epigenetic-modifying DNA-targeting system of any of claims 155-162, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160, 679, 000-160,679,200; chr6: 160, 679, 200-160,679,400; chr6: 160, 679, 400-160,679,600; or chr6: 160, 679, 500-160,679,700.

167. The epigenetic-modifying DNA-targeting system of any of claims 155-162 and 166, wherein at least one gRNA targets a target site for Lp(a) that comprises the hg38 genomic coordinates chr6: 160, 679,280-160, 679, 299.

168. The epigenetic-modifying DNA-targeting system of any of claims 155-162, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs:360MF-366426018Attorney No. 224742004040 31-35,256-258418-424, 528-533, 546, and 604 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

169. The epigenetic-modifying DNA-targeting system of any of claims 155-162 and 168, wherein the at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 31-35,256-258, 418-424, 528-533, 546, and 604.

170. The epigenetic-modifying DNA-targeting system of any of claims 155-162 and 168-169 wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in SEQ ID NO: 421, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

171. The epigenetic-modifying DNA-targeting system of any of claims 155-162 and 168-170, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in SEQ ID NO: 421.

172. The epigenetic-modifying DNA-targeting system of any of claims 155-171, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973.

173. The epigenetic-modifying DNA-targeting system of any of claims 155-172, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 512-521, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

174. The epigenetic-modifying DNA-targeting system of any of claims 155-173, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 512-521.

175. The epigenetic-modifying DNA-targeting system of any of claims 155-174, wherein at least one gRNA targets a target site for Lp(a) that is located between the hg38 genomic coordinates chr6: 160,663,920-160,664,656.361MF-366426018Attorney No. 224742004040 176. The epigenetic-modifying DNA-targeting system of any of claims 155-175, wherein at least one gRNA targets a target site for Lp(a) that is located is located within 250 bp the hg38 genomic coordinate chr6: 160,664,275.

177. The epigenetic-modifying DNA-targeting system of any of claims 155-176, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 1-10 and 522-524, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

178. The epigenetic-modifying DNA-targeting system of any of claims 155-177, wherein the at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 1-10 and 522-524.

179. The epigenetic-modifying DNA-targeting system of any of claims 155-178, wherein at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 4, 7 and 522-524, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

180. The epigenetic-modifying DNA-targeting system of any of claims 155-179, wherein the at least one gRNA targets a target site for Lp(a) that has the sequence set forth in any one of SEQ ID NOs: 4, 7 and 522-524.

181. The epigenetic-modifying DNA-targeting system of any one of claims 155-180, further comprising (c) at least a second gRNA that targets a target site of PCSK9 located within 500bp of the hg38 genomic coordinate chr1:55,039,548.

182. The epigenetic-modifying DNA-targeting system of claim 181, wherein the target site of PCSK9 is located within 110 bp of the hg38 genomic coordinate chr1:55,039,548.

183. The epigenetic-modifying DNA-targeting system of claim 181 or claim 182, wherein the target site forPCSK9 is within the coordinates chr1: 55,039,438-55,039,658.

184. The epigenetic-modifying DNA-targeting system of any of claims 181-183, wherein the target site of PCSK9 is located within 80 bp of the hg38 genomic coordinate chr1:55,039,548.362MF-366426018Attorney No. 224742004040 185. The epigenetic-modifying DNA-targeting system of any of claims 181-184, wherein the target site forPCSK9 is within the coordinates chr1: 55,039,470-55,039,597.

186. The epigenetic-modifying DNA-targeting system of any of claims 181-184, wherein the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58, 89-100409-412, 568, 569, and 574, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

187. The epigenetic-modifying DNA-targeting system of any of claims 181-186, wherein the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58,89-10, 409-412,, 568, 569, and 574.

188. The epigenetic-modifying DNA-targeting system of any of claims 181-187, wherein the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 48, 49, 52 and 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

189. The epigenetic-modifying DNA-targeting system of any of claims 181-188, wherein the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 48, 49, 52 and 89.

190. The epigenetic-modifying DNA-targeting system of any of claims 181-189, wherein the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

191. The epigenetic-modifying DNA-targeting system of any of claims 181-190, wherein the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48.

192. The epigenetic-modifying DNA-targeting system of any of claims 181-191, wherein the target site for PCSK9 has the sequence set forth in SEQ ID NO: 49, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

193. The epigenetic-modifying DNA-targeting system of any of claims 181-192, wherein the target site for PCSK9 has the sequence set forth in SEQ ID NO: 49.363MF-366426018Attorney No. 224742004040 194. The epigenetic-modifying DNA-targeting system of any of claims 155-193, wherein the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein.

195. The epigenetic-modifying DNA-targeting system of claim 194, wherein the dCas protein lacks nuclease activity.

196. The epigenetic-modifying DNA-targeting system of claim 194 or claim 195, wherein the dCas protein is a dCas9 protein.

197. The epigenetic-modifying DNA-targeting system of claim 194 or claim 195, wherein the dCas protein is a dCas12 protein.

198. The epigenetic-modifying DNA-targeting system of claim 196, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

199. The epigenetic-modifying DNA-targeting system of claim 198, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125.

200. The epigenetic-modifying DNA-targeting system of claim 198 or claim 199, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 126, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

201. The epigenetic-modifying DNA-targeting system of any of claims 198-200, wherein the dSaCas9 is set forth in SEQ ID NO: 126.

202. The epigenetic-modifying DNA-targeting system of claim 196, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

203. The epigenetic-modifying DNA-targeting system of claim 202, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 127.364MF-366426018Attorney No. 224742004040 204. The epigenetic-modifying DNA-targeting system of claim 202 or claim 203, wherein the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

205. The epigenetic-modifying DNA-targeting system of any of claims 202-204, wherein the dSpCas9 is set forth in SEQ ID NO: 128.

206. The epigenetic-modifying DNA-targeting system of any of claims 155-205, wherein the each of the at least one gRNA comprises a gRNA spacer that is complementary to the target site of the gene.

207. The epigenetic-modifying DNA-targeting system of any of claims 155-206, wherein the each of the at least one gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

208. The epigenetic-modifying DNA-targeting system of any of claims 155-207, wherein each of the at least one gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

209. The epigenetic-modifying DNA-targeting system of any of claims 155-208, wherein at least one gRNA that targets a target site of Lp(a) comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40,253-255, 417, 425-430, 534-539, 547, and 605 or a contiguous portion thereof of at least 14 nt.

210. The epigenetic-modifying DNA-targeting system of any of claims 155-209, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 36-40,253-255, 417, 425-430, 534-539, 547, and 605.

211. The epigenetic-modifying DNA-targeting system of any of claims 155-210, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in SEQ ID NO: 427.

212. The epigenetic-modifying DNA-targeting system of any of claims 155-211, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 577-586 or a contiguous portion thereof of at least 14 nt.365MF-366426018Attorney No. 224742004040213. The epigenetic -modifying DNA-targeting system of any of claims 155-212, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOs: 577-586.

214. The epigenetic-modifying DNA-targeting system of any of claims 155-213, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 11-20 and 587-589 or a contiguous portion thereof of at least 14 nt.

215. The epigenetic-modifying DNA-targeting system of any of claims 155-214, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOs: 11-20 and 587-589.

216. The epigenetic-modifying DNA-targeting system of any of claims 155-215, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 14, 17, and 587-589 or a contiguous portion thereof of at least 14 nt.

217. The epigenetic-modifying DNA-targeting system of any of claims 155-216, wherein at least one gRNA that targets a target site of Lp(a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOs: 14, 17, and 587-589.

218. The epigenetic -modifying DNA-targeting system of any of claims 181-217, wherein at least one gRNA that targets a target site of PCSK9 comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 59-71,101-112, 413-416, 570, 571, and 575, ora contiguous portion thereof of at least 14 nt; optionally wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:61 or a contiguous portion of at least 14 nt.

219. The epigenetic -modifying DNA-targeting system of any of claims 181-218, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 59-71,101-112, 413-416, 570, 571, and 575,; optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:61.366MF-366426018Attorney No. 224742004040 220. The epigenetic -modifying DNA-targeting system of any of claims 181-219, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 61, 62, 65 and 101.

221. The epigenetic -modifying DNA-targeting system of any of claims 181 -220, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in SEQ ID NO: 61.

222. The epigenetic -modifying DNA-targeting system of any of claims 181-221, wherein at least one gRNA that targets a target site of PCSK9 comprises the gRNA spacer sequence set forth in SEQ ID NO: 62.

223. The epigenetic-modifying DNA-targeting system of any of claims 202-222, wherein each of the least one gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 85, 259, 450 or 451.

224. The epigenetic-modifying DNA-targeting system of any of claims 202-223, wherein at least one gRNA comprises the sequence set forth in any one of SEQ ID NOS:41-45, 247-252, 439-440, 485-511, 540-545, 547, and 606-607.

225. The epigenetic-modifying DNA-targeting system of any of claims 202-224, wherein at least one gRNA is set forth in any one of SEQ ID NOS:41-45, 247-252, 439-440, 485-511, 540-545, 547, and 606-607.

226. The epigenetic-modifying DNA-targeting system of any of claims 202-225, wherein at least one gRNA that targets a target site of Lp(a) is set forth in SEQ ID NO: 508.

227. The epigenetic-modifying DNA-targeting system of any of claims 202-226, wherein at least one gRNA that targets a target site of Lp(a) comprises the sequence set forth in any one of SEQ ID NOs: 590-599.

228. The epigenetic-modifying DNA-targeting system of any of claims 202-227, wherein at least one gRNA that targets a target site of Lp(a) is set forth in any one of SEQ ID NOs: 590-599.367MF-366426018Attorney No. 224742004040 229. The epigenetic-modifying DNA-targeting system of any of claims 202-228, wherein at least one gRNA that targets a target site of Lp(a) comprises the sequence set forth in any one of SEQ ID NOs: 600-602, 607 and 608.

230. The epigenetic-modifying DNA-targeting system of any of claims 202-229, wherein at least one gRNA that targets a target site of Lp(a) is set forth in any one of SEQ ID NOs: 600-602, 607 and 608.

231. The epigenetic-modifying DNA-targeting system of any of claims 155-230, wherein each of the at least one gRNA comprises modified nucleotides for increased stability.

232. The epigenetic-modifying DNA-targeting system of any of claims 151-231, wherein the at least one transcriptional repressor domain is capable of reducing transcription of the Lp(a) gene in a cell.

233. The epigenetic-modifying DNA-targeting system of any of claims 151-232, wherein the at least one transcriptional repressor domain is capable of methylating or inducing methylation at the target site and / or a region surrounding the target site in the Lp(a) gene.

234. The epigenetic-modifying DNA-targeting system of any of claims 181-233, wherein the first gRNA and the second gRNA are selected from two different members of the group consisting of:(a) a gRNA targeting a target site of Lp(a) comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40,253-255, 417, 425-430, 534-539, 547, and 605 ora contiguous portion thereof of at least 14 nt; and(b) a gRNA targeting a target site of PCSK9 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 59-71,101-112, 413-416, 570, 571, and 575, ora contiguous portion thereof of at least 14 nt; optionally wherein the gRNA spacer sequence comprises the sequence set forth in SEQ ID NO:61 or a contiguous portion of at least 14 nt.

235. The epigenetic-modifying DNA-targeting system of any of claims 181-234, wherein the first gRNA and the second gRNA are selected from two different members of the group consisting of:(a) a gRNA targeting a target site of Lp(a) comprising the gRNA spacer sequence set forth in any one of SEQ ID NOS: 36-40,253-255, 417, 425-430, 534-539, 547, and 605; and368MF-366426018Attorney No. 224742004040 (b) a gRNA targeting a target site in PCSK9 comprising the gRNA spacer sequence set forth in any one of SEQ ID NOS: 59-71,101-112, 413-416, 570, 571, and 575; optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:61.

236. The epigenetic-modifying DNA-targeting system of any of claims 181-235, wherein the first gRNA and the second gRNA are selected from two different members of the group consisting of:(a) a gRNA targeting a target site of Lp(a) comprising the gRNA spacer sequence set forth in any one of SEQ ID NOS: 255, 417, and 425-430; and(b) a gRNA targeting a target site in PCSK9 comprising the gRNA spacer sequence set forth in any one of SEQ ID NOS: 61, 62, 65, and 101.

237. The epigenetic-modifying DNA-targeting system of any of claims 181-236, wherein: a) the first gRNA targets a target site of Lp(a) comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 421; and(b) the second gRNA targets a target site of PCSK9 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 62.

238. The epigenetic-modifying DNA-targeting system of any of claims 181-237, wherein each gRNA further comprises a scaffold sequence set forth in SEQ ID NO:85, 259, 450 or 451.

239. The epigenetic-modifying DNA-targeting system of any of claims 181-238, wherein the first gRNA and the second gRNA are selected from two different members of the group consisting of:(a) a gRNA targeting a target site in Lp(a) comprising the sequence set forth in any one of SEQ ID NOS:41-45, 247-252, 439-449, 485-511, 540-545, 547, and 606-607; and(b) a gRNA targeting a target site in PCSK9 comprising the sequence set forth in any one of SEQ ID NOS:72-84, 113-124, 431-438, 452, 454-484, 572, 573, 576, and 603; optionally wherein gRNA comprises the sequence set forth in SEQ ID NO: 74 or 431.

240. The epigenetic-modifying DNA-targeting system of any of claims 181-239, wherein the first gRNA and the second gRNA are selected from two different members of the group consisting of:(a) a gRNA targeting a target site in Lp(a) set forth in any one of SEQ ID NOS:41-45, 247-252, 439-449, 485-511, 540-545, 547, and 606-607; and(b) a gRNA targeting a target site inPCSK9 set forth in any one of SEQ ID NOS: 72-84,113-124, 431-438, 452, 454-484, 572, 573, 576, and 603, optionally wherein the gRNA is set forth in SEQ ID NO:74 or 431.369MF-366426018Attorney No. 224742004040241. The epigenetic-modifying DNA-targeting system of any of claims 181-240, wherein:(a) the first gRNA that targets a target site of Lp(a) is set forth in SEQ ID NO: 508; and (b) the second gRNA that targets a target site of PCSK9 is set forth in SEQ ID NO: 477 or SEQ ID NO: 478.

242. The epigenetic-modifying DNA-targeting system of any one of claims 181-241 further comprising a third gRNA that targets a second Lp(a) target site.

243. The epigenetic-modifying DNA-targeting system of claim 242, wherein the third gRNA is selected from:(a) a gRNA that targets a target site of Lp(a) comprising gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 577-586 or a contiguous portion thereof of at least 14 nt; or(b) a gRNA that targets a target site of Lp(a) comprising gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 11-20 and 587-589 or a contiguous portion thereof of at least 14 nt.

244. The epigenetic -modifying DNA-targeting system of any of claims 242 or claim 243, wherein the third gRNA is selected from:(a) a gRNA that targets a target site of Lp(a) comprising the sequence set forth in any one of SEQ ID NOs: 590-599; or(b) a gRNA that targets a target site of Lp(a) comprising sequence comprising the sequence set forth in any one of SEQ ID NOs: 600-602, 607 and 608.

245. The epigenetic-modifying DNA-targeting system of any of claims 181-244, wherein the at least one transcriptional repressor domain is capable of reducing transcription of the Lp(a) gene and the PCSK9 gene in a cell.

246. The epigenetic-modifying DNA-targeting system of any of claims 181-245, wherein the at least one transcriptional repressor domain is capable of methylating or inducing DNA methylation at the target site and / or a region surrounding the target site in Lp(a) gene and / or PCSK9 gene.

247. The epigenetic-modifying DNA-targeting system of any of claims 151-246, wherein the at least one transcriptional repressor domain comprises a DNA methyltransferase (DNMT).370MF-366426018Attorney No. 224742004040248. The epigenetic-modifying DNA-targeting system of any of claims 151-247, wherein the at least one transcriptional repressor domain comprises a DNMT3L domain or a variant or a functional portion thereof that exhibits transcriptional repressor activity.

249. The epigenetic-modifying DNA-targeting system of any of claims 151-248, wherein the at least one transcriptional repressor domain is a single transcriptional repressor domain.

250. The epigenetic-modifying DNA-targeting system of any of claims 151-249, wherein the single transcriptional repressor domain comprises a catalytically inactive DNA methyltransferase domain or a functional portion thereof.

251. The epigenetic-modifying DNA-targeting system of claim 250, wherein the catalytically inactive DNA methyltransferase domain or a functional portion thereof is capable of recruiting domains with DNA methyltransferase activity.

252. The epigenetic-modifying DNA-targeting system of any of claims 249-251, wherein the length of the single transcriptional repressor domain is less than 750 amino acids, 600 amino acids, 510 amino acids, 400 amino acids, or 300 amino in length.

253. The epigenetic-modifying DNA-targeting system of any of claims 249-252, wherein the single transcriptional repressor domain comprises a DNMT3L domain or functional portion thereof.

254. The epigenetic-modifying DNA-targeting system of any of claims 249-252, wherein the single transcriptional repressor domain is a DNMT3L domain or a functional portion thereof.

255. The epigenetic-modifying DNA-targeting system of claim 254, wherein the DNMT3L domain or a functional portion thereof is the only transcriptional repressor domain present.

256. The epigenetic-modifying DNA-targeting system of any of claims 249-255, wherein the single transcriptional repressor domain consists of a DNMT3L domain or functional portion thereof257. The epigenetic-modifying DNA-targeting of any of claims 248-256, wherein the fusion protein is devoid of any additional transcriptional repressor domains, optionally wherein the fusion371MF-366426018Attorney No. 224742004040 protein is devoid of a DNMT3A domain, a KRAB domain, a SID domain, or other heterochromatininducing domain.

258. The epigenetic-modifying DNA-targeting system of any of claims 248-257, wherein the DNMT3L domain or functional portion thereof is selected from one of the following species: Apodemus sylvaticus, Mus musculus, Homo sapiens, Equus caballus, Arvicanthis niloticus, Chinchilla lanigera, Desmodus rotundus, Echinops telfairi, Eptesicus fuscus, Fukomys damarensis, Globicephala melas, Jaculus jaculus, Mastomys coucha, Neofelis nebulosa, Neotoma lepida Ochotona princeps, Onychomys torridus, Perognathus longimembris pacificus, Phoca vitulina, Pteronotus mesoamericanus, and Saccopteryx leptura.

259. The epigenetic-modifying DNA-targeting system of any of claims 248-258, wherein the DNMT3L domain or functional portion thereof is selected from one of the following species: Apodemus sylvaticus, Mus musculus, Homo sapiens, Equus caballus, Neotoma lepida, Ochotona princeps, and Onychomys torridus.

260. The epigenetic -modifying DNA-targeting system of any of claims 248-259, wherein the DNMT3L domain or functional portion thereof is selected from one of the following species: Homo sapiens, Mus musculus, and Apodemus sylvaticus.

261. The epigenetic-modifying DNA-targeting system of any of claims 248-259, wherein the functional portion of the DNMT3L domain is a contiguous portion that is less than a full-length DNMT3L MTase-like domain and comprises at least 10 amino acids from a reference DNMT3L MTase-like domain, wherein the contiguous portion of at least 10 amino acids is involved in a DNMT3 A-DNMT3L interface.

262. The epigenetic-modifying DNA-targeting system of claim 261, wherein the contiguous portion is or comprises:e) the sequence set forth in WYX1FQFHRX2LQYAX3PX4X5 (SEQ ID NO: 293), wherein Xi is L or M, X2is L or I, X3is L or R, X4is K or R, and X5 is P or Q;f) the sequence set forth in X1DX2X3X4X5X6RFLX7 (SEQ ID NO: 294), wherein Xi is E or D, X2is L or Q, X3is D, E, or M, X4is V or T, X5is A or T, X6is S, T, or V, and X7is E or Q; g) the sequence set forth in WYXiFQFHRX2LQYAX3PX4X5X6SX7X8PFFWX9FXioDNLXiiLXi2Xi3Xi4DXi5Xi6Xi7Xi8Xi9 RFLX20 (SEQ ID NO: 302), wherein Xi is L or M, X2is L or I, X3is L or R, X4is K or R, X5is 372MF-366426018Attorney No. 224742004040 P or Q, X6is G or E, X7is P, Q, or absent, X8is R or Q, X9is M or I, Xw is V or M, Xu is V or L, X12 is N or T, Xi3is K or E, Xi4is E or D, Xi5is L or Q, Xi6is D, E, or M, Xi7is V or T, Xis is A or T, Xis is S, T, or V, and X2o is E or Q; orh) the sequence set forth in XiX2VRX3DVEX4WGPFDLX5YGX6TX7PLGX8X9CDRXioPXiiWYXi2FQFHRXi3LQYAXi4P X15X16X17SX18X19PFFWX2OFX21DNLX22LX23X24X25DX26X27X28X29X3ORFLX31 (SEQ ID NO: 306), wherein XI is D or N, X2 is T or V, X3 is K or R, X4 is E or K, X5 is V or L, X6 is A or S, X7 is P or Q, X8 is H or S, X9 is T or S, X10 is P or C, Xll is S or G, X12 is L orM, Xu is L or I, XMis L or R, Xu is K or R, Xi6is P or Q, Xi7is G or E, Xi8is P, Q, or absent, X19 is R or Q, X20 is M or I, X21 is V or M, X22 is V or L, X23is N or T, X24 is K or E, X25 is E or D, X2e is L or Q, X27is D, E, or M, X28is V or T, X29is A or T, X30is S, T, or V, and X3iis E or Q.

263. The epigenetic-modifying DNA-targeting system of claim 261 or claim 262, wherein the contiguous portion is comprises:e) the sequence set forth as amino acid residues 258-274 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134;f) the sequence set forth as amino acid residues 292-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134;g) the sequence set forth as amino acid residues 258-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134; orh) the sequence set forth as amino acid residues 226-303 from the reference DNMT3L MTase-like domain, corresponding to numbering of positions set forth in SEQ ID NO: 134.

264. The epigenetic -modifying DNA-targeting system of any of claim 261-263, wherein the contiguous portion is or comprises:e) the sequence set forth in any one of SEQ ID NOs: 296-298;f) the sequence set forth in any one of SEQ ID NOs: 299-301;g) the sequence set forth in any one of SEQ ID NOs: 303-305; orh) the sequence set forth in any one of SEQ ID NOs: 307-309.373MF-366426018Attorney No. 224742004040 265. The epigenetic-modifying DNA-targeting system of any one of claims 261-264, wherein the contiguous portion is or comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or at least 75 amino acids.

266. The epigenetic-modifying DNA-targeting system of any one of claims 261-265, wherein the reference DNMT3L MTase-like domain is or comprises the sequence set forth in any one of SEQ ID NOs: 272-274, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

267. The epigenetic-modifying DNA-targeting system of any one of claims 248-266, wherein the DNMT3L domain or functional portion thereof is a DNMT3L MTase-like domain or a portion of a DNMT3L MTase-like domain.

268. The epigenetic-modifying DNA-targeting system of any one of claims 248-267, wherein the DNMT3L domain or functional portion thereof is or comprises the sequence set forth in any one of SEQ ID NOs: 272-274 and 549-566, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

269. The epigenetic-modifying DNA-targeting system of any one of claims 248-267, wherein the DNMT3L domain or functional portion thereof is or comprises the sequence set forth in any one of SEQ ID NOs: 272-274 and 549-566.

270. The epigenetic-modifying DNA-targeting system of any one of claims 248-269, wherein the DNMT3L domain or functional portion thereof is or comprises the sequence set forth in SEQ ID NO: 274.

271. The epigenetic-modifying DNA-targeting system of any one of claims 161-270, wherein the at least one transcriptional repressor domain further comprises a DNMT3L ADD domain.

272. The epigenetic-modifying DNA-targeting system of claim 271, wherein the at least one transcriptional repressor domain comprises, from N-terminus to C-terminus:a) the DNMT3L ADD domain and the DNMT3L MTase-like domain; orb) the DNMT3L MTase-like domain and the DNMT3L ADD domain.374MF-366426018Attorney No. 224742004040 273. The epigenetic-modifying DNA-targeting system of claim 271 or claim 272, wherein the DNMT3L ADD domain is or comprises the sequence set forth in any one of SEQ ID NOs: 283, and 285-287.

274. The epigenetic-modifying DNA-targeting system of any one of claims 151-265 and 271- 273, wherein the at least one transcriptional repressor domain is or comprises the sequence set forth in any one of SEQ ID NOs: 284,288-290, and 567, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

275. The epigenetic-modifying DNA-targeting system of any one of claims 151-265 and 271- 274, wherein the at least one transcriptional repressor domain is or comprises:a) the sequences set forth in any one of SEQ ID NOs: 134, 171, and 291 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing; orb) the sequence set forth in any one of SEQ ID NOs: 281, 282, and 292 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

276. The epigenetic-modifying DNA-targeting system of any of claims 151-275, wherein the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 134, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 134.

277. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-276, wherein the at least one transcriptional repressor domain comprises a Krüppel-associated box (KRAB) domain.

278. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-277, wherein the at least one transcriptional repressor domain comprises a histone methyltransferase, or a repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase.

279. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-278, wherein the at least one transcriptional repressor domain comprises a DNA methyltransferase and a375MF-366426018Attorney No. 224742004040 repressor domain capable of recruiting heterochromatin-inducing factors, optionally wherein the heterochromatin-inducing factors include a histone methyltransferase.

280. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-279, wherein the at least one transcriptional repressor domain comprises a DNA methyltransferase and a histone methyltransferase.

281. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-280, wherein the transcriptional repressor domain is selected from the group consisting of a KRAB domain, a DNMT3 A domain, a DNMT3L domain, a DNMT3B domain, a DNMT3 A-DNMT3L fusion protein domain, an ERF repressor domain, an Mxil repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, an EZH2 repressor domain, a SunTag domain, or a variant or portion of any of the foregoing, or a combination of any of the foregoing.

282. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-281, wherein the transcriptional repressor domain is a KRAB domain, a DNMT3A domain, a DNMT3L domain, or a combination of any of the foregoing.

283. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-282, wherein the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 130 or SEQ ID NO: 527, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

284. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-283, wherein the at least one transcriptional repressor domain comprises a DNMT3 A domain or a variant or portion thereof that exhibits transcriptional repressor activity.

285. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-284, wherein the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 132, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32.

286. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-285, wherein the at least one transcriptional repressor domain is a DNMT3 A-DNMT3L fusion protein domain or a variant thereof that exhibits transcriptional repressor activity.376MF-366426018Attorney No. 224742004040287. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-283 and 286, wherein the at least one transcriptional repressor domain comprises the sequence set forth in SEQ ID NO: 136 or SEQ ID NO: 138, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

288. The epigenetic-modifying DNA-targeting system of any of claims 151-248 and 258-281, wherein the at least one transcriptional repressor domain comprises the sequence selected from any one of SEQ ID NOS: 130, 132, 134, 136, 138-146, and 527, or a domain thereof, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

289. The epigenetic-modifying DNA-targeting system of any of claims 151-288, wherein the at least one transcriptional repressor domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain (e.g. Cas protein).

290. The epigenetic-modifying DNA-targeting system of any of claims 151-289, wherein the fusion protein further comprises one or more nuclear localization signals (NLS).

291. The epigenetic-modifying DNA-targeting system of claim 290, wherein the fusion protein further comprises one or more linkers connecting two or more of: the DNA-binding domain, the at least one transcriptional repressor domain, and the one or more nuclear localization signals.

292. The epigenetic-modifying DNA-targeting system of claim 291, wherein the one or more linkers comprise the sequence set forth in SEQ ID NO: 407 or SEQ ID NO: 190, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

293. The epigenetic-modifying DNA-targeting system of claim 291 or claim 292, wherein the one or more linkers comprise the sequence set forth in SEQ ID NO: 190.

294. The epigenetic-modifying DNA-targeting system of any of claims 151-276 and 289-293, wherein the fusion protein is devoid of any domains with DNA methyltransferase activity, domains capable of recruiting heterochromatin inducing factors, and H3K4meO peptides.377MF-366426018Attorney No. 224742004040 295. The epigenetic-modifying DNA-targeting system of any of claims 194-294, wherein the fusion protein consists essentially of the dSpCas protein and the DNMT3L domain or functional portion thereof.

296. The epigenetic-modifying DNA-targeting system of any of claims 248-295, wherein the DNMT3L domain or functional portion thereof is the sole transcriptional repressor domain of the fusion protein.

297. The epigenetic-modifying DNA-targeting system of any of claims 248-276 and 289-296, wherein the fusion protein is devoid of any other transcriptional repressor domains, optionally wherein the fusion protein is devoid of a transcriptional repressor domain selected from a DNMT3 A domain, a KRAB domain, a SID domain, and other repressor motifs.

298. The epigenetic-modifying DNA-targeting system of any of claims 194-297, wherein the fusion protein comprises from N-terminus to C-terminus:c) the dpSpCas protein, the linker, and the DNMT3L domain or functional portion thereof;ord) the DNMT3L domain or functional portions thereof, the linker, and the dSpCas protein.

299. The epigenetic-modifying DNA-targeting system of any of claims 151-298, wherein the fusion protein comprises the sequence set forth in any one of SEQ ID NOS: 260-262, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

300. The epigenetic-modifying DNA-targeting system of any of claims 151-299, wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 262.

301. The epigenetic-modifying DNA-targeting system of any of claims 151-283 and 289-293, wherein the fusion protein comprises the sequence set forth in any one of SEQ ID NOS: 88, 148, 150, 152, and 279 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, optionally wherein the fusion protein is set forth in SEQ ID NO: 150.378MF-366426018Attorney No. 224742004040 302. The plurality of polynucleotides of any one of claims 1-150 or the epigenetic -modifying DNA-targeting system of any of claims 151-301 that is for repressing transcription of Lipoprotein(a) (Lp(a)) gene.

303. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of claim 302, wherein transcription of Lp(a) gene is repressed by a log2 fold-change less than or equal to -1.0.

304. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of claim 302 or claim 303 wherein repressed transcription of the Lp(a) gene in a cell or population of cells leads to a reduction of one or more of: apo(a) mRNA, apo(a) protein, and / or lipoprotein(a) (Lp(a)) particle levels.

305. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of any one of claims 302-304, wherein repressed transcription of the Lp(a) gene in a cell or population of cells leads to a reduction of low-density lipoprotein (LDL).

306. The plurality of polynucleotides or the epigenetic-modifying DNA targeting system of any one of claims 302-305, wherein the repression and / or reduction is relative to a control comprising a cell or population of cells not contacted with the plurality of polynucleotides or the epigenetic-modifying DNA-targeting system or is relative to a control comprising a cell or population of cells contacted with an otherwise identical plurality of polynucleotides or system but in which each guide nucleic acid is nontargeting with respect to the LP(a) gene.

307. The plurality of polynucleotides of any one of claims 3-150 or the epigenetic -modifying DNA-targeting system of any of claims 181-306 that is for repressing transcription of the Lp(a) gene and for repressing transcription of the PCSK9 gene.

308. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of claim 307, wherein transcription of the Lp(a) gene and transcription of the PCSK9 gene are each repressed by a log2 fold-change less than or equal to -1.0.

309. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of claim 307 or claim 308, wherein repressed transcription of the Lp(a) gene and the PCSK9 gene in a cell379MF-366426018Attorney No. 224742004040 or population of cells leads to a reduction of one or more of: apo(a) mRNA, apo(a) protein, lipoprotein(a) particle levels, PCSK9 mRNA, and / or PCSK9 protein.

310. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of any of claims 307-309, wherein repression of the Lp(a) gene and the PCSK9 gene in a cell or population of cells leads to a reduction of low-density lipoprotein (LDL).

311. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of claim 310, wherein the reduction of LDL resulting from repression of both the LP(a) gene and the PCSK9 gene is greater than the reduction of LDL resulting from repression of only the Lp(a) gene or only the PCSK9 gene.

312. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of claim 304, 305, 310 or 311, wherein the reduction of LDL occurs extracellularly.

313. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of any of claims 304, 305 or 310-312, wherein the cell or population of cells is a liver cell or comprises liver cells.

314. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of any of claims 304, 305 or 310-313, wherein the cell or population of cells is in a subject.

315. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of claim 314, wherein the reduction of LDL occurs in the subject or a fluid, tissue, or organ thereof.

316. The plurality of polynucleotides or the epigenetic -modifying DNA-targeting system of any of claims 304, 305 or 310-315, wherein the reduction of LDL occurs in the blood of a subject.

317. The plurality of polynucleotides or the epigenetic-modifying DNA targeting system of any one of claims 307-316, wherein the repression and / or reduction is relative to a control comprising a cell or population of cells not contacted with the plurality of polynucleotides or the epigenetic-modifying DNA-targeting system or is relative to a control comprising a cell or population of cells contacted with an otherwise identical plurality of polynucleotides or system but in which each guide nucleic acid is nontargeting with respect to the LP(a) gene and the PCSK9 gene.380MF-366426018Attorney No. 224742004040 318. A guide RNA (gRNA) that targets a target site of Lp(a), wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 or between the hg38 genomic coordinates chr6: 160,519,674 and 160,520,973.

319. The guide RNA of claim 318, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,800 and chr6: 160,679,650.

320. The guide RNA of claim 318 or claim 319, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,280 and chr6: 160,679,602.

321. The guide RNA of any of claims 318-320, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782.

322. The guide RNA of any of claims 318-321, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,050 and chr6: 160,679,650.

323. The guide RNA of any of claims 318-322, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679,000 and chr6: 160,679,350.

324. The guide RNA of any of claims 318-321, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 350 and chr6: 160,679,700.

325. The guide RNA of any of claims 318-321, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160, 679, 000-160,679,200; chr6: 160, 679, 200-160,679,400; chr6: 160, 679, 400-160,679,600; or chr6: 160, 679, 500-160,679,700.

326. The guide RNA of any of claims 318-321 and 325, wherein the target site for Lp(a) comprises the hg38 genomic coordinates chr6: 160, 679,280-160, 679, 299.

327. The gRNA of any of claims 318-321, wherein the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35,256-258, 418-424, 528-533, 546, and 604 a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

328. The gRNA of any of claims 318-321 and 327, wherein the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 31-35,256-258, 418-424, 528-533, 546, and 604.381MF-366426018Attorney No. 224742004040329. The gRNA of any of claims 318-321 and 327-328, wherein the target site for Lp(a) has the sequence set forth in SEQ ID NO: 421.

330. The guide RNA of claim 318, wherein the target site for Lp(a) is located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973.

331. The guide RNA of claim 318 or claim 330, wherein the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 512-521, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

332. The guide RNA of any of claims 318, 330 and 331, wherein the target site for Lp(a) has the sequence set forth in any one of SEQ ID NOs: 512-521.

333. The gRNA of any of claims 318-332, wherein the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

334. The gRNA of any of claims 318-333, wherein the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

335. The gRNA of any of claims 318-321 and 327-334, wherein the gRNA comprises a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOS: 36-40,253-255, 417, 435-430, 534-539, 547, and 605 or a contiguous portion thereof of at least 14 nt.

336. The gRNA of any of claims 318-321 and 327-335, wherein the gRNA comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 36-40, 253-255, 417425-430, 534-539, 547, and 605.

337. The gRNA of any of claims 318-321 and 327-336, wherein the gRNA comprises the gRNA spacer sequence set forth in SEQ ID NO: 427.

338. The gRNA of any of claims 318 and 329-332, wherein the gRNA comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 577-586.382MF-366426018Attorney No. 224742004040 339. The gRNA of any of claims 318-338, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO:85,259, 450 or 451.

340. The gRNA of any of claims 318-321 and 327-339, wherein the gRNA comprises the sequence set forth in any one of SEQ ID NOS: 41-45,247-252, -439-449, 485-511, 540-545, 547, and 606-607.

341. The gRNA of any of claims 318-321 and 327-340, wherein the gRNA is set forth in any one of SEQ ID NOS: 247-252, -439-449, 485-511, 540-545, 547, and 606-607.

342. The gRNA of any of claims 321-341, wherein the gRNA comprises modified nucleotides for increased stability.

343. A plurality of gRNAs comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a first target site of Lp(a) located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 or between the hg38 genomic coordinates chr6: 160,519,674 and 160,520,973, and the second gRNA targets a target site of PCSK9 located within 500bp of the hg38 genomic coordinate chr1:55,039,548.

344. The plurality of gRNAs of claim 350, wherein the first gRNA is selected from the gRNA of any of claims 321-342.

345. The plurality of gRNAs of claim 343, further comprising a third gRNA that targets a second Lp(a) target site.

346. The plurality of gRNAs of claim 345, wherein the first and second Lp(a) target sites are: a) a first target site located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and a second target site within 500 bp of the hg38 genomic coordinate chr6: 160,664,275;b) a first target site located between the hg38 genomic coordinates chr6: 160, 678,873 and chr6: 160,679,782 and a second target site located between the hg38 genomic coordinates chr6: 160,519,674 and chr6:160,520,973; orc) a first target site located between the hg38 genomic coordinates chr6: 160,519,674 and chr6: 160,520,973 and a second target site within 500 bp of the hg38 genomic coordinate chr6:160,664,275.383MF-366426018Attorney No. 224742004040 347. The plurality of gRNAs of any one of claims 343-346, wherein the target site of PCSK9 is located within 110 bp of the hg38 genomic coordinate chr1:55,039,548.

348. The plurality of gRNAs of any of claims 343-347, wherein the target site for PCSK9 is within the coordinates chr1: 55,039,438-55,039,658.

349. The plurality of gRNAs of any of claims 343-348, wherein the target site of PCSK9 is located within 80 bp of the hg38 genomic coordinate chr1:55, 039, 548.

350. The plurality of gRNAs of any of claims 343-349, wherein the target site for PCSK9 is within the coordinates chr1: 55,039,470-55,039,597.

351. The plurality of gRNAs of any of claims 343-350, wherein the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 46-58, 89-100, 409-412, 568, 569, and 574; optionally wherein the target site for PCSK9 has the sequence set forth in SEQ ID NO: 48.

352. The plurality of gRNAs of any of claims 343-351, wherein the target site for PCSK9 has the sequence set forth in any one of SEQ ID NOs: 48, 49, 52 and 89.

353. The plurality of gRNAs of any of claims 343-352, wherein the second gRNA:a) comprises the gRNA spacer sequence set forth in any one of SEQ ID NOS: 59-71,101-112, 413-416, 570, 571, and 575,; optionally wherein the gRNA spacer sequence is set forth in SEQ ID NO:61 or SEQ ID NO: 62; and / orb) is set forth in any one of SEQ ID NOS:72-84, 113-124, 431-438, 452, 454-484, 572, 573, 576, and 603; optionally wherein the gRNA is set forth in SEQ ID NO: 74,431, 477 or 478.

354. A Cas-guide RNA (gRNA) combination comprising:(a) a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof; and(b) the gRNA of any of claims 321-342 or the plurality of gRNAs of any of claim 343-354.

355. The Cas-gRNA combination of claim 354, wherein the Cas protein or variant thereof is a variant Cas protein that is a deactivated (dCas) protein.384MF-366426018Attorney No. 224742004040 356. The Cas-gRNA combination of claim 355, wherein the dCas protein lacks nuclease activity.

357. The Cas-gRNA combination of claim 355 or claim 356, wherein the dCas protein is a dCas9 protein.

358. The Cas-gRNA combination of claim 355 or claim 356, wherein the dCas protein is a dCas12 protein.

359. The Cas-gRNA combination of claim 357, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

360. The Cas-gRNA combination of claim 359, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 125.

361. The Cas-gRNA combination of claim 359 or claim 360, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 126, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

362. The Cas-gRNA combination of any of claims 359-361, wherein the dSaCas9 is set forth in SEQ ID NO: 126.

363. The Cas-gRNA combination of claim 357, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

364. The Cas-gRNA combination of claim 363, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 127.

365. The Cas-gRNA combination of 363 or claim 364, wherein the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.385MF-366426018Attorney No. 224742004040 366. The Cas-gRNA combination of any of claims 363-365, wherein the dSpCas9 is set forth in SEQ ID NO: 128.

367. A fusion protein comprising:a) a deactivated Clustered Regularly Interspaced Short Palindromic Repeats associated (dCas) protein; andb) an effector domain comprising a DNMT3L domain or functional portion thereof selected from one of the following species: Equus caballus, Arvicanthis niloticus, Chinchilla lanigera, Desmodus rotundus, Echinops telfairi, Eptesicus fuscus, Fukomys damarensis, Globicephala melas, Jaculus jaculus, Mastomys coucha, Neofelis nebulosa, Neotoma lepida Ochotona princeps, Onychomys torridus, Perognathus longimembris pacificus, Phoca vitulina, Pteronotus mesoamericanus, and Saccopteryx leptura.

368. The fusion protein of claim 367, wherein the fusion protein is devoid of any domains with DNA methyltransferase activity, domains capable of recruiting heterochromatin inducing factors, and H3K4meO peptides.

369. The fusion protein of claim 367 or claim 368, wherein the effector domain comprises a catalytically inactive DNA methyltransferase domain or portion thereof.

370. The fusion protein of any of claims 367-369, wherein the DNMT3L domain or functional portion thereof is a DNMT3L MTase-like domain or a portion of a DNMT3L MTase-like domain.

371. The fusion protein of claim 370, wherein the DNMT3L domain or functional portion thereof further comprises a DNMT3L ADD domain.

372. The fusion protein of any of claims 367-371, wherein the DNMT3L domain or functional portion thereof comprises any one of the sequences set forth in SEQ ID NOs: 549-567 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the foregoing.

373. The fusion protein of any of claims 367-372, wherein the DNMT3L domain or functional portion thereof is selected from one of the following species: Equus caballus, Neotoma lepida, Ochotona princeps, and Onychomys torridus.386MF-366426018Attorney No. 224742004040374. The fusion protein of any of claims 367-373, wherein the DNMT3L domain or functional portion thereof comprises any one of the sequences set forth in SEQ ID NOs: 549, 551, 550, and 552 or an amino acid sequence that has at least 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of any of the foregoing.

375. The fusion protein of any of claim 367-374, wherein the dCas protein is a dCas9 protein.

376. The fusion protein of claim 375, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

377. The fusion protein of claim 376, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 217.

378. The fusion protein of claim 376 or claim 377, wherein the dSpCas9 comprises the sequence set forth in SEQ ID NO: 128, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

379. The fusion protein of any one of claims 376-claim 378, wherein the dSpCas9 is set forth in SEQ ID NO: 128.

380. A polynucleotide encoding the epigenetic-modifying DNA-targeting system of any of claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the fusion protein of any of claims 367-379, or a portion or a component of any of the foregoing.

381. A polynucleotide encoding the epigenetic-modifying DNA-targeting system of any of claims 151-317.

382. A polynucleotide encoding the gRNA of any of claims 318-342.

383. A polynucleotide encoding the plurality of gRNAs of any of claims343-353.

384. A polynucleotide encoding the Cas-gRNA combination of any of claims 354-366.387MF-366426018Attorney No. 224742004040385. A polynucleotide encoding the fusion protein of any of claims 367-379.

386. A plurality of polynucleotides encoding the epigenetic-modifying DNA-targeting system of any of claims 151-317, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims354-366, or a portion or a component of any of the foregoing.

387. A plurality of polynucleotides encoding the epigenetic-modifying DNA-targeting system of any of claims 151-317.

388. A plurality of polynucleotides encoding the plurality of gRNAs of any of claims 343-353.

389. A plurality of polynucleotides encoding the Cas-gRNA combination of any one of claims 354-366.

390. A plurality of polynucleotides comprising:a) a polynucleotide encoding the fusion protein of the epigenetic -modifying DNA-targeting system of any of claims 155-317; andb) the gRNA of the epigenetic -modifying DNA-targeting system of any of claims 155-317.

391. A plurality of polynucleotides comprising:a) a polynucleotide encoding the fusion protein of the epigenetic -modifying DNA-targeting system of any of claims 181-371; andb) the at least first gRNA and the at least second gRNA of the epigenetic -modifying DNA-targeting system of any of claims 181-371.

392. The plurality of polynucleotides of claim 390 or claim 391, wherein the polynucleotide encoding the fusion protein is mRNA.

393. The plurality of polynucleotides of claim 392, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 156, or a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.388MF-366426018Attorney No. 224742004040 394. The plurality of polynucleotides of claim 392 or claim 393, wherein the mRNA has the sequence is set forth in SEQ ID NO: 153.

395. A vector comprising the polynucleotide of any of claims380-385.

396. A vector comprising the plurality of polynucleotides of any of claims 1-150 and 386-394.

397. The vector of claim 395 or claim 396, wherein the vector is a viral vector.

398. The vector of claim 397 wherein the vector is an adeno-associated virus (AAV) vector.

399. The vector of claim 398, wherein the vector is selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

400. The vector of claim 397, wherein the vector is a lentiviral vector.

401. The vector of claim 395 or claim 396, wherein the vector is a non-viral vector.

402. The vector of any of claims 395-404, wherein the vector exhibits hepatocyte tropism.

403. A method of decreasing transcription of Lp(a) in a cell or population of cells, the method comprising administering to a cell or population of cells the epigenetic-modifying DNA-targeting system of any of claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the polynucleotide of any of claims 380-385, the plurality of polynucleotides of any of claims 1-150, 302-317 and 386-394, the vector of any of claims 395-402, or a portion or a component of any of the foregoing.

404. The method of claim 403, wherein Lp(a) is epigenetically modified.

405. The method of claim 403 or claim 404, wherein the transcription of Lp(a) is decreased in comparison to a comparable cell or population of cells not subjected to the method.389MF-366426018Attorney No. 224742004040 406. The method of any of claims 403-405, wherein the transcription of Lp(a) is reduced by at least about 1.2-fold, 1.25-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.75-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold.

407. The method of any of claims 403-406, wherein the reduced transcription of Lp(a) leads to a reduction of Lp(a) and / or low-density lipoprotein (LDL).

408. A method of reducing Lp(a), the method comprising introducing into a cell or population of cells the epigenetic -modifying DNA-targeting system of any claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the polynucleotide of any of claims 380-385, the plurality of polynucleotides of any of claims 1-150, 302-317 and 386-394, the vector of any of claims 395-402, or a portion or a component of any of the foregoing.

409. A method of reducing LDL, the method comprising introducing into a cell or population of cells the epigenetic -modifying DNA-targeting system of any of claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the polynucleotide of any of claims 380-385, the plurality of polynucleotides of any of claims 1-150, 302-317 and 386-394, the vector of any of claims 395-402, or a portion or a component of any of the foregoing.

410. The method of any of claims 403-409, wherein the cell or population of cells is a liver cell or comprises liver cells.

411. The method of any of claims 403-409, wherein the cell or population of cells is in a subject and the method is carried out in vivo.

412. The method of claim 411, wherein Lp(a) and / or LDL is reduced in the subject or a fluid, tissue, or organ thereof.

413. The method of any of claims 408-412, wherein Lp(a) and / or LDL is reduced in the blood of a subject.

414. The method of any of claims 411-413, wherein the subject is a human.390MF-366426018Attorney No. 224742004040 415. The method of any of claims 411-413, wherein the subject has or is suspected of having a disease, condition, or disorder, optionally wherein the disease, condition or disorder is a cardiovascular disease.

416. The method of any of claims 411-415, wherein the subject has or is suspected of having one or more of: elevated levels of low-density lipoprotein in the blood, elevated levels of Lp(a) in the blood, increased risk of cardiovascular disease, increased risk of early -onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in a low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia.

417. The method of any of claims 411-416, wherein the subject has is or is suspected of having elevated levels of Lp(a) in the blood.

418. The method of any of claims 411-417, wherein the subject has is or is suspected of having familial hypercholesterolemia.

419. A pharmaceutical composition comprising the epigenetic-modifying DNA-targeting system of any of claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the polynucleotide of any of claims 380-385, the plurality of polynucleotides of any of claims 1-150, 302-317, and 386-394, the vector of any of claims 395-402, or a portion or a component of any of the foregoing.

420. The pharmaceutical composition of claim 419, for use in reducing Lp(a) and / or low-density lipoprotein (LDL) in a subject.

421. The pharmaceutical composition of claim 419, for use in treating a disease, condition, or disorder in a subject, optionally wherein the disease, condition or disorder is a cardiovascular disease.

422. Use of the pharmaceutical composition of claim 419 in the manufacture of a medicament for reducing Lp(a) and / or low-density lipoprotein (LDL) in a subject.

423. Use of the pharmaceutical composition of claim 419 in the manufacture of a medicament for treating a disease, condition, or disorder in a subject, optionally wherein the disease, condition or disorder is a cardiovascular disease.391MF-366426018Attorney No. 224742004040424. The pharmaceutical composition of claim 420 or claim 421 or the use of claim 422 or claim 423, wherein the subject has or is suspected of having a disease, condition, or disorder, optionally wherein the disease, condition or disorder is a cardiovascular disease.

425. The pharmaceutical composition or use of any of claims 419-424, wherein the subject has or is suspected of having one or more of: elevated levels of low -density lipoprotein in the blood, elevated levels of Lp(a) in the blood, increased risk of cardiovascular disease, increased risk of early -onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in a low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia.

426. The pharmaceutical composition or use of any of claims 419-425, wherein the subject has is or is suspected of having elevated levels of Lp(a) in the blood.

427. The pharmaceutical composition or use of any of claims 419-426, wherein the subject has is or is suspected of having familial hypercholesterolemia.

428. The pharmaceutical composition or use of any of claims 419-427, wherein the pharmaceutical composition is to be administered to the subject in vivo.

429. The pharmaceutical composition or use of claim 428, wherein the pharmaceutical composition is targeted to, or is to be administered to the liver of the subject.

430. The pharmaceutical composition or use of claim 428 or 429, wherein following administration of the pharmaceutical composition, the expression of Lp(a) is reduced in cells of the subject.

431. The pharmaceutical composition or use of any of claims 428-430, wherein following administration of the pharmaceutical composition, the expression of Lp(a) is reduced in liver cells of the subject.

432. The pharmaceutical composition or use of any of claims 419-431, wherein the pharmaceutical composition is for single dose infusion to the subject.392MF-366426018Attorney No. 224742004040 433. The pharmaceutical composition or use of any of claims 419-431, wherein the pharmaceutical composition is for repeated dose administration, optionally a plurality of times at regular intervals.

434. The pharmaceutical composition or use of any of claims 419-431 and 433, wherein the administration is a multiple dose administration comprising at least a first dose and a second dose.

435. The pharmaceutical composition or use of claim 434, wherein the first dose and the second dose are the same.

436. The pharmaceutical composition or use of claim 435, wherein the second dose is lower than the first dose, optionally wherein the second dose is 25% to 75% of the first dose (e.g., about 30%, about 40%, about 50%, about 60% or about 70%, or a percentage between any of the foregoing).

437. The pharmaceutical composition or use of claim 435, wherein the second dose is higher than the first dose, optionally wherein the second dose is 150% to 500% of the first dose (e.g., about 200%, about 300%, about 400% or about 500%, or a percentage between any of the foregoing).

438. A method for treating a disease, condition, or disorder associated with elevated Lp(a) in a subject in need thereof, comprising administering to the subject the epigenetic-modifying DNA-targeting system of any of claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the polynucleotide of any of claims 380-385, the plurality of polynucleotides of any of claims 1-150, 302-317, and 386-394, the vector of any of claims 395-402, or a portion or a component of any of the foregoing.

439. A method for treating a disease, condition, or disorder associated with elevated low-density lipoprotein (LDL) in a subject in need thereof, comprising administering to the subject the epigenetic -modifying DNA-targeting system of any of claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the polynucleotide of any of claims 380-385, the plurality of polynucleotides of any of claims 1-150, 302-317, and 386-394, the vector of any of claims 395-402, or a portion or a component of any of the foregoing.

440. The method of claim 439, wherein the disease, condition or disorder associated with elevated LDL is a cardiovascular disease.393MF-366426018Attorney No. 224742004040441. The method of claim 439 or claim 440, wherein the subject has or is suspected of having one or more of: elevated levels of low-density lipoprotein in the blood, elevated levels of Lp(a) in the blood, increased risk of cardiovascular disease, increased risk of early -onset cardiovascular disease, a mutation affecting cholesterol biosynthesis, a loss-of-function mutation in a low-density lipoprotein receptor (LDLR) gene, a loss-of-function mutation in APOB, a gain-of-function mutation in PCSK9, and familial hypercholesterolemia.

442. A method for treating a familial hypercholesterolemia in a subject, comprising administering to the subject the epigenetic -modifying DNA-targeting system of any of claims 151-317, the gRNA of any of claims 318-342, the plurality of gRNAs of any of claims 343-353, the Cas-gRNA combination of any of claims 354-366, the polynucleotide of any of claims 380-385, the plurality of polynucleotides of any of claims 1-150, 302-317, and 386-394, the vector of any of claims 395-402, or a portion or a component of any of the foregoing.

443. The method of any of claims 438-442, wherein the administration is a single dose infusion to the subject.

444. The method of any of claims 438-443, wherein the administration is repeated at least once, optionally a plurality of times at regular intervals.

445. The method of any of claims 438-442 and 444, wherein the administration is a multiple dose administration comprising at least a first dose and a second dose.

446. The method of claim 445, wherein the first dose and the second dose are the same.

447. The method of claim 445, wherein the second dose is lower than the first dose, optionally wherein the second dose is 25% to 75% of the first dose (e.g., about 30%, about 40%, about 50%, about 60% or about 70%, or a percentage between any of the foregoing).

448. The method of claim 445, wherein the second dose is higher than the first dose, optionally wherein the second dose is 150% to 500% of the first dose (e.g., about 200%, about 300%, about 400% or about 500%, or a percentage between any of the foregoing).394MF-366426018

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