Crispr-related methods and compositions targeting low-density lipoprotein receptor (LDLR)

Abstract

The present disclosure relates to genome editing systems and components for targeting, editing, and / or modulating the expression of a target nucleic acid sequence of interest, e.g., an LDLR target nucleic acid sequence in the 3' untranslated region (UTR) of the gene encoding the LDLR protein. The present disclosure is also directed to methods and applications thereof in connection with the treatment and / or management of hypercholesterolemia.

Description

Attorney Docket No. 126454-03020CRISPR-RELATED METHODS AND COMPOSITIONS TARGETING LOW-DENSITY LIPOPROTEIN RECEPTOR (LDLR)RELATED APPLICATIONS

[0001] The instant application claims priority to U. S. Provisional Application No.63 / 745,724, filed January 15, 2025; U. S. Provisional Application No. 63 / 805,160, filed May 13, 2025; U. S. Provisional Application No. 63 / 810,482, filed May 22, 2025; U. S. Provisional Application No. 63 / 872,782, filed August 29, 2025; U. S. Provisional Application No.63 / 872,843, filed August 29, 2025; U. S. Provisional Application No. 63 / 894,034, filed October 6, 2025; U. S. Provisional Application No. 63 / 894,039, filed October 6, 2025; U. S. Provisional Application No. 63 / 911,882, filed November 5, 2025; U. S. Provisional Application No.63 / 911,907, filed November 5, 2025; U. S. Provisional Application No. 63 / 920,708 filed November 19, 2025; U. S. Provisional Application No. 63 / 928,124 filed December 1, 2025; and U. S. Provisional Application No. 63 / 928,177, filed December 1, 2025.SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on January 14, 2026, is named “126454-03020. xml” and is 2,465,149 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD

[0003] The present disclosure is directed to CRISPR-related genome editing systems and components for targeting, editing and / or modulating the expression of a low-density lipoprotein receptor (LDLR) target nucleic acid sequence of interest, e.g., an LDLR target nucleic acid sequence in the 3’ untranslated region (UTR) of the gene encoding the LDLR protein. The present disclosure is also directed to methods and applications thereof in connection with the treatment and / or management of hypercholesterolemia.BACKGROUND

[0004] Familial hypercholesterolemia (FH), which can be heterozygous (HeFH) or homozygous (HoFH), is characterized by high levels of low-density lipoprotein cholesterol (LDL-C) in the blood (>160 mg / dL in people <20 years old, or >190 in people >20 years old)1ME1\59622102.v1Attorney Docket No. 126454-03020and an increased risk of atherosclerotic cardiovascular disease (ASCVD). FH has been associated with the formation of atherosclerotic plaques in the blood vessels that lead to coronary artery disease (CAD) and / or major adverse cardiovascular events (MACE) including, heart attack, stroke, aortic stenosis, peripheral vascular disease, and renal dysfunction. The current standard of care (SOC) for FH is lipoprotein apheresis, mitigation of other MACE risk factors, and drugs (e.g., statins, PCSK9 inhibitors), and fewer than 25% of patients achieve a target LDL of < 70 mg / dL. Therapies employing antisense oligonucleotides (ASO) and small interfering RNA (siRNA) have met with many challenges, including tolerability, dosing inconsistencies, area under-the-curve (AUC) reductions and poor medication adherence by the patient. There is therefore an unmet need in the art for the treatment and / or management of diseases, e.g., alternate approaches that address hypercholesterolemia to reduce the risk of MACE in an individual or treat an individual who has suffered from a MACE.

[0005] In addition to elevated LDL-C, up to 50% of patients with HeFH also present with elevated lipoprotein(a) (Lp(a)), an independent ASCVD risk factor. High levels of Lp(a) (>150 mg / dL) have been associated with the formation of atherosclerotic plaques in the blood vessels that lead to MACE. The presence of elevated LDL-C and Lp(a) exert a compounding effect on ASCVD risk, highlighting the need for aggressive LDL-lowering therapeutic strategies aimed at lowering both LDL-C and Lp(a) for treating familial hypercholesterolemia to reduce the risk of MACE. See, for example, WO2025 / 166323, the entire contents of which are expressly incorporated by reference herein in their entirety.

[0006] The human LDLR gene is located on Chromosome 19. The LDLR gene (ENSG00000130164) comprises 18 exons that encode the LDLR protein. Expression of LDLR, which primarily occurs on liver cells (hepatocytes), facilitates absorption of LDL into cells, thereby decreasing blood levels of LDL to maintain normal levels of 75-150 mg / dL. Individuals with a naturally-occurring 2.5 kb deletion in the 3’ UTR of LDLR express high levels of LDLR and exhibit low serum LDL levels (22-44 mg / dL). In contrast, LDLR deficiencies are associated with increased serum LDL. Individuals with Familial Hypercholesterolemia (FH) carry at least one loss-of-function variant in at least one of several genes, including, e.g., LDLR, APOB, LDLRAP1, and PCSK9. These individuals have increased plasma levels of total cholesterol (250-450 mg / dL) and are at higher risk of developing atherosclerotic cardiovascular disease, as compared to individuals with wild-type LDLR, APOB, LDLRAP1, and PCSK9. Currently there are no treatments available that genetically correct the decreased function of LDLR in these patients.2ME1\59622102.v1Attorney Docket No. 126454-03020SUMMARY

[0007] In one aspect, the present disclosure provides a composition comprising dual guide RNA (gRNA) molecules comprising targeting domains that flank the (AU-rich element 1) ARE1 element of a low-density lipoprotein receptor (LDLR) gene. In one embodiment, a composition comprises a first gRNA molecule comprising a first targeting domain that binds to a first target sequence of an LDLR gene, and a second gRNA molecule comprises a second targeting domain that binds to a second target sequence of an LDLR gene, wherein the first and second target sequences of the LDLR gene flank an ARE1 element of the LDLR gene. In one embodiment, the first gRNA molecule target sequence is located 5’ of the first AU-rich element (ARE1) of the Low Density Lipoprotein Receptor (LDLR) 3’ untranslated region (UTR); and the second gRNA molecule target sequence is located 3’ of the first AU-rich element (ARE1) of the LDLR 3’ UTR. In some embodiments, the first gRNA molecule target sequence is located within SEQ ID NO: 1482. In some embodiments, the first gRNA molecule target sequence comprises the ARE1 sequence (ATTTA). In some embodiments, the cleavage of the LDLR untranslated region (UTR) by a Cas protein complexed with such guide RNA molecules comprising targeting domains that bind to target sequences that flank the ARE1 element results in removal of the ARE1 element. Removal of the ARE1 element results in an increase in LDLR protein in the liver cell and / or liver, and a corresponding change of the levels of associated biomarkers, such as LDL-C, ApoB, and / or Lp(a). In other aspects, included herein are genome editing systems comprising the first and second gRNA molecules, RNP complexes comprising the first and second gRNA molecules, and delivery systems comprising the first and second gRNA molecules, each of which are described in more detail herein.

[0008] In some embodiments, administering the genome editing system, the RNP complex, or the delivery system disclosed herein induces an increase in an LDLR level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21 -fold, at least 22-fold, at least 23 -fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, or at least 30-fold. In some embodiments, administering the genome editing system, the RNP complex, or the delivery system disclosed herein induces an increase in an LDLR level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by at least 1.5-fold to at least 31-fold. In some embodiments, administering the genome editing system,3ME1\59622102.v1Attorney Docket No. 126454-03020the RNP complex, or the delivery system disclosed herein induces an increase in an LDLR level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by about 1.5-fold to about 30-fold, about 5-fold to about 30-fold, about 10-fold to about 30-fold, about 20-fold to about 30-fold, about 1.5-fold to about 25-fold, about 1.5-fold to about 20-fold, about 1.5-fold to about 15-fold, about 1.5-fold to about 10-fold, about 2-fold to about 30-fold, about 5-fold to about 30-fold, about 10-fold to about 20-fold, about 10-fold to about 30-fold. In one embodiment, the increase in the LDLR level occurs after editing of the cell, as compared to a cell that has not been edited, or as compared to the liver cell or liver prior to editing in the cell or the subject.

[0009] In some embodiments, the compositions disclosed herein provide increased uptake of LDL-C by a liver cell or the liver by about 1.5-fold to about 5-fold, e.g., after editing. In some embodiments, the compositions disclosed herein provide increased uptake of LDL-C by a liver cell or the liver by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, e.g., after editing. In some embodiments, the compositions disclosed herein provide increased uptake of LDL-C by a liver cell or the liver by about 1.5-fold to about 5-fold, about 1.5-fold to about 3.5-fold, or about 3.2-fold, e.g., after editing.

[0010] In some embodiments, administering the genome editing system, the RNP complex, or the delivery system disclosed herein decreases an LDL-cholesterol (LDL-C) level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by at least about 50% to at least about 95%, e.g., relative to the LDL-C level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject. In some embodiments, the LDL-C level is decreased by about 50%, by about 60%, by about 65%, by about 70%, by about 75%, by about 80%, by about 85%, or by about 90%. In some embodiments, the LDL-C level is decreased by about 50% to about 85%, by about 50% to about 75%, by about 50% to about 70%, by about 55% to about 70%, or by about 55% to about 65%. In some embodiments, the LDL-C level is decreased by at least 50%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, or by at least 90%.

[0011] In another embodiment, contacting the target cell with the genome editing system, the RNP complex, or the delivery system decreases an Apolipoprotein B (ApoB) level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by about at least about 30% to at least about 98%, e.g., relative to the ApoB level in the target cell prior to contacting the target cell with the genome editing system, the RNP complex, or the delivery system, or relative to a control cell. In some embodiments, a level of ApoB is 4ME1\59622102.v1Attorney Docket No. 126454-03020decreased by about 50% to about 150%. In some embodiments, a level of ApoB is decreased by about 60% to about 140%, by about 70% to about 130%, by about 80% to about 120%, by about 90% to about 110%, or about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, or about 120%.

[0012] In another embodiment, administering the genome editing system, the RNP complex, or the delivery system to a subject decreases a Lipoprotein(a) (“Lp(a)”) level in the subject. In one embodiment, administering the genome editing system, the RNP complex, or the delivery system decreases a Lipoprotein(a) level in a cell, tissue, or fluid of the subject. In one embodiment, the Lp(a) level is decreased in the serum or plasma of the subject. In one embodiment, the cell is a liver cell. In one embodiment, the tissue is a liver tissue. In one embodiment, the Lp(a) level is decreased by about at least about 5% to at least about 98%. In one embodiment, the Lp(a) level is decreased by about 25% to about 98%. In one embodiment, the Lp(a) level is decreased by about 50% to about 98%. In one embodiment, the Lp(a) level is decreased by about 75% to about 98%. In one embodiment, the Lp(a) level is decreased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 95%, about 97%, or about 98%. In one embodiment, the Lp(a) level is decreased by at least 5%, at least 10%, 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%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or at least 98%. In any of the previous embodiments, the Lp(a) level is decreased relative to a Lp(a) level in the target cell, tissue, or fluid (e.g., serum or plasma of a subject) prior to administering the genome editing system, the RNP complex, or the delivery system to the subject. In any of the previous embodiments, the Lp(a) level is decreased relative to an Lp(a) control level, or relative to an Lp(a) level in a control cell. In some embodiments, a level of Lp(a) is decreased by about 50% to about 98%. In some embodiments, a level of Lp(a) is decreased by about 50% to about 95%. In some embodiments, a level of Lp(a) is decreased by about 50% to about 90%. In some embodiments, a level of Lp(a) is decreased by about 60% to about 98%, by about 70% to about 98%, by about 80% to about 98%, by about 90% to about 98%.

[0013] In certain embodiments, administering the genome editing system, the RNP complex, or the delivery system reduces a serum or plasma Lp(a) level in the subject to 100 mg / dL or lower.5ME1\59622102.v1Attorney Docket No. 126454-03020

[0014] In another aspect, the present disclosure provides a guide RNA (gRNA) molecule comprising a targeting domain that targets a target sequence of a low -density lipoprotein receptor (LDLR) gene, wherein the target sequence comprises any target sequence nucleotide sequence disclosed herein. In one aspect, the present disclosure provides a guide RNA (gRNA) molecule comprising a targeting domain that targets a target sequence of a low-density lipoprotein receptor (LDLR) gene, wherein the targeting domain comprises any targeting domain nucleotide sequence disclosed herein. In one embodiment, the targeting domain comprises a nucleotide sequence that is not SEQ ID NO: 323 or SEQ ID NO: 543. In another aspect, the present disclosure provides a guide RNA (gRNA) molecule comprising any gRNA sequence disclosed herein.

[0015] In another aspect, the present disclosure provides a guide RNA (gRNA) molecule comprising a targeting domain that targets a target sequence of a low-density lipoprotein receptor (LDLR) gene, wherein the target sequence comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 61-304, 549-606, 665-750, 837-865, 1317-1323, 1365-1392, and 1461. In one aspect, the present disclosure provides a guide RNA (gRNA) molecule comprising a targeting domain that targets a target sequence of a low-density lipoprotein receptor (LDLR) gene, wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 305-548, 607-664, 751-836, 866-894, 1324-1330, 1393-1420, 1451-1456, 1462, 1464, 1466, 1468, 1470, 1474, and 1475. In one embodiment, the targeting domain comprises a nucleotide sequence that is not SEQ ID NO: 323 or SEQ ID NO: 543.

[0016] In one embodiment, the target sequence comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 61-74, 119, 123, 188, 275-302, 553, 589, 1317, 1318, and 1461; the targeting domain comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 305-318, 363, 367, 432, 519-542, 544-546, 611, 647, 1324, 1325, 1451-1454, 1456, 1462, 1464, 1466, 1470, 1474, and 1475; and / or the gRNA molecule comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 900-913, 958, 962, 1027, 1114-1138, 1139-1141, and 1148, 1184, 1331, 1332, 1449, 1450, 1457, 1458, 1460, 1463, 1465, 1467, 1471-1473, and 1476-1479.

[0017] In one aspect, disclosed herein is a gRNA molecule comprising a targeting domain that targets a target sequence of a low-density lipoprotein receptor (LDLR) gene, wherein the target sequence comprises a nucleotide sequence located within SEQ ID NO: 1482. In one embodiment, the target sequence comprises ATTTA.6ME1\59622102.v1Attorney Docket No. 126454-03020

[0018] In one embodiment, the gRNA molecule comprises one or more modifications. In one embodiment, the gRNA molecule comprises one or more modifications selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-methyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LNA), and any combination thereof. In one embodiment, the gRNA molecule comprises one or more modifications selected from the group consisting of a 2’ O-methyl modification, a phosphorothioate linkage, and any combination thereof.

[0019] In one aspect, the present disclosure provides a composition comprising: a first gRNA molecule comprising a first targeting domain that binds to a first target sequence of an LDLR gene, and a second gRNA molecule comprising a second targeting domain that binds to a second target sequence of an LDLR gene, wherein the first and second target sequences of the LDLR gene flank the first AU-rich element (ARE1 element) of the 3’ untranslated region (UTR) of the LDLR gene. In one embodiment, the first targeting domain comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 305-318, 1451, and 1454, and wherein the second targeting domain comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 519-542, 544-546, 1456, 1470, 1474, and 1475. In one embodiment, the first gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 900-913 and 1476-1479 and wherein the second gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1114- 1137, 1139-1141, 1449, 1450, 1460, and 1471.

[0020] In one embodiment, the composition increases LDLR expression in a target cell by at least about 2-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by at least about 3 -fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by at least about 4-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by at least about 5-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by at least about 6-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by at least about 7-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition.7ME1\59622102.v1Attorney Docket No. 126454-03020In one embodiment, the composition increases LDLR expression in a target cell by at least about 8-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by at least about 9-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by at least about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by about 6-fold to about 11 -fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by about 7-fold to about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by about 8-fold to about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by about 9-fold to about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition. In one embodiment, the composition increases LDLR expression in a target cell by about 9-fold to about 11 -fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the composition.

[0021] In one aspect, the present disclosure provides a genome editing system comprising at least one gRNA molecule as disclosed herein, or a nucleic acid encoding the gRNA molecule, and an RNA-guided nuclease, or a nucleic acid encoding the RNA-guided nuclease. In one embodiment, the genome editing system comprises a first gRNA molecule as herein disclosed, a second gRNA molecule as herein disclosed; and a RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, wherein the first gRNA molecule and the second gRNA molecule do not have the same nucleotide sequence.

[0022] In one aspect, the present disclosure provides a genome editing system comprising: a composition as herein disclosed, or a nucleic acid molecule encoding the first gRNA molecule and the second gRNA molecule of the composition as herein disclosed; and an RNA-guided nuclease, or a nucleic acid encoding the RNA-guided nuclease. In one embodiment, the nucleic acid encoding the RNA-guided nuclease is an RNA. In one embodiment, the RNA-guided nuclease is selected from the group consisting of Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Cas12h, Cas12i, CasX, CasY, and CasΦ In one embodiment, the RNA-guided nuclease is a 8ME1\59622102.v1Attorney Docket No. 126454-03020Cas9 nuclease. In one embodiment, the Cas9 nuclease is a Streptococcus pyogenes Cas9 (SpCas9) nuclease. In one embodiment, the RNA-guided nuclease is a Casl2a nuclease. In one embodiment, the Casl2a nuclease is a modified Casl2a nuclease. In one embodiment, the modified Casl2a nuclease is a modified Acidaminococcus sp. Casl2a (AsCasl2a) nuclease.

[0023] In one embodiment, the RNA-guided nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1338-1347. In one embodiment, the nucleic acid encoding the RNA-guided nuclease comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1350-1359 and 1480.

[0024] In one embodiment, the genome editing system comprises a first gRNA molecule as disclosed herein, a second gRNA molecule as disclosed herein, and a RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, wherein the first gRNA molecule and the second gRNA molecule do not have the same nucleotide sequence.

[0025] In one aspect, the present disclosure provides a genome editing system comprising a first gRNA molecule comprising a targeting domain selected from the group consisting of SEQ ID NOs: 305-318 and 1451-1454, a second gRNA molecule comprising a targeting domain selected form the group consisting of SEQ ID NOs: 519-542, 544-546, 1456, 1470, 1474, and 1475, and an RNA encoding an RNA-guided nuclease. In one embodiment, the first gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 900-913 and 1476-1479 and wherein the second gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1114-1137, 1139-1141, 1449, 1450, 1460, and 1471.

[0026] In one embodiment, the first gRNA molecule targeting domain and the second gRNA molecule targeting domain comprise, respectively, SEQ ID NO: 1452 and SEQ ID NO: 1475; SEQ ID NO: 305 and SEQ ID NO: 363, SEQ ID NO: 305 and SEQ ID NO: 367, SEQ ID NO: 305 and SEQ ID NO: 432, SEQ ID NO: 305 and SEQ ID NO: 519, SEQ ID NO: 305 and SEQ ID NO: 521, SEQ ID NO: 305 and SEQ ID NO: 524, SEQ ID NO: 305 and SEQ ID NO: 526, SEQ ID NO: 305 and SEQ ID NO: 527, SEQ ID NO: 305 and SEQ ID NO: 528, SEQ ID NO: 305 and SEQ ID NO: 529, SEQ ID NO: 305 and SEQ ID NO: 530, SEQ ID NO: 305 and SEQ ID NO: 531, SEQ ID NO: 305 and SEQ ID NO: 532, SEQ ID NO: 305 and SEQ ID NO: 535, SEQ ID NO: 305 and SEQ ID NO: 536, SEQ ID NO: 305 and SEQ ID NO: 537, SEQ ID NO: 305 and SEQ ID NO: 540, SEQ ID NO: 305 and SEQ ID NO: 543, SEQ ID NO: 305 and SEQ ID NO: 544, SEQ ID NO: 305 and SEQ ID NO: 545, SEQ ID NO: 306 and SEQ ID NO: 363, SEQ ID NO: 306 and SEQ ID NO: 367, SEQ ID NO: 306 and SEQ ID NO: 432, SEQ ID NO: 306 and SEQ ID NO: 519, SEQ ID NO: 306 and SEQ ID NO: 521, SEQ ID NO:9ME1\59622102.v1Attorney Docket No. 126454-03020306 and SEQ ID NO: 524, SEQ ID NO: 306 and SEQ ID NO: 526, SEQ ID NO: 306 and SEQ ID NO: 527, SEQ ID NO: 306 and SEQ ID NO: 528, SEQ ID NO: 306 and SEQ ID NO: 529, SEQ ID NO: 306 and SEQ ID NO: 530, SEQ ID NO: 306 and SEQ ID NO: 531, SEQ ID NO: 306 and SEQ ID NO: 532, SEQ ID NO: 306 and SEQ ID NO: 535, SEQ ID NO: 306 and SEQ ID NO: 536, SEQ ID NO: 306 and SEQ ID NO: 537, SEQ ID NO: 306 and SEQ ID NO: 540, SEQ ID NO: 306 and SEQ ID NO: 543, SEQ ID NO: 306 and SEQ ID NO: 544, SEQ ID NO: 306 and SEQ ID NO: 545, SEQ ID NO: 307 and SEQ ID NO: 363, SEQ ID NO: 307 and SEQ ID NO: 367, SEQ ID NO: 307 and SEQ ID NO: 432, SEQ ID NO: 307 and SEQ ID NO: 519, SEQ ID NO: 307 and SEQ ID NO: 521, SEQ ID NO: 307 and SEQ ID NO: 524, SEQ ID NO: 307 and SEQ ID NO: 526, SEQ ID NO: 307 and SEQ ID NO: 527, SEQ ID NO: 307 and SEQ ID NO: 528, SEQ ID NO: 307 and SEQ ID NO: 529, SEQ ID NO: 307 and SEQ ID NO: 530, SEQ ID NO: 307 and SEQ ID NO: 531, SEQ ID NO: 307 and SEQ ID NO: 532, SEQ ID NO: 307 and SEQ ID NO: 535, SEQ ID NO: 307 and SEQ ID NO: 536, SEQ ID NO: 307 and SEQ ID NO: 537, SEQ ID NO: 307 and SEQ ID NO: 540, SEQ ID NO: 307 and SEQ ID NO: 543, SEQ ID NO: 307 and SEQ ID NO: 544, SEQ ID NO: 307 and SEQ ID NO: 545, SEQ ID NO: 308 and SEQ ID NO: 363, SEQ ID NO: 308 and SEQ ID NO: 367, SEQ ID NO: 308 and SEQ ID NO: 432, SEQ ID NO: 308 and SEQ ID NO: 519, SEQ ID NO: 308 and SEQ ID NO: 521, SEQ ID NO: 308 and SEQ ID NO: 524, SEQ ID NO: 308 and SEQ ID NO: 526, SEQ ID NO: 308 and SEQ ID NO: 527, SEQ ID NO: 308 and SEQ ID NO: 528, SEQ ID NO: 308 and SEQ ID NO: 529, SEQ ID NO: 308 and SEQ ID NO: 530, SEQ ID NO: 308 and SEQ ID NO: 531, SEQ ID NO: 308 and SEQ ID NO: 532, SEQ ID NO: 308 and SEQ ID NO: 535, SEQ ID NO: 308 and SEQ ID NO: 536, SEQ ID NO: 308 and SEQ ID NO: 537, SEQ ID NO: 308 and SEQ ID NO: 540, SEQ ID NO: 308 and SEQ ID NO: 543, SEQ ID NO: 308 and SEQ ID NO: 544, SEQ ID NO: 308 and SEQ ID NO: 545, SEQ ID NO: 314 and SEQ ID NO: 363, SEQ ID NO: 314 and SEQ ID NO: 367, SEQ ID NO: 314 and SEQ ID NO: 432, SEQ ID NO: 314 and SEQ ID NO: 519, SEQ ID NO: 314 and SEQ ID NO: 521, SEQ ID NO: 314 and SEQ ID NO: 524, SEQ ID NO: 314 and SEQ ID NO: 526, SEQ ID NO: 314 and SEQ ID NO: 527, SEQ ID NO: 314 and SEQ ID NO: 528, SEQ ID NO: 314 and SEQ ID NO: 529, SEQ ID NO: 314 and SEQ ID NO: 530, SEQ ID NO: 314 and SEQ ID NO: 531, SEQ ID NO: 314 and SEQ ID NO: 532, SEQ ID NO: 314 and SEQ ID NO: 535, SEQ ID NO: 314 and SEQ ID NO: 536, SEQ ID NO: 314 and SEQ ID NO: 537, SEQ ID NO: 314 and SEQ ID NO: 540, SEQ ID NO: 314 and SEQ ID NO: 543, SEQ ID NO: 314 and SEQ ID NO: 544, SEQ ID NO: 314 and SEQ ID NO: 545, SEQ ID NO: 315 and SEQ ID NO: 363, SEQ ID NO: 315 and SEQ ID NO: 367, SEQ ID NO: 315 and SEQ ID NO: 432, SEQ ID NO: 315 and SEQ ID NO: 519, SEQ ID NO: 315 and SEQ 10ME1\59622102.v1Attorney Docket No. 126454-03020ID NO: 521, SEQ ID NO: 315 and SEQ ID NO: 524, SEQ ID NO: 315 and SEQ ID NO: 526, SEQ ID NO: 315 and SEQ ID NO: 527, SEQ ID NO: 315 and SEQ ID NO: 528, SEQ ID NO: 315 and SEQ ID NO: 529, SEQ ID NO: 315 and SEQ ID NO: 530, SEQ ID NO: 315 and SEQ ID NO: 531, SEQ ID NO: 315 and SEQ ID NO: 532, SEQ ID NO: 315 and SEQ ID NO: 535, SEQ ID NO: 315 and SEQ ID NO: 536, SEQ ID NO: 315 and SEQ ID NO: 537, SEQ ID NO: 315 and SEQ ID NO: 540, SEQ ID NO: 315 and SEQ ID NO: 543, SEQ ID NO: 315 and SEQ ID NO: 544, SEQ ID NO: 315 and SEQ ID NO: 545, SEQ ID NO: 316 and SEQ ID NO: 363, SEQ ID NO: 316 and SEQ ID NO: 367, SEQ ID NO: 316 and SEQ ID NO: 432, SEQ ID NO: 316 and SEQ ID NO: 519, SEQ ID NO: 316 and SEQ ID NO: 521, SEQ ID NO: 316 and SEQ ID NO: 524, SEQ ID NO: 316 and SEQ ID NO: 526, SEQ ID NO: 316 and SEQ ID NO: 527, SEQ ID NO: 316 and SEQ ID NO: 528, SEQ ID NO: 316 and SEQ ID NO: 529, SEQ ID NO: 316 and SEQ ID NO: 530, SEQ ID NO: 316 and SEQ ID NO: 531, SEQ ID NO: 316 and SEQ ID NO: 532, SEQ ID NO: 316 and SEQ ID NO: 535, SEQ ID NO: 316 and SEQ ID NO: 536, SEQ ID NO: 316 and SEQ ID NO: 537, SEQ ID NO: 316 and SEQ ID NO: 540, SEQ ID NO: 316 and SEQ ID NO: 543, SEQ ID NO: 316 and SEQ ID NO: 544, SEQ ID NO: 316 and SEQ ID NO: 545, SEQ ID NO: 317 and SEQ ID NO: 363, SEQ ID NO: 317 and SEQ ID NO: 367, SEQ ID NO: 317 and SEQ ID NO: 432, SEQ ID NO: 317 and SEQ ID NO: 519, SEQ ID NO: 317 and SEQ ID NO: 521, SEQ ID NO: 317 and SEQ ID NO: 524, SEQ ID NO: 317 and SEQ ID NO: 526, SEQ ID NO: 317 and SEQ ID NO: 527, SEQ ID NO: 317 and SEQ ID NO: 528, SEQ ID NO: 317 and SEQ ID NO: 529, SEQ ID NO: 317 and SEQ ID NO: 530, SEQ ID NO: 317 and SEQ ID NO: 531, SEQ ID NO: 317 and SEQ ID NO: 532, SEQ ID NO: 317 and SEQ ID NO: 535, SEQ ID NO: 317 and SEQ ID NO: 536, SEQ ID NO: 317 and SEQ ID NO: 537, SEQ ID NO: 317 and SEQ ID NO: 540, SEQ ID NO: 317 and SEQ ID NO: 543, SEQ ID NO: 317 and SEQ ID NO: 544, or SEQ ID NO: 317 and SEQ ID NO: 545.

[0027] In one embodiment, the first gRNA molecule targeting domain and the second gRNA molecule targeting domain comprise, respectively, SEQ ID NO: 1452 and SEQ ID NO: 1475; SEQ ID NO: 305 and SEQ ID NO: 532; SEQ ID NO: 316 and SEQ ID NO: 531; SEQ ID NO: 317 and SEQ ID NO: 532; SEQ ID NO: 318 and SEQ ID NO: 532; SEQ ID NO 305 and SEQ ID NO: 544; SEQ ID NO: 316 and SEQ ID NO: 544; SEQ ID NO: 317 and SEQ ID NO: 544; or SEQ ID NO: 318 and SEQ ID NO: 544. In one embodiment, the first gRNA molecule and the second gRNA molecule comprise, respectively, SEQ ID NO: 1477 and SEQ ID NO: 1450; SEQ ID NO: 900 and SEQ ID NO: 1127; SEQ ID NO: 911 and SEQ ID NO: 1126; SEQ ID NO: 912 and SEQ ID NO: 1127; SEQ ID NO: 913 and SEQ ID NO: 1127; SEQ ID NO: 900 and SEQ ID NO: 1139; SEQ ID NO: 911 and SEQ ID NO: 1139; SEQ ID NO:11ME1\59622102.v1Attorney Docket No. 126454-03020912 and SEQ ID NO: 1139; or SEQ ID NO: 913 and SEQ ID NO: 1139. In one embodiment, the first gRNA molecule and the second gRNA molecule comprise, respectively, targeting domain comprise, respectively, SEQ ID NO: 1452 and SEQ ID NO: 1475. In one embodiment, the first gRNA molecule and the second gRNA molecule comprise, respectively, SEQ ID NO: 1477 and SEQ ID NO: 1450.

[0028] In one embodiment, the first gRNA molecule comprises a targeting domain that targets a first target sequence, and wherein the second gRNA molecule comprises a targeting domain that targets a second target sequence, wherein the first target sequence and the second target sequence comprise a first and second nucleotide sequence, respectively, selected from the group consisting of SEQ ID NO: 73 and SEQ ID NO: 300; SEQ ID NO: 79 and SEQ ID NO: 299; SEQ ID NO: 553 and SEQ ID NO: 589; SEQ ID NO: 64 and SEQ ID NO: 277; SEQ ID NO: 64 and SEQ ID NO: 280; SEQ ID NO: 64 and SEQ ID NO: 284; SEQ ID NO: 64 and SEQ ID NO: 285; SEQ ID NO: 64 and SEQ ID NO: 300; SEQ ID NO: 70 and SEQ ID NO: 277; SEQ ID NO: 70 and SEQ ID NO: 280; SEQ ID NO: 70 and SEQ ID NO: 284; SEQ ID NO: 70 and SEQ ID NO: 285; SEQ ID NO: 70 and SEQ ID NO: 300; SEQ ID NO: 72 and SEQ ID NO: 277; SEQ ID NO: 72 and SEQ ID NO: 280; SEQ ID NO: 72 and SEQ ID NO: 284; SEQ ID NO: 72 and SEQ ID NO: 285; SEQ ID NO: 72 and SEQ ID NO: 300; SEQ ID NO: 73 and SEQ ID NO: 284; SEQ ID NO: 73 and SEQ ID NO: 285; and SEQ ID NO: 74 and SEQ ID NO: 300.

[0029] In one embodiment, the targeting domain comprises a first targeting domain and a second targeting domain, comprising a first and second nucleotide sequence, respectively, selected from the group consisting of SEQ ID NO: 1452 and SEQ ID NO: 1475; SEQ ID NO: 317 and SEQ ID NO: 544; SEQ ID NO: 1455 and SEQ ID NO: 1456; SEQ ID NO 1468 and SEQ ID NO: 1470; SEQ ID NO: 611 and SEQ ID NO: 647; SEQ ID NO: 308 and SEQ ID NO: 521; SEQ ID NO: 308 and SEQ ID NO: 524; SEQ ID NO: 308 and SEQ ID NO: 528; SEQ ID NO: 308 and SEQ ID NO: 529; SEQ ID NO: 308 and SEQ ID NO: 544; SEQ ID NO: 314 and SEQ ID NO: 521; SEQ ID NO: 314 and SEQ ID NO: 524; SEQ ID NO: 314 and SEQ ID NO: 28; SEQ ID NO: 314 and SEQ ID NO: 529; SEQ ID NO: 314 and SEQ ID NO: 544; SEQ ID NO: 316 and SEQ ID NO: 521; SEQ ID NO: 316 and SEQ ID NO: 521; SEQ ID NO: 316 and SEQ ID NO: 528; SEQ ID NO: 316 and SEQ ID NO: 529; SEQ ID NO: 316 and SEQ ID NO: 544; SEQ ID NO: 317 and SEQ ID NO: 528; SEQ ID NO: 317 and SEQ ID NO: 529; and SEQ ID NO: 318 and SEQ ID NO: 544.

[0030] In one embodiment, the first and second gRNA molecules comprise a first and second nucleotide sequence, respectively, selected from the group consisting of SEQ ID NO:12ME1\59622102.v1Attorney Docket No. 126454-030201477 and SEQ ID NO: 1450; SEQ ID NO: 900 and SEQ ID NO: 1139; SEQ ID NO: 1459 and SEQ ID NO: 1460; SEQ ID NO: 1469 and SEQ ID NO: 1471; SEQ ID NO: 1148 and SEQ ID NO: 1184; SEQ ID NO: 903 and SEQ ID NO: 1116; SEQ ID NO: 903 and SEQ ID NO: 1119; SEQ ID NO: 903 and SEQ ID NO: 1123; SEQ ID NO: 903 and SEQ ID NO: 1124; SEQ ID NO: 903 and SEQ ID NO: 1139; SEQ ID NO: 909 and SEQ ID NO: 1116; SEQ ID NO: 909 and SEQ ID NO: 1119; SEQ ID NO: 909 and SEQ ID NO: 1123; SEQ ID NO: 909 and SEQ ID NO: 1124; SEQ ID NO: 909 and SEQ ID NO: 1139; SEQ ID NO: 911 and SEQ ID NO: 1116; SEQ ID NO: 911 and SEQ ID NO: 1123; SEQ ID NO: 911 and SEQ ID NO: 1124; SEQ ID NO: 911 and SEQ ID NO: 1139; SEQ ID NO: 912 and SEQ ID NO: 1123; SEQ ID NO: 912 and SEQ ID NO: 1124; and SEQ ID NO: 913 and SEQ ID NO: 1139.

[0031] In one embodiment, the targeting domain comprises a first targeting domain and a second targeting domain, comprising a first and second nucleotide sequence, respectively, selected from the group consisting of SEQ ID NO: 1452 and SEQ ID NO: 1475. In one embodiment, the first and second gRNA molecules comprise a first and second nucleotide sequence, respectively, selected from the group consisting of SEQ ID NO: 1477 and SEQ ID NO: 1450. In one embodiment, the RNA guided nuclease is a Streptococcus pyogenes Cas9 (SpCas9) nuclease. In one aspect, the present disclosure provides a ribonucleoprotein (RNP) complex comprising the genome editing system as disclosed herein.

[0032] In one aspect, the present disclosure provides a delivery system for delivering the genome editing system as disclosed herein, wherein the delivery system comprises the at least one gRNA molecule and an RNA molecule encoding the RNA-guided nuclease. In one embodiment, the delivery system comprises a lipid nanoparticle (LNP) encapsulating the at least one gRNA molecule and the RNA molecule encoding the RNA-guided nuclease. In one embodiment, the LNP comprises an ionizable lipid, a polyethylene glycol (PEG) lipid, a helper lipid, a sterol, or any combination thereof.

[0033] In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 2-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 3 -fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 4-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 5-fold as compared 13ME1\59622102.v1Attorney Docket No. 126454-03020to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 6-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 7-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 8-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 9-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by at least about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by about 6-fold to about 11-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by about 7-fold to about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by about 8-fold to about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by about 9-fold to about 10-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system. In one embodiment, the genome editing system increases LDLR expression in a target cell by about 9-fold to about 11 -fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system.

[0034] In one aspect, the present disclosure provides a method of editing an LDLR gene in a target cell, the method comprising contacting the target cell with the genome editing system as disclosed herein, the RNP complex as disclosed herein, or the delivery system as disclosed herein. In one embodiment, the LNP comprises a targeting moiety. In one embodiment, the targeting moiety is one or more N-acetylgalactosamine (GalNAc) or GalNAc derivatives.14ME1\59622102.v1Attorney Docket No. 126454-03020

[0035] In one embodiment, the target cell is in vivo, ex vivo, or in vitro. In one embodiment, the target cell is in vivo. In one embodiment, the target cell is from a subject with heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH). In one embodiment, the target cell is a hepatocyte.

[0036] In one embodiment, contacting the target cell with the genome editing system, the RNP complex, or the delivery system increases an LDLR protein level in the target cell by at least about 5-fold to at least about 15-fold, relative to the LDLR protein level in the target cell prior to contacting the target cell with the genome editing system, the RNP complex, or the delivery system, or relative to a control cell. In one embodiment, the increase in LDLR protein level is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 18.5-fold, at least about 20-fold, at least about 25-fold, at least about 30 fold. In one embodiment, the increase in LDLR protein level is increased by at least about 9-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 6-fold to about 11 -fold. In one embodiment, the increase in LDLR protein level is increased by at least about 9-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 6-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 7-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 8-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 9-fold to about 11 -fold.

[0037] In one aspect, the present disclosure provides a method of treating a disease or a disorder in a subject, the method comprising administering to the subject the genome editing system as disclosed herein, the RNP complex as disclosed herein, or the delivery system as disclosed herein, thereby treating the disease or disorder. In one embodiment, the subject has heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH). In one embodiment, the disease or disorder is heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH). In one embodiment, administering the genome editing system, the RNP complex, or the delivery system increases an LDLR protein level in the subject, or in a cell, tissue, or fluid of the subject, by at least about 5-fold to at least about 15-fold, relative to the LDLR protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject. In one 15ME1\59622102.v1Attorney Docket No. 126454-03020embodiment, the increase in LDLR protein level is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 18.5-fold, at least about 20-fold, at least about 25-fold, at least about 30 fold. In one embodiment, the increase in LDLR protein level is increased by at least about 9-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 6-fold to about 11 -fold. In one embodiment, the increase in LDLR protein level is increased by at least about 9-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 6-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 7-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 8-fold to about 10-fold. In one embodiment, the increase in LDLR protein level is increased by at least about 9-fold to about 11 -fold.

[0038] In one embodiment, administering the genome editing system, the RNP complex, or the delivery system decreases an LDL-cholesterol (LDL-C) level in the subject, or in a cell, tissue, or fluid of the subject, by at least about 50% to at least about 95%, relative to the LDL-C level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject. In one embodiment, contacting the target cell with the genome editing system, the RNP complex, or the delivery system decreases an Apolipoprotein B (ApoB) level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by about at least about 30% to at least about 98% relative to the ApoB level in the target cell prior to contacting the target cell with the genome editing system, the RNP complex, or the delivery system, or relative to a control cell.

[0039] In one embodiment, administering the genome editing system, the RNP complex, or the delivery system decreases an Lipoprotein(a) (Lp(a)) level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in serum or plasma of a subject, or a liver cell or the liver of the subject, by about at least about 50% to at least about 95% relative to the Lp(a) level in the cell, tissue, or fluid of the subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control. In one embodiment, the control is an Lp(a) serum or plasma level from a control subject or population of control subjects.

[0040] In one embodiment, the first gRNA molecule comprises a first targeting domain that targets a first target sequence, wherein the first target sequence is a first nucleotide 16ME1\59622102.v1Attorney Docket No. 126454-03020sequence that is located 5’ to SEQ ID NO: 74 in the LDLR gene, and does not comprise SEQ ID NO: 74. In one embodiment, the first nucleotide sequence is SEQ ID NO: 73. In one embodiment, the second gRNA molecule comprises a second targeting domain that targets a second target sequence, wherein the second target sequence comprises a second nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 284, 285, 288, 289, or 300. In one embodiment, the second nucleotide sequence is SEQ ID NO: 300. In one embodiment, the first gRNA molecule comprises SEQ ID NO: 1477, and the second gRNA molecule comprises SEQ ID NO: 1450. In one embodiment, the first targeting domain comprises SEQ ID NO: 1452, and the second targeting domain comprises SEQ ID NO: 1475.

[0041] In one embodiment, contacting the target cell with, or administering to the subject, the genome editing system, the RNP complex, or the delivery system increases an LDLR protein level in the target cell, or in a cell, tissue, or fluid of the subject, by at least about 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold relative to a LDLR protein level in a target cell, or in a cell, tissue, or fluid of the subject, contacted with, or administered, a second genome editing system, RNP complex or delivery system which comprises a third gRNA molecule that comprises a third targeting domain that targets a third sequence, wherein the third target sequence is a third nucleotide sequence that comprises SEQ ID NO: 74, or that is located 3’ to SEQ ID NO: 74 in the LDLR gene. In one embodiment, the second genome editing system, RNP complex, or delivery system further comprises a fourth gRNA molecule that is the same as the second gRNA molecule. In one embodiment, the LDLR protein level is increased by at least about 2-fold. In one embodiment, the LDLR protein level is increased by at least about 3-fold. In one embodiment, the LDLR protein level is increased by at least about 4-fold. In one embodiment, the LDLR protein level is increased by at least about 5-fold. In one embodiment, the LDLR protein level is increased by at least about 6-fold. In one embodiment, the LDLR protein level is increased by about 2-fold to about 6-fold. In one embodiment, the LDLR protein level is increased by about 6-fold.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are intended to provide illustrative and schematic rather than comprehensive examples of certain aspects and embodiments of the present disclosure. The drawings are not intended to be limiting or binding to any particular theory or model. Without limiting the foregoing, nucleic acids and polypeptides can be depicted as linear sequences, or as schematic two- or three-dimensional structures; these depictions are intended17ME1\59622102.v1Attorney Docket No. 126454-03020to be illustrative rather than limiting or binding to any particular model or theory regarding their structure.

[0043] FIG. 1 depicts the architecture of the 3 ’ UTR of the human LDLR gene, including select regulatory sites and a naturally-occurring 2.5 kb deletion described in Example 1.

[0044] FIGs. 2A-2B depict the coverage of the human LDLR 3’ UTR by the gRNAs provided by the present disclosure for SpCas9 and AsCasl2a (WT, RR, and RVR) described in Example 1. The numbers on the graphic indicate DNA target sequence SEQ ID NOs.

[0045] FIG. 3 is a graph demonstrating ILL-seq data from duplicate experiments (dashed and open circles represent biological replicates) editing HepG2 cells with single disclosed AsCasl2a (WT) RNPs, as described in Example 2. The x-axis indicates DNA target sequence SEQ ID NO.

[0046] FIG 4 is a graph demonstrating UDiTaS data from duplicate experiments editing HepG2 cells with single disclosed AsCasl2a (WT) RNPs including a gRNA targeting the indicated DNA target sequence, as described in Example 2.

[0047] FIG. 5 is a graph demonstrating UDiTaS data from duplicate experiments editing HepG2 cells with dual disclosed AsCasl2a (WT) RNPs including gRNAs targeting the indicated DNA target sequences, as described in Example 2.

[0048] FIG. 6 is a graph demonstrating the surface expression level of LDLR on HepG2 cells edited with dual AsCasl2a (WT) RNPs including gRNAs targeting the indicated combinations of DNA target sequences, assessed by flow cytometry as described in Example 2.

[0049] FIG. 7A is a graph demonstrating dose-dependent effects of total RNA ( / .<., the sum of mRNA and gRNA) on frequency of edits to the LDLR 3’ UTR in HepG2 cells edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by UDiTaS as described in Example 3.

[0050] FIG. 7B is a graph demonstrating dose-dependent effects of total RNA (i.e., the sum of mRNA and gRNA) on LDLR mRNA levels in HepG2 cells edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by RTddPCR as described in Example 3.

[0051] FIG. 8A is a graph demonstrating dose-dependent effects of total RNA (z.e., the sum of mRNA and gRNA) on total LDLR protein levels in HepG2 cells edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by ELISA as described in Example 3.

[0052] FIG. 8B is a graph demonstrating dose-dependent effects of total RNA (i.e., the sum of mRNA and gRNA) on LDL-C uptake by HepG2 cells edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by flow cytometry as described in Example 3.18ME1\59622102.v1Attorney Docket No. 126454-03020

[0053] FIG. 8C is a histogram demonstrating the effect of 4E-4 mg / mL total RNA the sum of mRNA and gRNA) on LDL-C uptake by HepG2 cells edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by flow cytometry as described in Example 3.

[0054] FIG. 9A is a graph demonstrating dose-dependent effects of total RNA (z.e., the sum of mRNA and gRNA) on frequency of edits to LDLR 3’ UTR in primary human hepatocytes (PHHs) edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by UDiTaS as described in Example 4.

[0055] FIG. 9B is a graph demonstrating dose-dependent effects of total RNA (z.e., the sum of mRNA and gRNA) on LDLR mRNA levels in primary human hepatocytes (PHHs) edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by RTddPCR as described in Example 4.

[0056] FIG. 9C is a graph demonstrating dose-dependent effects of total RNA (z.e., the sum of mRNA and gRNA) on total LDLR protein levels in primary human hepatocytes (PHHs) edited with dual SpCas9 gRNAs, delivered by LNP, and assessed by ELISA as described in Example 4.

[0057] FIG. 10A is a graph demonstrating dose-dependent effects of total RNA (z.e., the sum of mRNA and gRNA) on total LDLR protein levels in HepG2 cells edited with dual-guide AsCasl2a (WT) systems including the indicated combinations of DNA target sequences. AsCasl2a mRNA and gRNA pairs were delivered by LNP and LDLR protein levels were assessed by ELISA as described in Example 5.

[0058] FIG. 10B is a graph demonstrating dose-dependent effects of total RNA (z.e., the sum of mRNA and gRNA) on total LDLR protein levels in primary human hepatocytes (PHHs) edited with dual -guide AsCasl2a (WT) systems including the indicated combinations of DNA target sequences. AsCasl2a mRNA and gRNA pairs were delivered by LNP and LDLR protein levels were assessed by ELISA described in Example 5.

[0059] FIG. HA is a graph demonstrating the frequency of indel generation in primary human hepatocytes (PHHs) cotransfected with mRNA encoding SpCas9 and single gRNAs proximal to the stop codon assessed by UDiTaS as described in Example 6. The x-axis depicts DNA target sequence and cut position within xe. LDLR 3’ UTR (in parentheses).

[0060] FIG. 11B is a graph demonstrating the frequency of indel generation in primary human hepatocytes (PHHs) cotransfected with mRNA encoding SpCas9 and single gRNAs distal to the stop codon assessed by UDiTaS as described in Example 6. The x-axis depicts DNA target sequence and cut position within xe. LDLR 3’ UTR (in parentheses).19ME1\59622102.v1Attorney Docket No. 126454-03020

[0061] FIGs. 12A-12D are graphs demonstrating the fold change in LDLR protein expression in primary human hepatocytes (PHHs) cotransfected with mRNA encoding SpCas9 and gRNA pairs, assessed by ELISA, in which one gRNA is always the specified “anchor gRNA,” which is proximal to the stop codon, as described in Example 6. The x-axis depicts DNA target sequence and cut position within xe. LDLR 3’ UTR (in parentheses).

[0062] FIGs. 13A-13D are graphs demonstrating the fold change in LDLR protein expression in primary human hepatocytes (PHHs) cotransfected with mRNA encoding SpCas9 and gRNA pairs, assessed by ELISA, in which one gRNA is always the specified “anchor gRNA,” which is distal to the stop codon and precedes the polyA sites, as described in Example 6. The x-axis depicts DNA target sequence and cut position within the LDLR 3’ UTR (in parentheses).

[0063] FIGs. 14A-14D are graphs demonstrating the fold change in LDLR protein expression in primary human hepatocytes (PHHs) cotransfected with mRNA encoding SpCas9 and gRNA pairs, assessed by ELISA, in which one gRNA is always the specified “anchor gRNA,” which is distal to the stop codon and follows the polyA sites, as described in Example 6. The x-axis depicts DNA target sequence and cut position within the LDLR 3’ UTR (in parentheses).

[0064] FIG. 15 is a chart demonstrating the fold upregulation of total LDLR protein levels in primary human hepatocytes (PHHs) cotransfected with mRNA encoding SpCas9 and combinations of gRNAs targeting the indicated DNA target sequences assessed by ELISA as described in Example 6.

[0065] FIG. 16A is a graph demonstrating the frequency and type of edits produced by AsCasl2a (WT) mRNA and gRNA pairs delivered by LNP in a population of primary human hepatocytes (PHHs) assessed by UDiTaS as described in Example 7. The indicated gRNA pair DNA target sequences are described in Table 24.

[0066] FIG. 16B is a graph demonstrating the fold change in total LDLR protein levels in primary human hepatocytes (PHHs) with AsCasl2a (WT) mRNA and gRNA pairs delivered by LNP assessed by ELISA as described in Example 7. The indicated gRNA pair DNA target sequences are described in Table 24.

[0067] FIG. 17A is a graph demonstrating dose-dependent effects of total RNA (i.e., the sum of mRNA and gRNA) on frequency and type of editing in a population of primary human hepatocytes (PHHs) edited with dual AsCas12a (WT) gRNAs delivered by LNP as assessed by UDiTaS, as described in Example 7. The indicated gRNA pair DNA target sequences are described in Table 24.20ME1\59622102.v1Attorney Docket No. 126454-03020

[0068] FIG. 17B is a graph demonstrating dose-dependent effects of total RNA ((*i.e.*, the sum of mRNA and gRNA) on total LDLR protein levels in primary human hepatocytes (PHHs) edited with dual AsCasl2a (WT) gRNAs delivered by LNP as assessed by ELISA, as described in Example 7. The indicated gRNA pair DNA target sequences are described in Table 24.

[0069] FIG. 17C is a graph demonstrating the correlation of frequency of edits (assessed by UDiTaS) and LDLR fold up-regulation (assessed by ELISA) in primary human hepatocytes (PHHs) edited with dual AsCasl2a (WT) gRNAs delivered by LNP as described in Example 7. The indicated gRNA pair DNA target sequences are described in Table 24.

[0070] FIG. 18A is a graph demonstrating the frequency and type of editing in a population of primary human hepatocytes (PHHs) edited with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as assessed by UDiTaS, as described in Example 8. The indicated gRNA pair DNA target sequences are described in Table 25 A.

[0071] FIG. 18B is a graph demonstrating fold change in total LDLR protein levels in primary human hepatocytes (PHHs) edited with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as assessed by ELISA, as described in Example 8. The indicated gRNA pair DNA target sequences are described in Table 25A.

[0072] FIG. 18C is a graph demonstrating the correlation of frequency of edits (assessed by UDiTaS) and LDLR fold up-regulation (assessed by ELISA) in primary human hepatocytes (PHHs) edited with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as described in Example 8. The indicated gRNA pair DNA target sequences are described in Table 25A.

[0073] FIG. 19 depicts the architecture of the 3’ UTR of the LDLR gene, including select regulatory sites and the Proximal and Proximal-ARE deletions induced by gRNA pairs disclosed herein.

[0074] FIG. 20A is a chart demonstrating the fold change in total LDLR protein levels in primary human hepatocytes (PHHs) edited with SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, assessed by ELISA as described in Example 9.

[0075] FIG. 20B is a graph demonstrating the fold-change in total LDLR protein levels in primary human hepatocytes (PHHs) after editing with SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, assessed by ELISA as described in Example 9.

[0076] FIG. 20C is a graph demonstrating total LDLR protein levels in primary human hepatocytes (PHHs) edited with SpCas9 gRNA pairs delivered by LNP and targeting the21ME1\59622102.v1Attorney Docket No. 126454-03020indicated combinations of DNA target sequences, assessed by ELISA as described in Example 9.

[0077] FIG. 20D is a graph demonstrating total LDLR protein levels in primary human hepatocytes (PHHs) edited with the Proximal-ARE SpCas9 gRNA pair delivered by LNP and assessed by ELISA, as described in Example 9.

[0078] FIG. 20E is a graph demonstrating the change in LDLR mRNA levels in HepG2 cells after editing with the indicated combination of SpCas9 gRNA pairs delivered by LNP and targeting the gRNA target sequences or a non-targeting control, and subsequent treatment with Actinomycin D (ActD), as described in Example 9.

[0079] FIG. 21 is a graph demonstrating frequency and type of editing in a population of primary human hepatocytes (PHHs) edited with SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, as assessed by UDiTaS as described in Example 9.

[0080] FIG. 22 A is a chart demonstrating the percent of mRNAs from primary human hepatocytes (PHHs) exhibiting large deletions after editing with SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, as assessed by RT-ddPCR as described in Example 9.

[0081] FIG. 22B is a chart demonstrating the sum percent of mRNAs from primary human hepatocytes (PHHs) exhibiting large deletions and inversions after editing with SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, as assessed by RT-ddPCR as described in Example 9.

[0082] FIG. 22C is a graph demonstrating the frequency of functional edits in in primary human hepatocytes (PHHs) after editing with SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, assessed by ELISA as described in Example 9.

[0083] FIG. 22D is a graph demonstrating the fold change in LDLR mRNA levels in primary human hepatocytes (PHHs) treated with varying doses of gRNA pairs delivered by LNP and targeting the indicated combinations of SpCas9 gRNA target sequences, as assessed by RT-ddPCR as described in Example 9.

[0084] FIG. 23A is a graph demonstrating the frequency and type of editing reflected in mRNA isolated from a population of primary human hepatocytes (PHHs) edited with SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, as assessed by RNA seq as described in Example 9.22ME1\59622102.v1Attorney Docket No. 126454-03020

[0085] FIG. 23B is a graph demonstrating the fold change in total LDLR protein levels in primary human hepatocytes (PHHs) treated with varying doses of SpCas9 gRNA pairs delivered by LNP and targeting the indicated combinations of DNA target sequences, as assessed by ELISA as described in Example 9.

[0086] FIG. 24 is a chart demonstrating the fold change in total LDLR protein levels in primary human hepatocytes (PHHs) edited with SpCas9 or AsCasl2a gRNA pairs targeting the indicated combinations of DNA target sequences, assessed by ELISA as described in Example 10.

[0087] FIG. 25A is a graph demonstrating the baseline expression level of LDLR protein in HepG2 and 1C10 cells as described in Example 11.

[0088] FIG. 25B is a graph demonstrating the baseline LDL-C uptake by HepG2 and 1C10 cells as described in Example 11.

[0089] FIG. 26A is a graph demonstrating the expression level of LDLR protein in HepG2 and 1C10 cells after editing with SpCas9 gRNAs delivered by LNP as described in Example 11.

[0090] FIG. 26B is a graph demonstrating the LDL-C uptake by HepG2 and 1C10 cells after editing with SpCas9 gRNAs delivered by LNP as described in Example 11.

[0091] FIG. 27A is a graph demonstrating the frequency and type of edits in mouse subjects treated with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as assessed by UDiTaS, as described in Example 12. The indicated gRNA pair target sequences are described in Tables 29C-29D

[0092] FIG. 27B is a graph demonstrating fold change in total Ldlr protein levels in mouse subjects treated with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as assessed by ELISA, as described in Example 12. The indicated gRNA pair target sequences are described in Tables 29C-29D

[0093] FIG. 28A is a graph demonstrating the percent reduction in LDL-C levels in mouse subjects treated with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as assessed using an LDL-C assay kit (BioTechne) as described in Example 12. The indicated DNA target sequences are described in Tables 29C-29D.

[0094] FIG. 28B is a graph demonstrating the correlation of Ldlr fold upregulation (assessed by ELISA and depicted in FIG. 27B) and LDL-C percent reduction (assessed using an LDL-C assay kit (BioTechne) as depicted in FIG. 28A) in mouse subjects treated with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as described in Example 12. The indicated DNA target sequences are described in Tables 29C-29D.23ME1\59622102.v1Attorney Docket No. 126454-03020

[0095] FIG. 29A is a graph demonstrating dose-dependent effects of total RNA ((*i.e.*, the sum of mRNA and gRNA) on frequency and type of editing in mouse liver cells from mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal region delivered by LNP as assessed by RT-ddPCR, as described in Example 13. The indicated DNA target sequences are described in Table 31 A.

[0096] FIG. 29B is a graph demonstrating dose-dependent effects of total RNA ((*i.e.*, the sum of mRNA and gRNA) on frequency and type of editing in mouse liver cells from mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP as assessed by RT-ddPCR, as described in Example 13. The indicated DNA target sequences are described in Table 31A.

[0097] FIG. 30A is a graph demonstrating dose-dependent effects of total RNA ((*i.e.*, the sum of mRNA and gRNA) on frequency and type of editing in mouse liver cells from mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal region delivered by LNP as assessed by Next-Generation Sequencing (NGS), as described in Example 13. The indicated DNA target sequences are described in Table 31A.

[0098] FIG. 30B is a graph demonstrating dose-dependent effects of total RNA ((*i.e.*, the sum of mRNA and gRNA) on frequency and type of editing in mouse liver cells from mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP as assessed by Next-Generation Sequencing (NGS), as described in Example 13. The indicated DNA target sequences are described in Table 31 A.

[0099] FIG. 31A is a graph demonstrating fold change in liver Ldlr protein levels in mouse subjects treated with dual SpCas9 gRNAs targeting the Proximal region delivered by LNP as assessed by ELISA, as described in Example 13. The indicated DNA target sequences are described in Table 31A.

[0100] FIG. 31B is a graph demonstrating fold change in liver Ldlr protein levels in mouse subjects treated with dual SpCas9 gRNAs targeting the Proximal-ARE region delivered by LNP as assessed by ELISA, as described in Example 13. The indicated DNA target sequences are described in Table 31 A.

[0101] FIG. 32A is a graph demonstrating change in serum LDL-C levels in mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal region delivered by LNP as described in Example 13. The indicated DNA target sequences are described in Table 31A.

[0102] FIG. 32B is a graph demonstrating change in serum LDL-C levels in mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered24ME1\59622102.v1Attorney Docket No. 126454-03020by LNP as described in Example 13. The indicated DNA target sequences are described in Table 31A

[0103] FIG. 33A is a graph demonstrating change in serum ApoB levels in mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal region delivered by LNP as described in Example 13. The indicated DNA target sequences are described in Table 31 A.

[0104] FIG. 33B is a graph demonstrating change in serum ApoB levels in mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP as described in Example 13. The indicated DNA target sequences are described in Table 31A.

[0105] FIG. 34A is a graph demonstrating the percent change in serum ApoB levels in wild-type mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal region delivered by LNP as described in Example 13. The indicated DNA target sequences are described in Table 31A.

[0106] FIG. 34B is a graph demonstrating the percent change in serum ApoB levels in wild-type mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP as described in Example 13. The indicated DNA target sequences are described in Table 31A.

[0107] FIG. 34C is a graph demonstrating the correlation frequency of edits (assessed by NGS and depicted in FIG. 30B) of Ldlr fold upregulation (assessed by ELISA and depicted in FIG. 31B) and ApoB percent reduction (as depicted in FIG.33B) in wild-type mouse subjects treated with dual SpCas9 gRNAs selected to induce the Proximal-ARE deletion delivered by LNP, as described in Example 13. The indicated DNA target sequences are described in Table 31A

[0108] FIG. 35A is a graph demonstrating the change in Ldlr mRNA levels in response to varying doses of SpCas9 gRNA target sequences delivered by LNP in wild-type mouse subj ects as described in Example 14.

[0109] FIG. 35B is a graph demonstrating the change in serum ApoB levels in response to varying doses of SpCas9 gRNA pairs delivered by LNP in wild-type mouse subjects as described in Example 14.

[0110] FIG. 36A is a graph demonstrating the frequency of functional edits in response to varying doses of SpCas9 gRNA pairs delivered by LNP in wild-type or Ldlr ~ mouse subjects as described in Example 14.

[0111] FIG. 36B is a graph demonstrating the change in LDLR protein levels in response to varying doses of SpCas9 gRNA pairs delivered by LNP in wild-type or Ldlr mouse subjects as described in Example 14.25ME1\59622102.v1Attorney Docket No. 126454-03020

[0112] FIG. 36C is a graph demonstrating the change in serum ApoB levels in response to varying doses of SpCas9 gRNA pairs delivered by LNP in wild-type or Ldlr ~ mouse subjects as described in Example 14.

[0113] FIG. 37A is a graph demonstrating the correlation frequency of edits (assessed by NGS and depicted in FIG. 36A) of Ldlr fold upregulation (assessed by ELISA and depicted in FIG. 36B) and ApoB percent reduction (as depicted in FIG. 36C) in Ldlr / +mouse subjects treated with dual SpCas9 gRNAs selected to induce the Proximal-ARE deletion delivered by LNP as described in Example 14.

[0114] FIG.37B is a graph demonstrating change in serum LDL-C levels in Ldlr / +mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP as measured by enzymatic LDL-C assay as described in Example 14.

[0115] FIG. 37C is a graph demonstrating the change in serum LDL-C levels in Ldlr+ / ~ mouse subjects treated with dual SpCas9 gRNAs targeting the Ldlr Proximal region delivered by LNP as measured an HPLC-based quantification method as described in Example 14.

[0116] FIG. 38A is an experimental design diagram for the experiment evaluating LDL-C levels in mouse subjects fed a regular or high-fat diet as described in Example 15.

[0117] FIG. 38B is a graph depicting absolute serum LDL-C concentrations in wild-type mice fed a regular or high-fat diet and treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP as described in Example 15.

[0118] FIG. 38C is a graph depicting the percent change in serum LDL-C levels in wildtype mice subjects fed a regular or high-fat diet and treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP at 11 weeks, as described in Example 15.

[0119] FIG. 38D is a graph depicting the absolute change in serum LDL-C levels in wildtype and Ldlr+ / ~ mice subj ects fed a high-fat diet and treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP at 11 weeks, as described in Example 15.

[0120] FIG. 38E is a graph demonstrating the change in LDLR protein levels in wild-type and Ldlr~ ~ mouse subjects fed a high-fat diet and treated with dual SpCas9 gRNAs targeting the Ldlr Proximal-ARE region delivered by LNP as described in Example 15.

[0121] FIG. 39 is a graph depicting mRNA fold upregulation and the frequency and type of editing in a population of cynomolgus hepatocytes edited with dual SpCas9 or AsCasl2a (WT) gRNAs delivered by LNP as assessed by RT-ddPCR, as described in Example 16. The indicated DNA target sequences are described in Table 33A.26ME1\59622102.v1Attorney Docket No. 126454-03020

[0122] FIG. 40 is a graph demonstrating the fold change in total LDLR protein levels in cynomolgus hepatocytes edited with dual SpCas9 or AsCasl2a gRNAs assessed by ELISA, as described in Example 16.

[0123] FIG. 41 is a study diagram illustrating the experiments and results identifying off-target editing of SpCas9 gRNAs as described in Example 17.

[0124] FIG. 42A is a graph demonstrating the fold change in LDLR protein levels in Heterozygous Familial Hypercholesterolemia (HeFH) patient-derived fibroblasts edited with the Proximal-ARE SpCas9 gRNA pair, delivered by LNP, and assessed by ELISA as described in Example 18.

[0125] FIG. 42B is a graph demonstrating the fold change in LDL-C uptake by Heterozygous Familial Hypercholesterolemia (HeFH) patient-derived fibroblasts edited with the Proximal-ARE SpCas9 gRNA pair, delivered by LNP, and assessed by flow cytometry as described in Example 18.

[0126] FIG. 43A is a graph demonstrating the normalized percent serum LDL-C remaining, relative to baseline, at multiple time points after administration of the SpCas9 Proximal-ARE gRNA pair delivered by LNP to cynomolgus monkey subjects as described in Example 19.

[0127] FIG. 43B is a graph demonstrating the normalized percent serum LDL-C remaining, relative to baseline, at multiple time points after administration of the SpCas9 Proximal-ARE gRNA pair delivered by LNP to cynomolgus monkey subjects as described in Example 19.

[0128] FIG. 43C is a graph demonstrating the correlation of functional editing and LDL-C percent reduction (as depicted in FIG. 43 A) in cynomolgus monkey subjects treated with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0129] FIG. 43D is a graph demonstrating the correlation of liver LDLR protein levels and LDL-C percent reduction (as depicted in FIG. 43 A) in cynomolgus monkey subjects treated with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0130] FIG. 43E is a graph depicting editing levels in liver of NHPs. Excisions, inversions, small indels, large resections and translocations in liver specimens of NHPs treated with a single IV dose at the indicated multiple dose levels as described in Example 19. Bars indicate total % editing and individual sub-bars the specific type of edit. NHP=3 at 1.5; 2 at 2mg / kg; 2 at 2 mg / kg and 3 at 4 mg / kg; ± SD.

[0131] FIG. 43F is a graph depicting LDLR protein levels in NHPs. LDLR protein levels are increased in the livers of NHPs treated as described in Example 19 in a dose-responsive 27ME1\59622102.v1Attorney Docket No. 126454-03020manner. Bars indicate mean values at each dose levels, NHP=3 at 1.5; 2 at 2 mg / kg; 2 at 2 mg / kg and 3 at 4 mg / kg; ± SD. Dotted line indicates mean of vehicle-treated animals (NHP=3).

[0132] FIG. 43G is a graph depicting ApoB reduction in NHPs as described in Example 19. Single IV administration at the indicated dose levels showed significant reduction of plasma ApoB, expressed as change from baseline levels of individual animals. NHP=3 at 1.5; 2 at 2mg / kg; 2 at 3 mg / kg and 3 at 4 mg / kg; ± SD.

[0133] FIG. 44A is a micrograph demonstrating staining for Cas9 mRNA (red) and nuclei (blue) in a liver sample from a cynomolgus monkey subject treated with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0134] FIG. 44B is a micrograph demonstrating staining for Cas9 mRNA (red) and nuclei (blue) in an ovary sample from a cynomolgus monkey subject treated with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0135] FIG. 45A is a micrograph demonstrating staining for Cas9 mRNA (red) and nuclei (blue) in a liver sample from a cynomolgus monkey subject after about 24 hours of treatment with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0136] FIG. 45B is a micrograph demonstrating staining for Cas9 protein (brown) and nuclei (blue) in a liver sample from a cynomolgus monkey subject after about 24 hours of treatment with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0137] FIG. 45C is a micrograph demonstrating staining for Cas9 mRNA (red) and nuclei (blue) in an ovary sample from a cynomolgus monkey subject after about 24 hours of treatment with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0138] FIG. 45D is a micrograph demonstrating staining for Cas9 protein (brown) and nuclei (blue) in an ovary sample from a cynomolgus monkey subject after about 24 hours of treatment with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0139] FIG. 45E is a micrograph demonstrating staining for LDLR protein (brown) and nuclei (blue) in a liver sample from a cynomolgus monkey subject after about 24 hours of treatment with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0140] FIG. 46 is a graph demonstrating the percent of indels generated (assessed using UDiTaS) in multiple tissue samples collected from a cynomolgus monkey (non-human primate) subject after about 24 hours of treatment with dual SpCas9 gRNAs delivered by LNP as described in Example 19.

[0141] FIG. 47A is a graph demonstrating the percent Lp(a) protein reduction using an immunoturbidimetry assay relative to baseline, at multiple time points (pre-dose, pre-dose, 24 hours, 48 hours, day 5, day 8, day 15, day 22, and day 29) after administration of the SpCas928ME1\59622102.v1Attorney Docket No. 126454-03020Proximal-ARE gRNA pair delivered by LNP (doses of 1.5 mpk, 2 mpk, 3 mpk, and 4 mpk) to cynomolgus monkey subjects as described in Example 19.

[0142] FIG. 47B is a graph demonstrating the percent Lp(a) protein reduction using an immunoturbidimetry assay (pre-dose averaged), at multiple time points (pre-dose, pre-dose, 24 hours, 48 hours, day 5, day 8, day 15, day 22, and day 29) after administration of the SpCas9 Proximal-ARE gRNA pair delivered by LNP (doses of 1.5 mpk, 2 mpk, 3 mpk, and 4 mpk) to cynomolgus monkey subjects as described in Example 19.DETAILED DESCRIPTION

[0143] The present disclosure relates to genome editing systems (e.g., RNA-guided nuclease-related, e.g, CRISPR / Cas-related, genome editing systems), compositions, delivery vehicles, and methods for targeting an LDLR nucleic acid sequence, editing a target LDLR nucleic acid sequence, or modulating expression of an LDLR gene, and applications thereof. The present disclosure also provides genome editing systems, compositions, vectors, and methods for editing cells using CRISPR / Cas-related components to edit a target LDLR gene. The present disclosure also provides lipid nanoparticle (LNP) facilitated delivery of genome editing systems and methods for editing cells using CRISPR / Cas-related components delivered via LNP to edit a target LDLR gene. Some aspects of the present disclosure provide pharmaceutical compositions, cells, cell populations, methods, strategies, and treatment modalities that are useful in the context of treating and / or managing a disease, e.g, hyperlipidemia or hypercholesterolemia.

[0144] The subject matter of the present disclosure is described with reference to the Figures. It should be understood that numerous specific details, relationships, and methods are set forth in this Detailed Description, Examples, and accompanying Figures to provide a more complete understanding of the subject matter disclosed herein. For purposes of clarity of disclosure and not by way of limitation, the Detailed Description is divided into the following subsections:1. Definitions and Abbreviations2. Genome editing systems3. Guide RNA (gRNA) molecules4. Guide RNA design5. RNA-guided nucleases6. Genome editing strategies29ME1\59622102.v1Attorney Docket No. 126454-030207. Implementation of genome editing systems: delivery, formulations, and routes of administration8. Examples1. Definitions and Abbreviations

[0145] Unless otherwise specified, each of the following terms has the meaning associated with it in this section.

[0146] The indefinite articles “a” and “an” refer to at least one of the associated noun and are used interchangeably with the terms “at least one” and “one or more.” For example, “a module” means at least one module, or one or more modules.

[0147] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, (*i.e.*, the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0148] The conjunctions “or” and “and / or” are used interchangeably as non-exclusive disjunctions.

[0149] The phrase “consisting essentially of’ means that the species recited are the predominant species, but that other species can be present in trace amounts or amounts that do not affect structure, function or behavior of the subject composition. For instance, a composition that consists essentially of a particular species will generally comprise 90%, 95%, 96%, or more of that species.

[0150] “Domain” is used to describe a segment of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.

[0151] An “indel” is an insertion and / or deletion in a nucleic acid sequence. An indel can be the product of the repair of a DNA double strand break, such as a double strand break formed by a genome editing system of the present disclosure. An indel is most commonly formed when a break is repaired by an “error prone” repair pathway such as the nonhom ologous end joining (NHEJ) pathway described below.

[0152] Gene conversion” refers to the alteration of a DNA sequence by incorporation of an endogenous homologous sequence (e.g., a homologous sequence within a gene array).30ME1\59622102.v1Attorney Docket No. 126454-03020“Gene correction” refers to the alteration of a DNA sequence by incorporation of an exogenous homologous sequence, such as an exogenous single-or double stranded donor template DNA. Gene conversion and gene correction are products of the repair of DNA double-strand breaks by HDR pathways such as those described below.

[0153] Indels, gene conversion, gene correction, and other genome editing outcomes are typically assessed by sequencing (most commonly by “next-gen” or “sequencing-by-synthesis” methods, though Sanger sequencing can still be used) and are quantified by the relative frequency of numerical changes (e.g., ±1, ±2 or more bases) at a site of interest among all sequencing reads. DNA samples for sequencing can be prepared by a variety of methods known in the art and can involve the amplification of sites of interest by polymerase chain reaction (PCR), the capture of DNA ends generated by double strand breaks, as in the GUTDE-seq process described in Tsai et al. (Nat. Biotechnol. 34(5): 483 (2016), incorporated by reference herein), the UDiTaS method described in Giannoukos et al. (BMC Genomics. 19(1): 212 (2018), incorporated by reference herein), or by other means well known in the art. Genome editing outcomes can also be assessed by in situ hybridization methods such as the FiberComb™ system commercialized by Genomic Vision (Bagneux, France), and by any other suitable methods known in the art.

[0154] “Alt-HDR,” “alternative homology-directed repair,” or “alternative HDR” are used interchangeably to refer to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g., a sister chromatid, or an exogenous nucleic acid, e.g., a template nucleic acid). Alt-HDR is distinct from canonical HDR in that the process utilizes different pathways from canonical HDR, and can be inhibited by the canonical HDR mediators, RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of a single-stranded or nicked homologous nucleic acid template, whereas canonical HDR generally involves a double-stranded homologous template.

[0155] “Canonical HDR,” “canonical homology-directed repair” or “cHDR” refer to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g, a sister chromatid, or an exogenous nucleic acid, e.g., a template nucleic acid). Canonical HDR typically acts when there has been significant resection at the double strand break, forming at least one single stranded portion of DNA. In a normal cell, cHDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. The process requires RAD51 and BRCA2, and the homologous nucleic acid is typically double stranded.31ME1\59622102.v1Attorney Docket No. 126454-03020

[0156] Unless indicated otherwise, the term “HDR” as used herein encompasses both canonical HDR and alt-HDR.

[0157] “Non-homologous end joining” or “NHEJ” refers to ligation mediated repair and / or non-template mediated repair including canonical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), which in turn includes microhomology -mediated end joining (MMEJ), singlestrand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

[0158] “Replacement” or “replaced,” when used with reference to a modification of a molecule e.g., a nucleic acid or protein), does not require a process limitation but merely indicates that the replacement entity is present.

[0159] “Knock-out” or “knockout” refers to an inactivating mutation in a target gene, wherein the product of the target gene comprises a loss of function.

[0160] As used herein, “upregulate” refers to an increase in expression and / or abundance of a target gene product, for example by mutation of a target gene.

[0161] Gain of function” refers to a mutation in a target gene, wherein the protein product of the gene has an increase in a functional activity. This may be achieved, e.g. through an increase in abundance and / or a change in the capacity of the protein product to carry out a function.

[0162] Gene product” refers to biochemical products resulting from the expression of the gene and includes the RNA or protein that is encoded by the gene.

[0163] As used herein, “expression” refers to the amount of gene product (e.g., LDLR) produced through the transcription and / or translation of a gene.

[0164] On-target site” refers to the exact genomic sequence or locus within a gene of interest for which a guide RNA / RNA-guided nuclease was designed to target. “Off-target site” refers to a genomic sequence or locus that is not the on-target site and may be (or is found to be) edited by the guide RNA / RNA guided nuclease.

[0165] As used herein, the term “hairpin” or “hairpin region” refers to a nucleic acid secondary structure comprising a stem-loop structure. A guide-RNA of the present disclosure may comprise one or more hairpins, or hairpin regions, that are not part of its targeting domain.

[0166] As used herein, the term “pseudoknot” refers to a nucleic acid secondary structure comprising at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. Exemplary pseudoknot sequences are bolded in the two gRNA sequences shown below;32ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrCrUrGrArGrArUrGrCrCrArGrC rUrGrUrCrCrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU [SEQ ID NO: 24]; mU*rArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrCrUrGrArGrArUrGrCrCrArG rCrUrGrUrCrCrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrA*mU [SEQ ID NO: 25].

[0167] As used herein, the term “locked nucleic acid” or “LNA” refers to a modified RNA nucleotide in which the ribose moiety is modified with a bridge connecting the 2' oxygen and 4' carbon. An exemplary nucleic acid including a LNA is shown below, where the “+” indicates a locked nucleotide; / 5IdT / +A+T+G+T+G+T+T+T+T+T+G+T+C+A+A+A+A+G+A+C+C+T+T+T+TrUrArArU rUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArArCrCrUrCrCrUrGrGrCrCrArGrArUrUrCrU rC / 3InvdT / [SEQ ID NO: 26],

[0168] Subject” means a human or non-human animal. A human subject can be any age (e.g., an infant, child, young adult, or adult), and can suffer from a disease, or can be in need of alteration of a gene. Alternatively, the subject can be an animal, which term includes, but is not limited to, mammals, birds, fish, reptiles, amphibians, and more particularly non-human primates (NHP), rodents (such as mice, rats, hamsters, etc.), rabbits, guinea pigs, dogs, cats, and so on. In certain embodiments of this disclosure, the subject is livestock, e.g, a cow, a horse, a sheep, or a goat. In certain embodiments, the subject is poultry.

[0169] As used herein a “therapeutically effective amount” refers to the amount of a cell and / or composition that when administered to a subject for treating a disease, is sufficient to beneficially affect such treatment for the disease.

[0170] Treat,” “treating,” and “treatment” mean the treatment of a disease in a subject (e.g, a human subject), including one or more of inhibiting the disease, z.e., arresting or preventing its development or progression; relieving the disease, ie., causing regression of the disease state; relieving one or more symptoms of the disease; and curing the disease.

[0171] “Prevent,” “preventing,” and “prevention” refer to the prevention of a disease in a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease.

[0172] A “kit” refers to any collection of two or more components that together constitute a functional unit that can be employed for a specific purpose. By way of illustration (and not limitation), one kit according to this disclosure can include a guide RNA complexed or able to complex with an RNA-guided nuclease, and accompanied by (e.g., suspended in, or 33ME1\59622102.v1Attorney Docket No. 126454-03020suspendable in) a pharmaceutically-acceptable carrier. The kit can be used to introduce the complex into, for example, a cell or a subject, for the purpose of causing a desired genomic alteration in such cell or subject. The components of a kit can be packaged together, or they can be separately packaged. Kits according to this disclosure also optionally include directions for use (DFU) that describe the use of the kit e.g., according to a method of this disclosure. The DFU can be physically packaged with the kit, or it can be made available to a user of the kit, for instance by electronic means.

[0173] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” mean any chain of two or more nucleotide bases (also called “nucleotides”). The polynucleotides, nucleotide sequences, nucleic acids etc. can be chimeric mixtures or derivatives or modified versions thereof, singlestranded or double-stranded. They can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide sequence typically carries genetic information, including, but not limited to, the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

[0174] Conventional International Union of Pure and Applied Chemistry (IUPAC) notation is used in nucleotide sequences presented herein, as shown in Table 1, below (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10; 13(9):3021-30, incorporated by reference herein). It should be noted, however, that “T” denotes “Thymine or Uracil” in those instances where a sequence can be encoded by either DNA or RNA, for example in a gRNA, for example in a gRNA targeting domain. The prefix r (for ribo) may be used for emphasis or clarity.Table 1: IUPAC nucleic acid notationCharacter BaseA AdenineT Thymine or UracilG GuanineC CytosineU UracilK G or T / UM A or CR A or GY C or T / U34ME1\59622102.v1Attorney Docket No. 126454-03020s C orGw A or T / UB C, G or T / UV A, C or GH A, C or T / UD A, G or T / UN A, C, G or T / U

[0175] The terms “protein,” “peptide” and “polypeptide” are used interchangeably to refer to a sequential chain of amino acids linked together via peptide bonds. The terms include individual proteins, groups or complexes of proteins that associate together, as well as fragments or portions, variants, derivatives and analogs of such proteins. Peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left, and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three-letter abbreviations can be used.

[0176] The term “variant” refers to an entity such as a polypeptide, polynucleotide or small molecule that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity.

[0177] As used herein, the term “promoter” refers to a region (z.e., a DNA sequence) of a genome that initiates the transcription of a gene.

[0178] As used herein, the term “3’ untranslated region” or “3’ UTR” refers to a region of a gene that is transcribed, does not form part of the protein-coding portion of a messenger RNA (mRNA), and is located to the 3’ of the protein-coding portion of the messenger RNA (mRNA), i.e. after the stop codon.

[0179] The term “endogenous,” as used herein in the context of nucleic acids (e.g., genes, protein-encoding genomic regions, promoters) or proteins, refers to a native nucleic acid or protein in its natural location, e.g., within the genome of a cell. In contrast, the term “exogenous,” as used herein in the context of nucleic acids, e.g., expression constructs, cDNAs, indels, and nucleic acid vectors, or proteins, refers to nucleic acids or proteins that have artificially been introduced into the cell. For example, an exogenous nucleic acid may be35ME1\59622102.v1Attorney Docket No. 126454-03020introduced into the genome of a cell using, for example, gene-editing or genetic engineering techniques, e.g., CRISPR-based editing techniques.

[0180] The terms “RNA-guided nuclease” and “RNA-guided nuclease molecule” are used interchangeably herein. In some embodiments, the RNA-guided nuclease is an RNA-guided DNA endonuclease enzyme. In some embodiments, the RNA-guided nuclease is a CRISPR nuclease. Non-limiting examples of RNA-guided nucleases are listed in Table 2 below, and the methods and compositions disclosed herein can use any combination of RNA-guided nucleases disclosed herein, or known to those of ordinary skill in the art. Those of ordinary skill in the art will be aware of additional nucleases and nuclease variants suitable for use in the context of the present disclosure, and it will be understood that the present disclosure is not limited in this respect.Table 2: Exemplary RNA-Guided NucleasesNuclease Length PAM Reference(aminoacid)SpCas9 1368 NGG Cong etal., Science. 2013;339(6121):819-23 SaCas9 1053 NNGRRT Ran etal., Nature. 2015; 520(7546): 186-91.(KKH) Kleinstivere / aZ, NatBiotechnol. 2015;33(12):1293- 1067 NNNRRTSaCas9 1298AsCasl2a1353 TTTV Zetsche etal., NatBiotechnol. 2017;35(l):31-34. (AsCpfl)LbCasl2a1274 TTTV Zetsche et al., Cell. 2015;163(3):759-71.(LbCpfl)CasX 980 TTC Burstein etal., Nature. 2017;542(7640):237-241. CasY 1200 TA Burstein etal., Nature. 2017;542(7640):237-241. Cas12hl 870 RTR Yan e / aZ, Science. 2019;363(6422):88-91.Cas12il 1093 TTN Yan e / aZ, Science. 2019;363(6422):88-91.Cas12cl unknown TG Yan e / aZ, Science. 2019;363(6422):88-91.Cas12c2 unknown TN Yan e / aZ, Science. 2019;363(6422):88-91. eSpCas9 1423 NGG Chen etal., Nature. 2017;550(7676):407-410. Cas9-HF1 1367 NGG Chen etal., Nature. 2017;550(7676):407-410. HypaCas9 1404 NGG Chen etal., Nature. 2017;550(7676):407-410. dCas9- 1623 NGG U. S. Patent No. 9,322,037FoklSniper- 1389 NGG Lee et al., Nat Commun. 2018;9(l):3048.Cas9NGG, NG,xCas9 1786 Wang etal., Plant Bi otechnol J. 2018; pbi.13053.GAA, GATAaCas12b 1129 TTN Teng e / a / . Cell Discov. 2018;4:63.evoCas9 1423 NGG Casini etal., NatBiotechnol. 2018;36(3):265-271.36ME1\59622102.v1Attorney Docket No. 126454-03020SpCas9- Nishimasu et al., Science. 2018;361(6408):1259- 1423 NGNG 1262.VRQR 1368 NGA Li et a!., The CRISPR Journal, 2018; 01:01 VRER 1372 NGCG Kleinstiver etal., Nature. 2016;529(7587):490-5. NmeCas9 1082 NNNGATT Amrani etal., Genome Biol. 2018; 19(1):214.CjCas9 984 NNNNRYA Kim etal., Nat Commun. 2017;8: 14500.BhCas12b 1108 ATTN Strecker et al., Nat Commun. 2019 Jan 22;10(l):212.BhCas12b 1108 ATTN Strecker et al., Nat Commun. 2019 Jan V4 22;10(l):212.CasO Pausch et al., Science 2020;369(6501):333-337.

[0181] Additional suitable RNA-guided nucleases, e.g., Cas9 and Cast 2 nucleases, will be apparent to the skilled artisan in view of the present disclosure, and the disclosure is not limited by the exemplary suitable nucleases provided herein. In some embodiment, a suitable nuclease is a Cas9 or Cas12a (Cpf1) nuclease. In some embodiments, the disclosure also embraces nuclease variants, e.g., Cas9 or Casl2a nuclease variants. A nuclease variant refers to a nuclease comprising an amino acid sequence characterized by one or more amino acid substitutions, deletions, or additions as compared to the wild type (WT) amino acid sequence of the nuclease. As used herein, AsCasl2a (WT) generally refers to the M537R, H800A, and F870L triple mutant (e.g., SEQ ID NO: 1348, but could also refer to ‘true’ wild-type (e.g., Uniprot Identifier U2UMQ6). Suitable nucleases and nuclease variants may also comprise purification tags (e.g., polyhistidine tags) and signaling peptides, e.g., comprising or consisting of a nuclear localization signal sequence (NLS). Some non-limiting examples of suitable nucleases and nuclease variants are described in more detail elsewhere herein, and also comprise those described in PCT application PCT / US2019 / 22374, filed March 14, 2019, and entitled “ Systems and Methods for the Treatment of Hemoglobinopathies,” the entire contents of which are incorporated herein by reference.

[0182] In some embodiments, the RNA-guided nuclease is a Streptococcus pyogenes Cas9 (SpCas9). In some embodiments, the RNA-guided nuclease is an SpCas9 variant. In some embodiments, the RNA-guided nuclease is an Acidaminococcus sp. Casl2a (AsCasl2a or AsCpfl). In some embodiments, the RNA-guided nuclease is an AsCasl2a variant. Suitable nuclease variants, including suitable SpCas9 or AsCasl2a variants will be known or apparent to those of ordinary skill in the art based on the present disclosure, and include, but are not limited to, the SpCas9 or AsCasl2a variants disclosed herein or otherwise known in the art. For example, in some embodiments, the RNA-guided nuclease is an Acidaminococcus sp.37ME1\59622102.v1Attorney Docket No. 126454-03020Casl2a RR variant (AsCasl2a-RR). In certain embodiments, the RNA-guided nuclease is a Cas12a RVR variant. These and other variants are described in PCT patent application PCT / US2017 / 028420 and Guo et al., NatBiotechnol. 2017 Aug; 35(8): 789-792, each of which is incorporated by reference herein for all purposes. Exemplary SpCas9 variants comprising mutations corresponding to the D10A mutation are depicted in SEQ ID NOs 1341, 1343, and 1346. Exemplary SpCas9 variants comprising mutations corresponding to the D10A / H840A mutation are depicted in SEQ ID NOs 1342, 1344, and 1347. Exemplary AsCas 12a RR variants (comprising mutations corresponding to the S542R / K607R mutations) are depicted in SEQ ID NOs: 50 and 51. Exemplary AsCasl2a RVR variants (comprising mutations corresponding to the S542R / K548V / N552R mutations) are depicted in SEQ ID NOs: 52 and 53. Additionally, or alternatively, suitable Casl2a variants include those having an M537R substitution, an H800A substitution, and / or an F870L substitution, or any combination thereof (numbering scheme according to AsCas 12a wild-type sequence).

[0183] In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a DNA modifying enzyme for targeted nucleotide alteration, commonly referred to as a “base editor.” In certain embodiments, the base editor is a cytosine base editor. In certain embodiments, the base editor is an adenosine base editor.

[0184] In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a DNA modifying enzyme fused to a reverse transcriptase for targeted nucleotide insertion, deletion, or substitution, commonly referred to as a “prime editor.”

[0185] The term “targeting moiety” or “targeting modification” refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment. Some exemplary targeting moieties include, but are not limited to, antibodies, antigens, carbohydrate base moieties, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. Carbohydrate based targeting moieties include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g., GalNAc? and GalNAcs; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, multivalent fucose, glycosylated polyaminoacids and lectins. The term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.38ME1\59622102.v1Attorney Docket No. 126454-03020

[0186] The terms “low-density lipoprotein receptor” and “LDLR” refer to gene ENSG00000130164 on Chromosome 19 of the Homo sapiens genome, or its accepted homologues in an animal species, i.e. animal as defined under “subject” above. These terms also refer to the protein coding product of this gene. LDLR (italicized) is used to denote nucleic acid (e.g. the chromosomal gene or an mRNA transcript thereof) and LDLR (not italicized) is used to denote the protein product of the gene.

[0187] The term “Icelandic mutation” refers to a 2.5 kb deletion in the 3 ’ UTR of the LDLR gene characterized in an Icelandic family (Bjornsson et al. Circ Genom Precis Med. 2021 Feb; 14(1): e003029 [Bjornsson], incorporated by reference herein).

[0188] As used herein, the term “functional edit” or “functional editing” refers to the type of molecular edit events required for LDLR upregulation, for example, excision (or deletion) and inversion events.2. Genome editing systems

[0189] Various genome editing systems known in the art can be used for the methods disclosed herein. Non-limiting examples of genome editing systems that can be used with the presently-disclosed subject matter include, but are not limited to CRISPR systems, zinc-finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, meganuclease (MN) systems, MegaTAL systems, other targeted endonuclease systems, and other chimeric endonuclease systems.

[0190] In certain embodiments, the genome editing system has RNA-guided DNA editing activity. In certain embodiments, the genome editing system includes at least two components adapted from naturally-occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex that is capable of associating with a specific nucleic acid sequence and optionally editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and / or a point mutation.

[0191] Naturally-occurring CRISPR systems are organized evolutionarily into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun; 9(6): 467-477 [Makarova], incorporated by reference herein), and while genome editing systems of the present disclosure can adapt components of any type or class of naturally-occurring CRISPR system, the embodiments presented herein are generally adapted from Class 2, and type II or V CRISPR systems. Class 2 systems, which encompass types II and V, are characterized by relatively large, multidomain RNA-guided nuclease proteins (e.g., Cas9 or Casl2a) and one or 39ME1\59622102.v1Attorney Docket No. 126454-03020more guide RNAs (e.g., a crRNA and, optionally, a tracrRNA) that form ribonucleoprotein (RNP) complexes that associate with ( / .<., target) and cleave specific loci complementary to a targeting (or spacer) sequence of the crRNA. Genome editing systems according to the present disclosure similarly target and optionally edit cellular DNA sequences but differ significantly from CRISPR systems occurring in nature. For example, the unimolecular guide RNAs described herein do not occur in nature, and both guide RNAs and RNA-guided nucleases according to this disclosure can incorporate any number of non-naturally occurring modifications.

[0192] Genome editing systems disclosed herein can be delivered into a cell by electroporation. Other non-viral approaches can also be employed for genome editing of target cells disclosed herein. For example, a nucleic acid molecule can be introduced into cells / subjects by administering the nucleic acid by lipofection (Feigner et al., Proc. Natl. Acad. Sci. U. S. A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Lipid nanoparticles (LNPs) or liposomes are also contemplated for delivery of nucleic acid molecules into a cell. In some embodiments, the genome editing systems disclosed herein are delivered in vivo to a subject by administration of lipid nanoparticles (LNPs) containing one or more components of the genome editing system.

[0193] Genome editing system disclosed herein can be delivered into subjects or cells using viral vectors, e.g., retroviral vectors, gamma-retroviral vectors or lentiviral vectors. Combinations of a retroviral vector and an appropriate packaging line are suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virusproducing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art. Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate40ME1\59622102.v1Attorney Docket No. 126454-03020growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0194] Genome editing systems can be implemented e.g., administered or delivered to a cell or a subject) in a variety of ways, and different implementations can be suitable for distinct applications. For instance, a genome editing system is implemented, in certain embodiments, as a protein / RNA complex (a ribonucleoprotein complex, or RNP complex), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and / or an encapsulating agent, such as a lipid or polymer micro- or nanoparticle, micelle, liposome, etc. In certain embodiments, a genome editing system is implemented as one or more nucleic acids encoding the RNA-guided nuclease and / or guide RNA components described above (optionally with one or more additional components). In certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus. In certain embodiments, the genome editing system is implemented as a combination of any of the foregoing. Additional or modified implementations that operate according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.

[0195] It should be noted that the genome editing systems of the present disclosure can be targeted to a single specific nucleotide sequence or can be targeted to — and capable of editing in parallel — two or more specific nucleotide sequences through the use of two or more guide RNAs. The use of multiple gRNAs is referred to as “multiplexing” throughout this disclosure, and can be employed to target multiple, unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain and, in some cases, to generate specific edits within such target domain. For example, International Patent Publication No. WO 2015 / 138510 by Maeder et al. (Maeder), which is incorporated by reference herein, describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene that results in the creation of a cryptic splice site, which in turn reduces or eliminates the function of the gene. The genome editing system of Maeder utilizes two guide RNAs targeted to sequences on either side of (i.e., flanking) the point mutation, and forms DSBs that flank the mutation. This, in turn, promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site, and restoring normal gene function.

[0196] Genome editing systems can, in some instances, form double strand breaks that are repaired by cellular DNA double-strand break mechanisms such as NHEJ or HDR. These mechanisms are described throughout the literature, for example by Davis & Maizels, PNAS,41ME1\59622102.v1Attorney Docket No. 126454-03020111(10):E924-932, March 11, 2014 (Davis) (describing Alt-HDR); Frit et al. DNA Repair 17(2014) 81-97 (Frit) (describing Alt-NHEJ); and Iyama and Wilson III, DNA Repair (Amst.) 2013-Aug; 12(8): 620-636 (Iyama) (describing canonical HDR and NHEJ pathways generally).

[0197] Where genome editing systems operate by forming DSBs, such systems optionally comprise one or more components that promote or facilitate a particular mode of double-strand break repair or a particular repair outcome. For instance, Cotta-Ramusino also describes genome editing systems in which a single stranded oligonucleotide “donor template” is added; the donor template is incorporated into a target region of cellular DNA that is cleaved by the genome editing system and can result in a change in the target sequence.

[0198] In certain embodiments, genome editing systems modify a target sequence, or modify expression of a gene in or near the target sequence, without causing double-strand breaks, e.g., by causing single-strand breaks or no cleavage ( / .<., no strand breaks). For example, a genome editing system can comprise an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression. As one example, an RNA-guided nuclease can be connected to (e.g., fused to) a cytidine deaminase functional domain, and can operate by generating targeted C-to-A substitutions. An RNA-guided nuclease can also, for example, be connected to (e.g. fused to) an adenosine deaminase functional domain. Exemplary nuclease / deaminase fusions are described in Komor et al. Nature 533, 420-424 (19 May 2016) (Komor) and Kantor et al., Int. J. Mol. Sci. 21(17) 6240 (2020), which are hereby incorporated by reference in their entirety. Further non-limiting examples of suitable base editors, variants thereof, and strategies for preparing RNA-guided nucleases comprising the same are described in PCT applications: PCT / US2020 / 016664, filed February 4, 2020; PCT / US2020 / 018192, filed February 13, 2020; PCT / US2020 / 049975, field September 9, 2020; PCT / US2022 / 012054, filed January 11, 2022; and PCT / US2022 / 078655, filed October 25, 2022, the entire contents of each of which are incorporated herein by reference.

[0199] Alternatively, a genome editing system can utilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a dead Cas9 (dCas9), and can operate by forming stable complexes on one or more targeted regions of cellular DNA, thereby recruiting other functional domains and / or interfering with functions involving the targeted region(s) including, without limitation, mRNA transcription, chromatin remodeling, among others.

[0200] In certain embodiments, the RNA-guided nucleases of the present disclosure can comprise a polymerase domain (e.g., a reverse transcriptase domain). In certain42ME1\59622102.v1Attorney Docket No. 126454-03020embodiments, the RNA-guided nuclease may use a gRNA with a primer binding sequence and / or a template for the polymerase domain.

[0201] In certain embodiments, the RNA-guided nuclease may be a prime editor (PE), where the PE is an RNA-guided nuclease with nickase activity that is fused to a reverse transcriptase domain. In certain embodiments, the PE may use a prime editing gRNA (pegRNA), where the pegRNA is a gRNA with a primer binding sequence (PBS) and a donor template, e.g, added at one of the termini, e.g, the 3' end. In certain embodiments, a PE:pegRNA complex binds to the target DNA, and the nickase domain of the prime editor nicks only one strand, generating a flap. The PBS, located on the pegRNA, binds to the DNA flap and the edited RNA sequence is reverse transcribed using the reverse transcriptase domain of the prime editor. The edited strand is incorporated into the DNA at the end of the nicked flap, and the target DNA is repaired with the new reverse transcribed DNA. The original DNA segment is removed by a cellular endonuclease. Additional methods employing RNA-guided nucleases and polymerases for template mediated gene editing are described in PCT publications: WO 2020 / 191233, WO 2020 / 191248, WO 2021226558, WO2023283246, WO 2023 / 235501, and WO 2023 / 076898, each of which are incorporated by reference for all purposes herein.3. Guide RNA (gRNA) molecules

[0202] The terms “guide molecule,” “guide RNA” and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 or a Cas12a (Cpf1) to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing). gRNAs and their component parts are described throughout the literature, for instance in Briner et al. (Molecular Cell 56(2), 333-339, October 23, 2014 (Briner), which is incorporated by reference), and in Cotta-Ramusino. The guide molecule can be an RNA molecule. The guide molecule can also comprise one or more nucleotides other than RNA nucleotides, for example, the guide molecule can be a DNA / RNA hybrid molecule, and / or the guide molecule can comprise one or more modified nucleotides (including, but not limited to, one or more modified DNA or RNA nucleotides).43ME1\59622102.v1Attorney Docket No. 126454-03020

[0203] In bacteria and archaea, type II CRISPR systems generally comprise an RNA-guided nuclease protein such as Cas9, a CRISPR RNA (crRNA) that comprises a 5’ region that is complementary to a foreign sequence, and a trans-activating crRNA (tracrRNA) that comprises a 5’ region that is complementary to, and forms a duplex with, a 3’ region of the crRNA. This duplex can facilitate the formation of — and is necessary for the activity of — the Cas / gRNA complex. As type II CRISPR systems were adapted for use in gene editing, it was discovered that the crRNA and tracrRNA could be joined into a single unimolecular or chimeric guide RNA, in one non-limiting example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3’ end) and the tracrRNA (at its 5’ end) (Mali et al. Science. 2013 Feb 15; 339(6121): 823-826 (Mali); Jiang et al. Nat Biotechnol. 2013 Mar; 31(3): 233-239 (Jiang); and Jinek et al., 2012 Science Aug. 17; 337(6096): 816-821 (Jinek), all of which are incorporated by reference herein.)

[0204] Guide RNAs, whether unimolecular or modular, comprise a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. Targeting domains are referred to by various names in the literature, including without limitation “guide sequences” (Hsu et al., Nat Biotechnol. 2013 Sep; 31(9): 827-832, (Hsu), incorporated by reference herein), “complementarity regions” (Cotta-Ramusino), “spacers” (Briner) and generically as “crRNAs” (Jiang). Irrespective of the names they are given, targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5’ terminus of in the case of a Cas9 gRNA, and at or near the 3’ terminus in the case of a Casl2a gRNA.

[0205] In addition to the targeting domains, gRNAs typically (but not necessarily, as discussed below) comprise a plurality of domains that can influence the formation or activity of gRNA / Cas9 complexes. For instance, as mentioned above, the duplexed structure formed by first and secondary complementarity domains of a gRNA (also referred to as a repeatantirepeat duplex) interacts with the recognition (REC) lobe of Cas9 and can mediate the formation of Cas9 / gRNA complexes. (Nishimasu et al., Cell 156, 935-949, February 27, 2014 (Nishimasu 2014) and Nishimasu et al., Cell 162, 1113-1126, August 27, 2015 (Nishimasu 2015), both incorporated by reference herein). It should be noted that the first and / or second complementarity domains can contain one or more poly-A tracts, which can be recognized by RNA polymerases as a termination signal. The sequence of the first and second complementarity domains are, therefore, optionally modified to eliminate these tracts and promote the complete in vitro transcription of gRNAs, for instance through the use of A-G 44ME1\59622102.v1Attorney Docket No. 126454-03020swaps as described in Briner, or A-U swaps. These and other similar modifications to the first and second complementarity domains are within the scope of the present disclosure.

[0206] Along with the first and second complementarity domains, Cas9 gRNAs typically comprise two or more additional duplexed regions that are involved in nuclease activity in vivo but not necessarily in vitro (Nishimasu 2015). A first stem-loop near the 3’ portion of the second complementarity domain is referred to variously as the “proximal domain” (Cotta-Ramusino), “stem loop 1” (Nishimasu 2014 and 2015), and the “nexus” (Briner). One or more additional stem loop structures are generally present near the 3’ end of the gRNA, with the number varying by species: S. pyogenes gRNAs typically comprise two 3’ stem loops (for a total of four stem loop structures including the repeat: anti -repeat duplex), while S. aureus and other species have only one (for a total of three stem loop structures). A description of conserved stem loop structures (and gRNA structures more generally) organized by species is provided in Briner.

[0207] While the foregoing description has focused on gRNAs for use with Cas9, it should be appreciated that other RNA-guided nucleases have been (or can in the future be) discovered or invented which utilize gRNAs that differ in some ways from those described to this point. For instance, Casl2a (also known as Cpfl; “CRISPR from Prevotella and Franciscella 1”) is an RNA-guided nuclease that does not require a tracrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771 October 22, 2015 (Zetsche I), incorporated by reference herein). A gRNA for use in a Casl2a genome editing system generally comprises a targeting domain and a complementarity domain (alternately referred to as a “handle”). It should also be noted that, in gRNAs for use with Casl2a, the targeting domain is usually present at or near the 3’ end, rather than the 5’ end as described above in connection with Cas9 gRNAs (the handle is at or near the 5’ end of a Casl2a gRNA).

[0208] Those of skill in the art will appreciate that, although structural differences can exist between gRNAs from different prokaryotic species, or between Casl2a and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that comprises one or more chemical modifications and / or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs can be described solely in terms of their targeting domain sequences.45ME1\59622102.v1Attorney Docket No. 126454-03020

[0209] More generally, skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA-guided nuclease, and not only those gRNAs that are compatible with a particular RNA-guided nuclease, e.g., a particular species of Cas9 or Casl2a. By way of illustration, the term gRNA can, in certain embodiments, comprise a gRNA for use with any RNA-guided nuclease occurring in a Class 2 CRISPR system, such as a type II or type V CRISPR system, or an RNA-guided nuclease derived or adapted therefrom.

[0210] In some embodiments, the guide RNA used comprises a modification as compared to the standard gRNA scaffold. Such modifications may comprise, for example, chemical modifications of a part of the gRNA, e.g., of a nucleobase or backbone moiety. In some embodiments, such a modification may also comprise the presence of one or more DNA nucleotide within the gRNA, e.g., within or outside of the targeting domain. In some embodiments, the modification may comprise an extension of the gRNA scaffold, e.g., by addition of 1-100 nucleotides, including RNA and / or DNA nucleotides at the 3’ or the 5’ terminus of the guide RNA, e.g., at the terminus distal to the targeting domain.

[0211] In certain embodiments, a gRNA complexed to an unmodified or modified Cas12a protein may be modified to increase the editing efficiency of a target nucleic acid. In certain embodiments, the modified gRNA may comprise one or more modifications including a phosphorothioate (PS2) linkage modification, a 2’-O-methyl modification (non-limiting exemplary modifications are illustrated in FIG. 3), one or more or a stretch of additional nucleotides (e.g., RNA or deoxyribonucleic acid (DNA) nucleotides) not found in a corresponding native gRNA (also referred herein as a “gRNA extension”), or combinations thereof.

[0212] In some embodiments, a gRNA used herein, e.g., a Casl2a gRNA, comprises one or more or a stretch of additional ribonucleic acid or deoxyribonucleic acid (DNA) bases outside of the spacer region, also referred to herein as a “gRNA extension.” In some embodiments, a gRNA used herein comprises a gRNA extension that comprises one or more or a stretch of DNA bases, referred to herein as a “DNA extension”. In some embodiments, a gRNA used herein comprises a DNA extension at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof. In certain embodiments, the DNA extension may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,46ME1\59622102.v1Attorney Docket No. 126454-0302056, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 DNA bases long. For example, in certain embodiments, the DNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 DNA bases long. In certain embodiments, the DNA extension may comprise one or more DNA bases selected from adenine (A), guanine (G), cytosine (C), or thymine (T). In certain embodiments, the DNA extension comprises the same DNA bases. For example, the DNA extension may comprise a stretch of adenine (A) bases. In certain embodiments, the DNA extension may comprise a stretch of thymine (T) bases. In certain embodiments, the DNA extension comprises a combination of different DNA bases. In certain embodiments, a DNA extension may comprise or consist of a sequence set forth in Table 3. In certain embodiments, a gRNA used herein comprises a DNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof. In certain embodiments, a gRNA including a DNA extension may comprise a sequence set forth in Table 3 that comprises a DNA extension. Without wishing to be bound by theory, it is contemplated that any DNA extension may be used herein, so long as it does not hybridize to the target nucleic acid being targeted by the gRNA. In some embodiments the DNA extension additionally exhibits an increase in editing efficiency, e.g., via changes to gRNA stability, uptake, and / or activity, at the target nucleic acid site relative to a gRNA which does not comprise such a DNA extension.

[0213] In some embodiments, a gRNA used herein, e.g., a Casl2a gRNA, comprises a gRNA extension that comprises one or more or a stretch of ribonucleic acid (RNA) bases, also referred to herein as an “RNA extension.” In some embodiments, a gRNA used herein comprises an RNA extension at the 5’ end of the gRNA, the 3’ end of the gRNA, or a combination thereof. In certain embodiments, the RNA extension may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 RNA bases long. For example, in certain embodiments, the RNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 RNA bases long. In certain embodiments, the RNA extension may comprise one or more RNA bases selected from adenine (rA), guanine (rG), cytosine (rC), or uracil (rU), in which the “r” represents RNA, 2’-hydroxy. In certain embodiments, the RNA extension comprises the same 47ME1\59622102.v1Attorney Docket No. 126454-03020RNA bases. For example, the RNA extension may comprise a stretch of adenine (rA) bases. In certain embodiments, the RNA extension comprises a combination of different RNA bases. In certain embodiments, an RNA extension may comprise or consist of a sequence set forth in Table 3. In certain embodiments, a gRNA used herein comprises an RNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof. In certain embodiments, a gRNA including an RNA extension may comprise a sequence set forth in Table 3 that comprises an RNA extension. gRNAs including an RNA extension at the 5’ end of the gRNA may comprise a sequence disclosed herein. gRNAs including an RNA extension at the 3 ’ end of the gRNA may comprise a sequence disclosed herein.

[0214] It is contemplated that gRNAs used herein may also comprise an RNA extension and a DNA extension. In certain embodiments, the RNA extension and DNA extension may both be at the 5’ end of the gRNA, the 3’ end of the gRNA, or a combination thereof. In certain embodiments, the RNA extension is at the 5’ end of the gRNA and the DNA extension is at the 3’ end of the gRNA. In certain embodiments, the RNA extension is at the 3’ end of the gRNA and the DNA extension is at the 5’ end of the gRNA.

[0215] It is further contemplated that gRNAs used herein may comprise a gRNA extension that is a hybrid extension that comprises both deoxyribonucleic acid and ribonucleic acid moi eties.

[0216] In some embodiments a gRNA which comprises a modification, e.g., a DNA extension at the 5’ end, is complexed with a RNA-guided nuclease, e.g., an AsCas 12a nuclease, to form an RNP complex, (such RNP complex formation occurring either prior to delivery of a composition described herein to a subject or following such delivery, e.g., in a cell after expression of an mRNA encoding the RNA-guided nuclease), which then edits a target cell (e.g., a liver cell). Exemplary suitable 5’ extensions for guide RNAs, e.g., Casl2a guide RNAs are provided in the table below:Table 3: gRNA 5’ ExtensionsSEQ 5’ extension sequence 5’ modification ID NO1 rCrUrUrUrU +5 RNA2 rArArGrArCrCrUrUrUrU +10 RNA48ME1\59622102.v1Attorney Docket No. 126454-030203 rArUrGrUrGrUrUrUrUrUrGrUrCrArArArArGrArCrCrUrUrUr +25 RNAU4 rArGrGrCrCrArGrCrUrUrGrCrCrGrGrUrUrUrUrUrUrArGrUr +60 RNA CrGrUrGrCrUrGrCrUrUrCrArUrGrUrGrUrUrUrUrUrGrUrCr ArArArArGrArCrCrUrUrUrU5 CTTTT +5 DNA6 AAGACCTTTT +10 DNA7 ATGTGTTTTTGTCAAAAGACCTTTT +25 DNA8 AGGCCAGCTTGCCGGTTTTTTAGTCGTGCTGCTTCATG +60 DNATG9 TTTTTGTCAAAAGACCTTTT +20 DNA10 GCTTCATGTGTTTTTGTCAAAAGACCTTTT +30 DNA11 GCCGGTTTTTTAGTCGTGCTGCTTCATGTGTTTTTGTCA +50 DNA AAAGACCTTTT12 TAGTCGTGCTGCTTCATGTGTTTTTGTCAAAAGACCTT +40 DNATT13 C*C*GAAGTTTTCTTCGGTTTT +20 DNA + 2xPS 14T*T*TTTCCGAAGTTTTCTTCGGTTTT+25 DNA + 2xPS 15 A*A* CGCTTTTTCCGAAGTTTTCTTCGGTTTT +30 DNA + 2xPS 16 G*C*GTTGTTTTCAACGCTTTTTCCGAAGTTTTCTTCGG +41 DNA + 2xPS TTTT17 G*G*CTTCTTTTGAAGCCTTTTTGCGTTGTTTTCAACGC +62 DNA + 2xPS TTTTTCCGAAGTTTTCTTCGGTTTT18 A*T*GTGTTTTTGTCAAAAGACCTTTT +25 DNA + 2xPS 19 AAAAAAAAAAAAAAAAAAAAAAAAA +25 A20 TTTTTTTTTTTTTTTTTTTTTTTTT +25 T21 mA*mU*rGrUrGrUrUrUrUrUrGrUrCrArArArArGrArCrCrUr +25 RNA + 2xPS UrUrU22 mA*mA*rAr Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar Ar PolyA RNA + ArArA 2xPS23 mU*mU*rUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUr PolyU RNA +UrUrU 2xPSAll bases are in upper case Lowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bonds except as noted:represents phosphorothioate modification“PS” represents phosphorothioate modification

[0217] Additional suitable gRNA modifications will be apparent to those of ordinary skill in the art based on the present disclosure. Suitable gRNA modifications comprise, for example, those described in PCT application PCT / US2018 / 054027, filed on Oct. 2, 2018, and entitled “MODIFIED CPF1 GUIDE RNA;” in PCT application PCT / US2015 / 000143, filed on Dec. 3, 2015, and entitled “GUIDE RNA WITH CHEMICAL MODIFICATIONS;” in PCT application PCT / US2016 / 026028, filed Apr. 5, 2016, and entitled “CHEMICALLY MODIFIED GUIDE RNAS FOR CRISPR / C AS-MEDIATED GENE REGULATION;” and in49ME1\59622102.v1Attorney Docket No. 126454-03020PCT application PCT / US2016 / 053344, filed on Sep. 23, 2016, and entitled “NUCLEASE-MEDIATED GENOME EDITING OF PRIMARY CELLS AND ENRICHMENT THEREOF;” the entire contents of each of which are incorporated herein by reference. Without being bound by theory, in certain embodiments of genome editing systems of the present disclosure, the one or more modifications of the gRNA enhance binding affinity of the gRNA molecule to RNA-guided nuclease of the genome editing system, e.g., a Casl2a nuclease.4. Guide RNA design

[0218] Methods for selection and validation of target sequences as well as off-target analyses have been described previously, e.g., in Mali; Hsu; Fu et al., 2014 Nat Biotechnol 32(3): 279-84, Heigwer et al., 2014 Nat methods 11(2): 122-3; Bae et al. (2014) Bioinformatics 30(10): 1473-5; Xiao A et al. (2014) Bioinformatics 30(8): 1180-1182; and Fennel et al (2021) CRISPR J 4(2): 264-274. Each of these references is incorporated by reference herein. In certain non-limiting embodiments, gRNA design can involve the use of a software tool to optimize the choice of potential target sequences corresponding to a user’s target sequence, e.g., to minimize total off-target activity across the genome. These and other guide selection methods are described in detail in Maeder and Cotta-Ramusino.

[0219] In certain embodiments, one or more or all of the nucleotides in a gRNA are modified. Strategies for modifying a gRNA are described in WO2019 / 152519, published Aug.8, 2019, the entire contents of which are expressly incorporated herein by reference.

[0220] Non-limiting examples of guide RNAs suitable for certain embodiments embraced by the present disclosure are provided herein, for example, in the Tables below. Those of ordinary skill in the art will be able to envision suitable guide RNA sequences for a specific nuclease, e.g., a Cas9 or Casl2a nuclease, from the disclosure of the targeting domain sequence, either as a DNA or RNA sequence. For example, a guide RNA comprising a targeting domain consisting of RNA nucleotides would comprise the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and thus contain uracil instead of thymidine nucleotides. For example, a guide RNA comprising a targeting domain sequence consisting of RNA nucleotides and described by the DNA sequence TTCCTTTCCTCGTGAAGGATA (SEQ ID NO: 552) would have a targeting domain of the corresponding RNA sequence rUrUrCrCrUrUrUrCrCrUrCrGrUrGrArArGrGrArUrA (SEQ ID NO: 610). As will be apparent to the skilled artisan, such a targeting domain would be linked to a suitable guide RNA scaffold, e.g., a crRNA scaffold sequence or a chimeric 50ME1\59622102.v1Attorney Docket No. 126454-03020crRNA / tracrRNA scaffold sequence. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For AsCasl2a, for example, a suitable scaffold sequence comprises the sequence rUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU (SEQ ID NO: 27), added to the 5’-terminus of the targeting domain. In the example above, this would result in a Casl2a guide RNA of the sequence; rUrUrCrCrUrUrUrCrCrUrCrGrUrGrArArGrGrArUrArUrArArUrUrUrCrUrArCrUrCrUrUrG rUrArGrArU (SEQ ID NO: 28). Those of skill in the art would further understand how to modify such a guide RNA. For example, adding a 25-mer DNA extension (e.g., SEQ ID NO: 7) would result, for example, in a guide RNA of the sequence ATGTGTTTTTGTCAAAAGACCTTTTrUrUrCrCrUrUrUrCrCrUrCrGrUrGrArArGrGrArU rArUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU (SEQ ID NO: 29). It will be understood that the exemplary targeting domain sequences provided herein are not limiting, and additional suitable sequences, e.g, variants of the specific sequences disclosed herein, will be apparent to the skilled artisan based on the present disclosure in view of the general knowledge in the art.

[0221] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting LDLR (LDLR gRNA). In some embodiments, LDLR cDNA sequence is SEQ ID NO: 1483: GTGCAATCGCGGGAAGCCAGGGTTTCCAGCTAGGACACAGCAGGTCGTGATCCG GGTCGGGACACTGCCTGGCAGAGGCTGCGAGCATGGGGCCCTGGGGCTGGAAAT TGCGCTGGACCGTCGCCTTGCTCCTCGCCGCGGCGGGGACTGCAGTGGGCGACA GATGCGAAAGAAACGAGTTCCAGTGCCAAGACGGGAAATGCATCTCCTACAAGT GGGTCTGCGATGGCAGCGCTGAGTGCCAGGATGGCTCTGATGAGTCCCAGGAGA CGTGCTTGTCTGTCACCTGCAAATCCGGGGACTTCAGCTGTGGGGGCCGTGTCAA CCGCTGCATTCCTCAGTTCTGGAGGTGCGATGGCCAAGTGGACTGCGACAACGG CTCAGACGAGCAAGGCTGTCCCCCCAAGACGTGCTCCCAGGACGAGTTTCGCTG CCACGATGGGAAGTGCATCTCTCGGCAGTTCGTCTGTGACTCAGACCGGGACTGC TTGGACGGCTCAGACGAGGCCTCCTGCCCGGTGCTCACCTGTGGTCCCGCCAGCT TCCAGTGCAACAGCTCCACCTGCATCCCCCAGCTGTGGGCCTGCGACAACGACCC CGACTGCGAAGATGGCTCGGATGAGTGGCCGCAGCGCTGTAGGGGTCTTTACGT GTTCCAAGGGGACAGTAGCCCCTGCTCGGCCTTCGAGTTCCACTGCCTAAGTGGC GAGTGCATCCACTCCAGCTGGCGCTGTGATGGTGGCCCCGACTGCAAGGACAAA TCTGACGAGGAAAACTGCGCTGTGGCCACCTGTCGCCCTGACGAATTCCAGTGCT CTGATGGAAACTGCATCCATGGCAGCCGGCAGTGTGACCGGGAATATGACTGCA51ME1\59622102.v1Attorney Docket No. 126454-03020AGGACATGAGCGATGAAGTTGGCTGCGTTAATGTGACACTCTGCGAGGGACCCA ACAAGTTCAAGTGTCACAGCGGCGAATGCATCACCCTGGACAAAGTCTGCAACA TGGCTAGAGACTGCCGGGACTGGTCAGATGAACCCATCAAAGAGTGCGGGACCA ACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGAT CGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATG CGAAGATATCGATGAGTGTCAGGATCCCGACACCTGCAGCCAGCTCTGCGTGAA CCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCA CACGAAGGCCTGCAAGGCTGTGGGCTCCATCGCCTACCTCTTCTTCACCAACCGG CACGAGGTCAGGAAGATGACGCTGGACCGGAGCGAGTACACCAGCCTCATCCCC AACCTGAGGAACGTGGTCGCTCTGGACACGGAGGTGGCCAGCAATAGAATCTAC TGGTCTGACCTGTCCCAGAGAATGATCTGCAGCACCCAGCTTGACAGAGCCCAC GGCGTCTCTTCCTATGACACCGTCATCAGCAGAGACATCCAGGCCCCCGACGGGC TGGCTGTGGACTGGATCCACAGCAACATCTACTGGACCGACTCTGTCCTGGGCAC TGTCTCTGTTGCGGATACCAAGGGCGTGAAGAGGAAAACGTTATTCAGGGAGAA CGGCTCCAAGCCAAGGGCCATCGTGGTGGATCCTGTTCATGGCTTCATGTACTGG ACTGACTGGGGAACTCCCGCCAAGATCAAGAAAGGGGGCCTGAATGGTGTGGAC ATCTACTCGCTGGTGACTGAAAACATTCAGTGGCCCAATGGCATCACCCTAGATC TCCTCAGTGGCCGCCTCTACTGGGTTGACTCCAAACTTCACTCCATCTCAAGCAT CGATGTCAACGGGGGCAACCGGAAGACCATCTTGGAGGATGAAAAGAGGCTGG CCCACCCCTTCTCCTTGGCCGTCTTTGAGGACAAAGTATTTTGGACAGATATCAT CAACGAAGCCATTTTCAGTGCCAACCGCCTCACAGGTTCCGATGTCAACTTGTTG GCTGAAAACCTACTGTCCCCAGAGGATATGGTTCTCTTCCACAACCTCACCCAGC CAAGAGGAGTGAACTGGTGTGAGAGGACCACCCTGAGCAATGGCGGCTGCCAGT ATCTGTGCCTCCCTGCCCCGCAGATCAACCCCCACTCGCCCAAGTTTACCTGCGC CTGCCCGGACGGCATGCTGCTGGCCAGGGACATGAGGAGCTGCCTCACAGAGGC TGAGGCTGCAGTGGCCACCCAGGAGACATCCACCGTCAGGCTAAAGGTCAGCTC CACAGCCGTAAGGACACAGCACACAACCACCCGACCTGTTCCCGACACCTCCCG GCTGCCTGGGGCCACCCCTGGGCTCACCACGGTGGAGATAGTGACAATGTCTCA CCAAGCTCTGGGCGACGTTGCTGGCAGAGGAAATGAGAAGAAGCCCAGTAGCGT GAGGGCTCTGTCCATTGTCCTCCCCATCGTGCTCCTCGTCTTCCTTTGCCTGGGGG TCTTCCTTCTATGGAAGAACTGGCGGCTTAAGAACATCAACAGCATCAACTTTGA CAACCCCGTCTATCAGAAGACCACAGAGGATGAGGTCCACATTTGCCACAACCA GGACGGCTACAGCTACCCCTCGAGACAGATGGTCAGTCTGGAGGATGACGTGGC GTGAACATCTGCCTGGAGTCCCGTCCCTGCCCAGAACCCTTCCTGAGACCTCGCC52ME1\59622102.v1Attorney Docket No. 126454-03020GGCCTTGTTTTATTCAAAGACAGAGAAGACCAAAGCATTGCCTGCCAGAGCTTTG TTTTATATATTTATTCATCTGGGAGGCAGAACAGGCTTCGGACAGTGCCCATGCA ATGGCTTGGGTTGGGATTTTGGTTTCTTCCTTTCCTCGTGAAGGATAAGAGAAAC AGGCCCGGGGGGACCAGGATGACACCTCCATTTCTCTCCAGGAAGTTTTGAGTTT CTCTCCACCGTGACACAATCCTCAAACATGGAAGATGAAAGGGGAGGGGATGTC AGGCCCAGAGAAGCAAGTGGCTTTCAACACACAACAGCAGATGGCACCAACGG GACCCCCTGGCCCTGCCTCATCCACCAATCTCTAAGCCAAACCCCTAAACTCAGG AGTCAACGTGTTTACCTCTTCTATGCAAGCCTTGCTAGACAGCCAGGTTAGCCTT TGCCCTGTCACCCCCGAATCATGACCCACCCAGTGTCTTTCGAGGTGGGTTTGTA CCTTCCTTAAGCCAGGAAAGGGATTCATGGCGTCGGAAATGATCTGGCTGAATCC GTGGTGGCACCGAGACCAAACTCATTCACCAAATGATGCCACTTCCCAGAGGCA GAGCCTGAGTCACTGGTCACCCTTAATATTTATTAAGTGCCTGAGACACCCGGTT ACCTTGGCCGTGAGGACACGTGGCCTGCACCCAGGTGTGGCTGTCAGGACACCA GCCTGGTGCCCATCCTCCCGACCCCTACCCACTTCCATTCCCGTGGTCTCCTTGCA CTTTCTCAGTTCAGAGTTGTACACTGTGTACATTTGGCATTTGTGTTATTATTTTG CACTGTTTTCTGTCGTGTGTGTTGGGATGGGATCCCAGGCCAGGGAAAGCCCGTG TCAATGAATGCCGGGGACAGAGAGGGGCAGGTTGACCGGGACTTCAAAGCCGTG ATCGTGAATATCGAGAACTGCCATTGTCGTCTTTATGTCCGCCCACCTAGTGCTTC CACTTCTATGCAAATGCCTCCAAGCCATTCACTTCCCCAATCTTGTCGTTGATGGG TATGTGTTTAAAACATGCACGGTGAGGCCGGGCGCAGTGGCTCACGCCTGTAATC CCAGCACTTTGGGAGGCCGAGGCGGGTGGATCATGAGGTCAGGAGATCGAGACC ATCCTGGCTAACACGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCG GGCGTGGTGGCGGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGA ATGGTGTGAACCCGGGAAGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCA GTCCGCAGTCTGGCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAC AAAAAAAAACCATGCATGGTGCATCAGCAGCCCATGGCCTCTGGCCAGGCATGG CGAGGCTGAGGTGGGAGGATGGTTTGAGCTCAGGCATTTGAGGCTGTCGTGAGC TATGATTATGCCACTGCTTTCCAGCCTGGGCAACATAGTAAGACCCCATCTCTTA AAAAATGAATTTGGCCAGACACAGGTGCCTCACGCCTGTAATCCCAGCACTTTGG GAGGCTGAGCTGGATCACTTGAGTTCAGGAGTTGGAGACCAGGCCTGAGCAACA AAGCGAGATCCCATCTCTACAAAAACCAAAAAGTTAAAAATCAGCTGGGTACGG TGGCACGTGCCTGTGATCCCAGCTACTTGGGAGGCTGAGGCAGGAGGATCGCCT GAGCCCAGGAGGTGGAGGTTGCAGTGAGCCATGATCGAGCCACTGCACTCCAGC CTGGGCAACAGATGAAGACCCTATTTCAGAAATACAACTATAAAAAAATAAATA53ME1\59622102.v1Attorney Docket No. 126454-03020AATCCTCCAGTCTGGATCGTTTGACGGGACTTCAGGTTCTTTCTGAAATCGCCGT GTTACTGTTGCACTGATGTCCGGAGAGACAGTGACAGCCTCCGTCAGACTCCCGC GTGAAGATGTCACAAGGGATTGGCAATTGTCCCCAGGGACAAAACACTGTGTCC CCCCCAGTGCAGGGAACCGTGATAAGCCTTTCTGGTTTCGGAGCACGTAAATGCG TCCCTGTACAGATAGTGGGGATTTTTTGTTATGTTTGCACTTTGTATATTGGTTGA AACTGTTATCACTTATATATATATATATACACACATATATATAAAATCTATTTATT TTTGCAAACCCTGGTTGCTGTATTTGTTCAGTGACTATTCTCGGGGCCCTGTGTAG GGGGTTATTGCCTCTGAAATGCCTCTTCTTTATGTACAAAGATTATTTGCACGAAC TGGACTGTGTGCAACGCTTTTTGGGAGAATGATGTCCCCGTTGTATGTATGAGTG GCTTCTGGGAGATGGGTGTCACTTTTTAAACCACTGTATAGAAGGTTTTTGTAGC CTGAATGTCTTACTGTGATCAATTAAATTTCTTAAATGAACCAA

[0222] In some embodiments, the target sequence of an LDLR gene comprises or consists of a nucleotide sequence set forth in SEQ ID NOs: 61-304; 549-606; 665-750; or 837-865 (Tables 4, 7, 10, 13). In certain embodiments, the target sequence of an LDLR gene comprises or consists of the nucleotide sequence set forth in SEQ ID NOs: 61, 72, 73, 74, 288, 300, 552, 553, 576, 585, 589, or 602. In certain embodiments, the target sequence of an LDLR gene comprises or consists of the nucleotide sequence set forth in SEQ ID NOs: 552 or 585. In some embodiments, the targeting domain of the gRNA molecule comprises or consists of a nucleotide sequence set forth in SEQ ID NOs: 305-548; 607-664; 751-836; or 866-894 (Tables 5, 8, 11, 14) In certain embodiments, the targeting domain of the gRNA targeting an LDLR gene is SEQ ID NO: 610, or SEQ ID NO: 643. In some embodiments, the gRNA molecule targeting an LDLR gene comprises or consists of a nucleotide sequence set forth in SEQ ID NOs: 900-1316, or 1331-1337 (Tables 6, 9, 12, and 15). In certain embodiments, the gRNA molecule targeting an LDLR gene comprises or consists of the sequence set forth in SEQ ID NOs: 900-913, 1114-1138, or 1139-1141. An exemplary LDLR gene target sequence, gRNA targeting domain, scaffold sequence, and DNA extension are set forth in Table 16A-16B.Table 4: LDLR Target Sequences for SpCas9SEQ ID NO Target Sequence (DNA)61 ACGTGGCGTGAACATCTGCC62 CTGGGCAGGGACGGGACTCC63 GAGGTCTCAGGAAGGGTTCT64 CGAGGTCTCAGGAAGGGTTC65 AACCCTTCCTGAGACCTCGC54ME1\59622102.v1Attorney Docket No. 126454-0302066 GGCCGGCGAGGTCTCAGGAA 67 AGGCCGGCGAGGTCTCAGGA 68 AACAAGGCCGGCGAGGTCTC 69 TGAATAAAACAAGGCCGGCG 70 GTCTTTGAATAAAACAAGGC 71 CTCTGTCTTTGAATAAAACA 72 AGCTCTGGCAGGCAATGCTT 73 ATATAAAACAAAGCTCTGGC 74 AAATATATAAAACAAAGCTC 75 ATTCATCTGGGAGGCAGAAC 76 CTGGGAGGCAGAACAGGCTT 77 TTCGGACAGTGCCCATGCAA 78 ACAGTGCCCATGCAATGGCT 79 CAGTGCCCATGCAATGGCTT 80 GCCCATGCAATGGCTTGGGT 81 CCCATGCAATGGCTTGGGTT 82 CCCAACCCAAGCCATTGCAT 83 TCCCAACCCAAGCCATTGCA 84 GTTTCTTCCTTTCCTCGTGA 85 CTCTTATCCTTCACGAGGAA 86 TGTTTCTCTTATCCTTCACG 87 AGGATAAGAGAAACAGGCCC 88 GGATAAGAGAAACAGGCCCG 89 GATAAGAGAAACAGGCCCGG 90 ATAAGAGAAACAGGCCCGGG 91 GAAACAGGCCCGGGGGGACC 92 GGTGTCATCCTGGTCCCCCC 93 AGGTGTCATCCTGGTCCCCC 94 GAGAAATGGAGGTGTCATCC 95 TGACACCTCCATTTCTCTCC 96 AACTTCCTGGAGAGAAATGG 97 CAAAACTTCCTGGAGAGAAA 98 GAGAGAAACTCAAAACTTCC 99 GTTTGAGGATTGTGTCACGG 100 CGTGACACAATCCTCAAACA 101 CATGTTTGAGGATTGTGTCA 102 CCTCAAACATGGAAGATGAA 103 CTCAAACATGGAAGATGAAA 104 TCAAACATGGAAGATGAAAG 105 CCTTTCATCTTCCATGTTTG 106 GTCAGGCCCAGAGAAGCAAG 107 TGAAAGCCACTTGCTTCTCT 108 TTGAAAGCCACTTGCTTCTC109 TTTCAACACACAACAGCAGA55ME1\59622102.v1Attorney Docket No. 126454-03020110 ACAACAGCAGATGGCACCAA 111 CAACAGCAGATGGCACCAAC 112 ATGGCACCAACGGGACCCCC 113 GCAGGGCCAGGGGGTCCCGT 114 GTGGATGAGGCAGGGCCAGG 115 AGAGATTGGTGGATGAGGCA 116 GGCTTAGAGATTGGTGGATG 117 GGGTTTGGCTTAGAGATTGG 118 TAGGGGTTTGGCTTAGAGAT 119 TAAGCCAAACCCCTAAACTC 120 GACTCCTGAGTTTAGGGGTT 121 ACGTTGACTCCTGAGTTTAG 122 CACGTTGACTCCTGAGTTTA 123 ACACGTTGACTCCTGAGTTT 124 GCAAGCCTTGCTAGACAGCC 125 GCTAACCTGGCTGTCTAGCA 126 TGACAGGGCAAAGGCTAACC 127 ATTCGGGGGTGACAGGGCAA 128 GTCATGATTCGGGGGTGACA 129 GGTCATGATTCGGGGGTGAC 130 CTGGGTGGGTCATGATTCGG 131 ACTGGGTGGGTCATGATTCG 132 CACTGGGTGGGTCATGATTC 133 ACACTGGGTGGGTCATGATT 134 GACCCACCCAGTGTCTTTCG 135 CCACCCAGTGTCTTTCGAGG 136 CACCCAGTGTCTTTCGAGGT 137 CACCTCGAAAGACACTGGGT 138 AACCCACCTCGAAAGACACT 139 AAACCCACCTCGAAAGACAC 140 GTTTGTACCTTCCTTAAGCC 141 TACCTTCCTTAAGCCAGGAA 142 ACCTTCCTTAAGCCAGGAAA 143 TCCCTTTCCTGGCTTAAGGA 144 TGAATCCCTTTCCTGGCTTA 145 TAAGCCAGGAAAGGGATTCA 146 AGGAAAGGGATTCATGGCGT 147 GACGCCATGAATCCCTTTCC 148 TCATGGCGTCGGAAATGATC 149 AAATGATCTGGCTGAATCCG 150 TGATCTGGCTGAATCCGTGG 151 GTTTGGTCTCGGTGCCACCA 152 TTGGTGAATGAGTTTGGTCT153 CATCATTTGGTGAATGAGTT56ME1\59622102.v1Attorney Docket No. 126454-03020154 CAAATGATGCCACTTCCCAG 155 CTCTGGGAAGTGGCATCATT 156 CAGGCTCTGCCTCTGGGAAG 157 AGAGGCAGAGCCTGAGTCAC 158 AGTGACTCAGGCTCTGCCTC 159 TTAAGGGTGACCAGTGACTC 160 CAGGCACTTAATAAATATTA 161 TATTAAGTGCCTGAGACACC 162 CCTGAGACACCCGGTTACCT 163 CCAAGGTAACCGGGTGTCTC 164 ACCCGGTTACCTTGGCCGTG 165 TCCTCACGGCCAAGGTAACC 166 GTCCTCACGGCCAAGGTAAC 167 ACCTTGGCCGTGAGGACACG 168 GCCACGTGTCCTCACGGCCA 169 GTGCAGGCCACGTGTCCTCA 170 AGGACACGTGGCCTGCACCC 171 ACGTGGCCTGCACCCAGGTG 172 TGCACCCAGGTGTGGCTGTC 173 TGACAGCCACACCTGGGTGC 174 GTGTCCTGACAGCCACACCT 175 GGTGTCCTGACAGCCACACC 176 TGGCTGTCAGGACACCAGCC 177 CGGGAGGATGGGCACCAGGC 178 GGGTCGGGAGGATGGGCACC 179 TGGGTAGGGGTCGGGAGGAT 180 GGAAGTGGGTAGGGGTCGGG 181 CCTACCCACTTCCATTCCCG 182 ACGGGAATGGAAGTGGGTAG 183 CACGGGAATGGAAGTGGGTA 184 CCACGGGAATGGAAGTGGGT 185 GAGACCACGGGAATGGAAGT 186 GGAGACCACGGGAATGGAAG 187 GTGCAAGGAGACCACGGGAA 188 AGAAAGTGCAAGGAGACCAC 189 GAGAAAGTGCAAGGAGACCA 190 CTCTGAACTGAGAAAGTGCA 191 AGTTGTACACTGTGTACATT 192 TGTTTTCTGTCGTGTGTGTT 193 TTCTGTCGTGTGTGTTGGGA 194 TCTGTCGTGTGTGTTGGGAT 195 GTGTGTTGGGATGGGATCCC 196 ACACGGGCTTTCCCTGGCCT197 GACACGGGCTTTCCCTGGCC57ME1\59622102.v1Attorney Docket No. 126454-03020198 TCATTGACACGGGCTTTCCC 199 AAGCCCGTGTCAATGAATGC 200 AGCCCGTGTCAATGAATGCC 201 GCCCGTGTCAATGAATGCCG 202 TCCCCGGCATTCATTGACAC 203 GTCCCCGGCATTCATTGACA 204 AATGAATGCCGGGGACAGAG 205 ATGAATGCCGGGGACAGAGA 206 TGCCGGGGACAGAGAGGGGC 207 AACCTGCCCCTCTCTGTCCC 208 ACAGAGAGGGGCAGGTTGAC 209 CAGAGAGGGGCAGGTTGACC 210 CGATCACGGCTTTGAAGTCC 211 GTTCTCGATATTCACGATCA 212 GGCGGACATAAAGACGACAA 213 AAGTGGAAGCACTAGGTGGG 214 TAGAAGTGGAAGCACTAGGT 215 ATAGAAGTGGAAGCACTAGG 216 TGCATAGAAGTGGAAGCACT 217 TGGAGGCATTTGCATAGAAG 218 TGGGGAAGTGAATGGCTTGG 219 GATTGGGGAAGTGAATGGCT 220 GACAAGATTGGGGAAGTGAA 221 TTCCCCAATCTTGTCGTTGA 222 TCCCCAATCTTGTCGTTGAT 223 ACCCATCAACGACAAGATTG 224 TACCCATCAACGACAAGATT 225 GTATGTGTTTAAAACATGCA 226 TGTTTAAAACATGCACGGTG 227 TAAAACATGCACGGTGAGGC 228 AAAACATGCACGGTGAGGCC 229 GGCTGCTGATGCACCATGCA 230 CATGGTGCATCAGCAGCCCA 231 CATCAGCAGCCCATGGCCTC 232 CATGCCTGGCCAGAGGCCAT 233 GCCTCTGGCCAGGCATGGCG 234 GCCTCGCCATGCCTGGCCAG 235 CATCTCTTAAAAAATGAATT 236 AAATGAATTTGGCCAGACAC 237 TATAGTTGTATTTCTGAAAT 238 CTCCAGTCTGGATCGTTTGA 239 TCCAGTCTGGATCGTTTGAC 240 CGTCAAACGATCCAGACTGG241 TCCCGTCAAACGATCCAGAC58ME1\59622102.v1Attorney Docket No. 126454-03020242 GGATCGTTTGACGGGACTTC 243 ACATCAGTGCAACAGTAACA 244 GGAGGCTGTCACTGTCTCTC 245 TTCACGCGGGAGTCTGACGG 246 ATCTTCACGCGGGAGTCTGA 247 TCCCGCGTGAAGATGTCACA 248 CCCGCGTGAAGATGTCACAA 249 CCCTTGTGACATCTTCACGC 250 TCCCTTGTGACATCTTCACG 251 GTGAAGATGTCACAAGGGAT 252 AGGGATTGGCAATTGTCCCC 253 GGGATTGGCAATTGTCCCCA 254 GACACAGTGTTTTGTCCCTG 255 GGACACAGTGTTTTGTCCCT 256 GGGACACAGTGTTTTGTCCC 257 CACTGTGTCCCCCCCAGTGC 258 ACTGTGTCCCCCCCAGTGCA 259 CACGGTTCCCTGCACTGGGG 260 TCACGGTTCCCTGCACTGGG 261 ATCACGGTTCCCTGCACTGG 262 TATCACGGTTCCCTGCACTG 263 TTATCACGGTTCCCTGCACT 264 CTTATCACGGTTCCCTGCAC 265 GGAACCGTGATAAGCCTTTC 266 GTGATAAGCCTTTCTGGTTT 267 GAAACCAGAAAGGCTTATCA 268 TACGTGCTCCGAAACCAGAA 269 ATGCGTCCCTGTACAGATAG 270 TGCGTCCCTGTACAGATAGT 271 GCGTCCCTGTACAGATAGTG 272 AAATCCCCACTATCTGTACA 273 AAAATCCCCACTATCTGTAC 274 TATGTTTGCACTTTGTATAT 275 CTATTTATTTTTGCAAACCC 276 CTGAACAAATACAGCAACCA 277 ACTGAACAAATACAGCAACC 278 ATTTGTTCAGTGACTATTCT 279 TTTGTTCAGTGACTATTCTC 280 TTGTTCAGTGACTATTCTCG 281 CTATTCTCGGGGCCCTGTGT 282 TATTCTCGGGGCCCTGTGTA 283 ATTCTCGGGGCCCTGTGTAG 284 TTCTCGGGGCCCTGTGTAGG285 AGGCAATAACCCCCTACACA59ME1\59622102.v1Attorney Docket No. 126454-03020286 GAGGCAATAACCCCCTACAC 287 ATAAAGAAGAGGCATTTCAG 288 AATCTTTGTACATAAAGAAG 289 CAAAGATTATTTGCACGAAC 290 GGACTGTGTGCAACGCTTTT 291 GACTGTGTGCAACGCTTTTT 292 GTCCCCGTTGTATGTATGAG 293 AGCCACTCATACATACAACG 294 AAGCCACTCATACATACAAC 295 GAAGCCACTCATACATACAA 296 TTGTATGTATGAGTGGCTTC 297 TGTATGTATGAGTGGCTTCT 298 TATGAGTGGCTTCTGGGAGA 299 ATGAGTGGCTTCTGGGAGAT 300 TACAAAAACCTTCTATACAG 301 TTGATCACAGTAAGACATTC 302 TGCATCGAGTTTAGACAAAT 303 TTAGCGTTGGCCACCCGCGG 304 AAATTAGCGTTGGCCACCCG 1317 TTGAAATTCGAATTCTGCCT 1318 GTGCGTTGCTTTGAGTGGGT 1365 ACAGATGACAGAAGGAAGTG 1366 TTCCTTCTGTCATCTGTTTG 1367 TCTGTCATCTGTTTGTGGAT 1368 ATCCACAAACAGATGACAGA 1369 GTTTGTGGATTGGACTCCCC 1370 GGATTGGACTCCCCAGGAGA 1371 GGGAAGACTCCCAGAACTCC 1372 GTGTATGAATGGATGAGCAG 1373 AGTGTATGAATGGATGAGCA 1374 ATATTTGTCGAGTGTATGAA 1375 CACAAGCCTACGGCAGAGCA 1376 GTAGGCTTGTGGGACACTAC 1377 TAGGCTTGTGGGACACTACA 1378 TGTAGTGTCCCACAAGCCTA 1379 AGGCTTGTGGGACACTACAG 1380 AAAGAAATGAAGTCTTGATA 1381 GAAATGAAGTCTTGATACGG 1382 AATCTCCCTGTCTGTCCTTA 1383 TCAGGTCCTTAAGGACAGAC 1384 AAAATGATGTCAGGTCCTTA 1385 CCTCAAGTCGTCTACCGCCT 1386 CCGAGGCGGTAGACGACTTG1387 TTCTCTAATGAAGTAACATT60ME1\59622102.v1Attorney Docket No. 126454-030201388 CAGAAAAGCAACTGGCAGGA 1389 GGGACAGAAAAGCAACTGGC 1390 TATGGGGACAGAAAAGCAAC 1391 TTTTGTATCTATTCTAAAAA1392 TGTATCTATTCTAAAAATGG1461 GAGCAACACAGAGCCCAGGGTable 5: LDLR Targeting Sequences for SpCas9SEQ ID NO gDNA targeting Domain (RNA)305 TArCrGrUrGrGrCrGrUrGrArArCrArUrCrUrGrCrC 306 TCrUrGrGrGrCrArGrGrGrArCrGrGrGrArCrUrCrC 307 TGrArGrGrUrCrUrCrArGrGrArArGrGrGrUrUrCrU 308 TCrGrArGrGrUrCrUrCrArGrGrArArGrGrGrUrUrC 309 TArArCrCrCrUrUrCrCrUrGrArGrArCrCrUrCrGrC 310 TGrGrCrCrGrGrCrGrArGrGrUrCrUrCrArGrGrArA 311 TArGrGrCrCrGrGrCrGrArGrGrUrCrUrCrArGrGrA 312 TArArCrArArGrGrCrCrGrGrCrGrArGrGrUrCrUrC 313 TUrGrArArUrArArArArCrArArGrGrCrCrGrGrCrG 314 TGrUrCrUrUrUrGrArArUrArArArArCrArArGrGrC 315 TCrUrCrUrGrUrCrUrUrUrGrArArUrArArArArCrA 316 TArGrCrUrCrUrGrGrCrArGrGrCrArArUrGrCrUrU 317 TArUrArUrArArArArCrArArArGrCrUrCrUrGrGrC 318 TArArArUrArUrArUrArArArArCrArArArGrCrUrC 319 TArUrUrCrArUrCrUrGrGrGrArGrGrCrArGrArArC 320 TCrUrGrGrGrArGrGrCrArGrArArCrArGrGrCrUrU 321 TUrUrCrGrGrArCrArGrUrGrCrCrCrArUrGrCrArA 322 TArCrArGrUrGrCrCrCrArUrGrCrArArUrGrGrCrU 323 TCrArGrUrGrCrCrCrArUrGrCrArArUrGrGrCrUrU 324 TGrCrCrCrArUrGrCrArArUrGrGrCrUrUrGrGrGrU 325 TCrCrCrArUrGrCrArArUrGrGrCrUrUrGrGrGrUrU 326 TCrCrCrArArCrCrCrArArGrCrCrArUrUrGrCrArU 327 TUrCrCrCrArArCrCrCrArArGrCrCrArUrUrGrCrA 328 rGrUrUrUrCrUrUrCrCrUrUrUrCrCrUrCrGrUrGrA 329 TCrUrCrUrUrArUrCrCrUrUrCrArCrGrArGrGrArA 330 rUrGrUrUrUrCrUrCrUrUrArUrCrCrUrUrCrArCrG 331 TArGrGrArUrArArGrArGrArArArCrArGrGrCrCrC 332 TGrGrArUrArArGrArGrArArArCrArGrGrCrCrCrG 333 TGrArUrArArGrArGrArArArCrArGrGrCrCrCrGrG 334 TArUrArArGrArGrArArArCrArGrGrCrCrCrGrGrG 335 TGrArArArCrArGrGrCrCrCrGrGrGrGrGrGrArCrC 336 rGrGrUrGrUrCrArUrCrCrUrGrGrUrCrCrCrCrCrC337 TArGrGrUrGrUrCrArUrCrCrUrGrGrUrCrCrCrCrC61ME1\59622102.v1Attorney Docket No. 126454-03020338 TGrArGrArArArUrGrGrArGrGrUrGrUrCrArUrCrC 339 rUrGrArCrArCrCrUrCrCrArUrUrUrCrUrCrUrCrC 340 TArArCrUrUrCrCrUrGrGrArGrArGrArArArUrGrG 341 TCrArArArArCrUrUrCrCrUrGrGrArGrArGrArArA 342 TGrArGrArGrArArArCrUrCrArArArArCrUrUrCrC 343 TGrUrUrUrGrArGrGrArUrUrGrUrGrUrCrArCrGrG 344 TCrGrUrGrArCrArCrArArUrCrCrUrCrArArArCrA 345 TCrArUrGrUrUrUrGrArGrGrArUrUrGrUrGrUrCrA 346 TCrCrUrCrArArArCrArUrGrGrArArGrArUrGrArA 347 TCrUrCrArArArCrArUrGrGrArArGrArUrGrArArA 348 TUrCrArArArCrArUrGrGrArArGrArUrGrArArArG 349 rCrCrUrUrUrCrArUrCrUrUrCrCrArUrGrUrUrUrG 350 TGrUrCrArGrGrCrCrCrArGrArGrArArGrCrArArG 351 TUrGrArArArGrCrCrArCrUrUrGrCrUrUrCrUrCrU 352 TUrUrGrArArArGrCrCrArCrUrUrGrCrUrUrCrUrC 353 TUrUrUrCrArArCrArCrArCrArArCrArGrCrArGrA 354 TArCrArArCrArGrCrArGrArUrGrGrCrArCrCrArA 355 TCrArArCrArGrCrArGrArUrGrGrCrArCrCrArArC 356 TArUrGrGrCrArCrCrArArCrGrGrGrArCrCrCrCrC 357 TGrCrArGrGrGrCrCrArGrGrGrGrGrUrCrCrCrGrU 358 TGrUrGrGrArUrGrArGrGrCrArGrGrGrCrCrArGrG 359 TArGrArGrArUrUrGrGrUrGrGrArUrGrArGrGrCrA 360 TGrGrCrUrUrArGrArGrArUrUrGrGrUrGrGrArUrG 361 TGrGrGrUrUrUrGrGrCrUrUrArGrArGrArUrUrGrG 362 TUrArGrGrGrGrUrUrUrGrGrCrUrUrArGrArGrArU 363 TUrArArGrCrCrArArArCrCrCrCrUrArArArCrUrC 364 TGrArCrUrCrCrUrGrArGrUrUrUrArGrGrGrGrUrU 365 TArCrGrUrUrGrArCrUrCrCrUrGrArGrUrUrUrArG 366 TCrArCrGrUrUrGrArCrUrCrCrUrGrArGrUrUrUrA 367 TArCrArCrGrUrUrGrArCrUrCrCrUrGrArGrUrUrU 368 TGrCrArArGrCrCrUrUrGrCrUrArGrArCrArGrCrC 369 TGrCrUrArArCrCrUrGrGrCrUrGrUrCrUrArGrCrA 370 TUrGrArCrArGrGrGrCrArArArGrGrCrUrArArCrC 371 TArUrUrCrGrGrGrGrGrUrGrArCrArGrGrGrCrArA 372 TGrUrCrArUrGrArUrUrCrGrGrGrGrGrUrGrArCrA 373 TGrGrUrCrArUrGrArUrUrCrGrGrGrGrGrUrGrArC 374 TCrUrGrGrGrUrGrGrGrUrCrArUrGrArUrUrCrGrG 375 TArCrUrGrGrGrUrGrGrGrUrCrArUrGrArUrUrCrG 376 TCrArCrUrGrGrGrUrGrGrGrUrCrArUrGrArUrUrC 377 TArCrArCrUrGrGrGrUrGrGrGrUrCrArUrGrArUrU 378 TGrArCrCrCrArCrCrCrArGrUrGrUrCrUrUrUrCrG379 TCrCrArCrCrCrArGrUrGrUrCrUrUrUrCrGrArGrG62ME1\59622102.v1Attorney Docket No. 126454-03020380 rCrArCrCrCrArGrUrGrUrCrUrUrUrCrGrArGrGrU 381 TCrArCrCrUrCrGrArArArGrArCrArCrUrGrGrGrU 382 TArArCrCrCrArCrCrUrCrGrArArArGrArCrArCrU 383 TArArArCrCrCrArCrCrUrCrGrArArArGrArCrArC 384 rGrUrUrUrGrUrArCrCrUrUrCrCrUrUrArArGrCrC 385 TUrArCrCrUrUrCrCrUrUrArArGrCrCrArGrGrArA 386 TArCrCrUrUrCrCrUrUrArArGrCrCrArGrGrArArA 387 TUrCrCrCrUrUrUrCrCrUrGrGrCrUrUrArArGrGrA 388 rUrGrArArUrCrCrCrUrUrUrCrCrUrGrGrCrUrUrA 389 TUrArArGrCrCrArGrGrArArArGrGrGrArUrUrCrA 390 TArGrGrArArArGrGrGrArUrUrCrArUrGrGrCrGrU 391 TGrArCrGrCrCrArUrGrArArUrCrCrCrUrUrUrCrC 392 TUrCrArUrGrGrCrGrUrCrGrGrArArArUrGrArUrC 393 TArArArUrGrArUrCrUrGrGrCrUrGrArArUrCrCrG 394 TUrGrArUrCrUrGrGrCrUrGrArArUrCrCrGrUrGrG 395 TGrUrUrUrGrGrUrCrUrCrGrGrUrGrCrCrArCrCrA 396 TUrUrGrGrUrGrArArUrGrArGrUrUrUrGrGrUrCrU 397 TCrArUrCrArUrUrUrGrGrUrGrArArUrGrArGrUrU 398 TCrArArArUrGrArUrGrCrCrArCrUrUrCrCrCrArG 399 TCrUrCrUrGrGrGrArArGrUrGrGrCrArUrCrArUrU 400 TCrArGrGrCrUrCrUrGrCrCrUrCrUrGrGrGrArArG 401 TArGrArGrGrCrArGrArGrCrCrUrGrArGrUrCrArC 402 TArGrUrGrArCrUrCrArGrGrCrUrCrUrGrCrCrUrC 403 TUrUrArArGrGrGrUrGrArCrCrArGrUrGrArCrUrC 404 TCrArGrGrCrArCrUrUrArArUrArArArUrArUrUrA 405 TUrArUrUrArArGrUrGrCrCrUrGrArGrArCrArCrC 406 TCrCrUrGrArGrArCrArCrCrCrGrGrUrUrArCrCrU 407 TCrCrArArGrGrUrArArCrCrGrGrGrUrGrUrCrUrC 408 TArCrCrCrGrGrUrUrArCrCrUrUrGrGrCrCrGrUrG 409 TUrCrCrUrCrArCrGrGrCrCrArArGrGrUrArArCrC 410 TGrUrCrCrUrCrArCrGrGrCrCrArArGrGrUrArArC 411 TArCrCrUrUrGrGrCrCrGrUrGrArGrGrArCrArCrG 412 TGrCrCrArCrGrUrGrUrCrCrUrCrArCrGrGrCrCrA 413 TGrUrGrCrArGrGrCrCrArCrGrUrGrUrCrCrUrCrA 414 TArGrGrArCrArCrGrUrGrGrCrCrUrGrCrArCrCrC 415 TArCrGrUrGrGrCrCrUrGrCrArCrCrCrArGrGrUrG 416 TUrGrCrArCrCrCrArGrGrUrGrUrGrGrCrUrGrUrC 417 TUrGrArCrArGrCrCrArCrArCrCrUrGrGrGrUrGrC 418 TGrUrGrUrCrCrUrGrArCrArGrCrCrArCrArCrCrU 419 TGrGrUrGrUrCrCrUrGrArCrArGrCrCrArCrArCrC 420 TUrGrGrCrUrGrUrCrArGrGrArCrArCrCrArGrCrC421 TCrGrGrGrArGrGrArUrGrGrGrCrArCrCrArGrGrC63ME1\59622102.v1Attorney Docket No. 126454-03020422 TGrGrGrUrCrGrGrGrArGrGrArUrGrGrGrCrArCrC 423 TUrGrGrGrUrArGrGrGrGrUrCrGrGrGrArGrGrArU 424 TGrGrArArGrUrGrGrGrUrArGrGrGrGrUrCrGrGrG 425 rCrCrUrArCrCrCrArCrUrUrCrCrArUrUrCrCrCrG 426 TArCrGrGrGrArArUrGrGrArArGrUrGrGrGrUrArG 427 TCrArCrGrGrGrArArUrGrGrArArGrUrGrGrGrUrA 428 TCrCrArCrGrGrGrArArUrGrGrArArGrUrGrGrGrU 429 TGrArGrArCrCrArCrGrGrGrArArUrGrGrArArGrU 430 TGrGrArGrArCrCrArCrGrGrGrArArUrGrGrArArG 431 TGrUrGrCrArArGrGrArGrArCrCrArCrGrGrGrArA 432 TArGrArArArGrUrGrCrArArGrGrArGrArCrCrArC 433 TGrArGrArArArGrUrGrCrArArGrGrArGrArCrCrA 434 TCrUrCrUrGrArArCrUrGrArGrArArArGrUrGrCrA 435 TArGrUrUrGrUrArCrArCrUrGrUrGrUrArCrArUrU 436 rUrGrUrUrUrUrCrUrGrUrCrGrUrGrUrGrUrGrUrU 437 TUrUrCrUrGrUrCrGrUrGrUrGrUrGrUrUrGrGrGrA 438 TUrCrUrGrUrCrGrUrGrUrGrUrGrUrUrGrGrGrArU 439 TGrUrGrUrGrUrUrGrGrGrArUrGrGrGrArUrCrCrC 440 TArCrArCrGrGrGrCrUrUrUrCrCrCrUrGrGrCrCrU 441 TGrArCrArCrGrGrGrCrUrUrUrCrCrCrUrGrGrCrC 442 TUrCrArUrUrGrArCrArCrGrGrGrCrUrUrUrCrCrC 443 TArArGrCrCrCrGrUrGrUrCrArArUrGrArArUrGrC 444 TArGrCrCrCrGrUrGrUrCrArArUrGrArArUrGrCrC 445 TGrCrCrCrGrUrGrUrCrArArUrGrArArUrGrCrCrG 446 TUrCrCrCrCrGrGrCrArUrUrCrArUrUrGrArCrArC 447 TGrUrCrCrCrCrGrGrCrArUrUrCrArUrUrGrArCrA 448 TArArUrGrArArUrGrCrCrGrGrGrGrArCrArGrArG 449 TArUrGrArArUrGrCrCrGrGrGrGrArCrArGrArGrA 450 TUrGrCrCrGrGrGrGrArCrArGrArGrArGrGrGrGrC 451 rArArCrCrUrGrCrCrCrCrUrCrUrCrUrGrUrCrCrC 452 TArCrArGrArGrArGrGrGrGrCrArGrGrUrUrGrArC 453 TCrArGrArGrArGrGrGrGrCrArGrGrUrUrGrArCrC 454 TCrGrArUrCrArCrGrGrCrUrUrUrGrArArGrUrCrC 455 TGrUrUrCrUrCrGrArUrArUrUrCrArCrGrArUrCrA 456 TGrGrCrGrGrArCrArUrArArArGrArCrGrArCrArA 457 TArArGrUrGrGrArArGrCrArCrUrArGrGrUrGrGrG 458 TUrArGrArArGrUrGrGrArArGrCrArCrUrArGrGrU 459 TArUrArGrArArGrUrGrGrArArGrCrArCrUrArGrG 460 TUrGrCrArUrArGrArArGrUrGrGrArArGrCrArCrU 461 TUrGrGrArGrGrCrArUrUrUrGrCrArUrArGrArArG 462 TUrGrGrGrGrArArGrUrGrArArUrGrGrCrUrUrGrG463 TGrArUrUrGrGrGrGrArArGrUrGrArArUrGrGrCrU64ME1\59622102.v1Attorney Docket No. 126454-03020464 TGrArCrArArGrArUrUrGrGrGrGrArArGrUrGrArA 465 rUrUrCrCrCrCrArArUrCrUrUrGrUrCrGrUrUrGrA 466 rUrCrCrCrCrArArUrCrUrUrGrUrCrGrUrUrGrArU 467 TArCrCrCrArUrCrArArCrGrArCrArArGrArUrUrG 468 TUrArCrCrCrArUrCrArArCrGrArCrArArGrArUrU 469 TGrUrArUrGrUrGrUrUrUrArArArArCrArUrGrCrA 470 TUrGrUrUrUrArArArArCrArUrGrCrArCrGrGrUrG 471 TUrArArArArCrArUrGrCrArCrGrGrUrGrArGrGrC 472 TArArArArCrArUrGrCrArCrGrGrUrGrArGrGrCrC 473 TGrGrCrUrGrCrUrGrArUrGrCrArCrCrArUrGrCrA 474 TCrArUrGrGrUrGrCrArUrCrArGrCrArGrCrCrCrA 475 TCrArUrCrArGrCrArGrCrCrCrArUrGrGrCrCrUrC 476 TCrArUrGrCrCrUrGrGrCrCrArGrArGrGrCrCrArU 477 TGrCrCrUrCrUrGrGrCrCrArGrGrCrArUrGrGrCrG 478 TGrCrCrUrCrGrCrCrArUrGrCrCrUrGrGrCrCrArG 479 TCrArUrCrUrCrUrUrArArArArArArUrGrArArUrU 480 TArArArUrGrArArUrUrUrGrGrCrCrArGrArCrArC 481 TUrArUrArGrUrUrGrUrArUrUrUrCrUrGrArArArU 482 TCrUrCrCrArGrUrCrUrGrGrArUrCrGrUrUrUrGrA 483 TUrCrCrArGrUrCrUrGrGrArUrCrGrUrUrUrGrArC 484 TCrGrUrCrArArArCrGrArUrCrCrArGrArCrUrGrG 485 TUrCrCrCrGrUrCrArArArCrGrArUrCrCrArGrArC 486 TGrGrArUrCrGrUrUrUrGrArCrGrGrGrArCrUrUrC 487 TArCrArUrCrArGrUrGrCrArArCrArGrUrArArCrA 488 TGrGrArGrGrCrUrGrUrCrArCrUrGrUrCrUrCrUrC 489 TUrUrCrArCrGrCrGrGrGrArGrUrCrUrGrArCrGrG 490 TArUrCrUrUrCrArCrGrCrGrGrGrArGrUrCrUrGrA 491 TUrCrCrCrGrCrGrUrGrArArGrArUrGrUrCrArCrA 492 TCrCrCrGrCrGrUrGrArArGrArUrGrUrCrArCrArA 493 rCrCrCrUrUrGrUrGrArCrArUrCrUrUrCrArCrGrC 494 rUrCrCrCrUrUrGrUrGrArCrArUrCrUrUrCrArCrG 495 TGrUrGrArArGrArUrGrUrCrArCrArArGrGrGrArU 496 TArGrGrGrArUrUrGrGrCrArArUrUrGrUrCrCrCrC 497 TGrGrGrArUrUrGrGrCrArArUrUrGrUrCrCrCrCrA 498 TGrArCrArCrArGrUrGrUrUrUrUrGrUrCrCrCrUrG 499 TGrGrArCrArCrArGrUrGrUrUrUrUrGrUrCrCrCrU 500 TGrGrGrArCrArCrArGrUrGrUrUrUrUrGrUrCrCrC 501 rCrArCrUrGrUrGrUrCrCrCrCrCrCrCrArGrUrGrC 502 TArCrUrGrUrGrUrCrCrCrCrCrCrCrArGrUrGrCrA 503 TCrArCrGrGrUrUrCrCrCrUrGrCrArCrUrGrGrGrG 504 TUrCrArCrGrGrUrUrCrCrCrUrGrCrArCrUrGrGrG505 TArUrCrArCrGrGrUrUrCrCrCrUrGrCrArCrUrGrG65ME1\59622102.v1Attorney Docket No. 126454-03020506 rUrArUrCrArCrGrGrUrUrCrCrCrUrGrCrArCrUrG 507 rUrUrArUrCrArCrGrGrUrUrCrCrCrUrGrCrArCrU 508 rCrUrUrArUrCrArCrGrGrUrUrCrCrCrUrGrCrArC 509 TGrGrArArCrCrGrUrGrArUrArArGrCrCrUrUrUrC 510 TGrUrGrArUrArArGrCrCrUrUrUrCrUrGrGrUrUrU 511 TGrArArArCrCrArGrArArArGrGrCrUrUrArUrCrA 512 TUrArCrGrUrGrCrUrCrCrGrArArArCrCrArGrArA 513 TArUrGrCrGrUrCrCrCrUrGrUrArCrArGrArUrArG 514 TUrGrCrGrUrCrCrCrUrGrUrArCrArGrArUrArGrU 515 TGrCrGrUrCrCrCrUrGrUrArCrArGrArUrArGrUrG 516 TArArArUrCrCrCrCrArCrUrArUrCrUrGrUrArCrA 517 TArArArArUrCrCrCrCrArCrUrArUrCrUrGrUrArC 518 TUrArUrGrUrUrUrGrCrArCrUrUrUrGrUrArUrArU 519 rCrUrArUrUrUrArUrUrUrUrUrGrCrArArArCrCrC 520 TCrUrGrArArCrArArArUrArCrArGrCrArArCrCrA 521 TArCrUrGrArArCrArArArUrArCrArGrCrArArCrC 522 TArUrUrUrGrUrUrCrArGrUrGrArCrUrArUrUrCrU 523 rUrUrUrGrUrUrCrArGrUrGrArCrUrArUrUrCrUrC 524 TUrUrGrUrUrCrArGrUrGrArCrUrArUrUrCrUrCrG 525 TCrUrArUrUrCrUrCrGrGrGrGrCrCrCrUrGrUrGrU 526 TUrArUrUrCrUrCrGrGrGrGrCrCrCrUrGrUrGrUrA 527 TArUrUrCrUrCrGrGrGrGrCrCrCrUrGrUrGrUrArG 528 TUrUrCrUrCrGrGrGrGrCrCrCrUrGrUrGrUrArGrG 529 TArGrGrCrArArUrArArCrCrCrCrCrUrArCrArCrA 530 TGrArGrGrCrArArUrArArCrCrCrCrCrUrArCrArC 531 TArUrArArArGrArArGrArGrGrCrArUrUrUrCrArG 532 TArArUrCrUrUrUrGrUrArCrArUrArArArGrArArG 533 TCrArArArGrArUrUrArUrUrUrGrCrArCrGrArArC 534 TGrGrArCrUrGrUrGrUrGrCrArArCrGrCrUrUrUrU 535 TGrArCrUrGrUrGrUrGrCrArArCrGrCrUrUrUrUrU 536 TGrUrCrCrCrCrGrUrUrGrUrArUrGrUrArUrGrArG 537 TArGrCrCrArCrUrCrArUrArCrArUrArCrArArCrG 538 TArArGrCrCrArCrUrCrArUrArCrArUrArCrArArC 539 TGrArArGrCrCrArCrUrCrArUrArCrArUrArCrArA 540 TUrUrGrUrArUrGrUrArUrGrArGrUrGrGrCrUrUrC 541 TUrGrUrArUrGrUrArUrGrArGrUrGrGrCrUrUrCrU 542 TUrArUrGrArGrUrGrGrCrUrUrCrUrGrGrGrArGrA 543 TArUrGrArGrUrGrGrCrUrUrCrUrGrGrGrArGrArU 544 TUrArCrArArArArArCrCrUrUrCrUrArUrArCrArG 545 TUrUrGrArUrCrArCrArGrUrArArGrArCrArUrUrC 546 TUrGrCrArUrCrGrArGrUrUrUrArGrArCrArArArU547 TUrUrArGrCrGrUrUrGrGrCrCrArCrCrCrGrCrGrG66ME1\59622102.v1Attorney Docket No. 126454-03020548 rArArArUrUrArGrCrGrUrUrGrGrCrCrArCrCrCrG 1324 TUrUrGrArArArUrUrCrGrArArUrUrCrUrGrCrCrU 1325 TGrUrGrCrGrUrUrGrCrUrUrUrGrArGrUrGrGrGrU 1393 TArCrArGrArUrGrArCrArGrArArGrGrArArGrUrG 1394 rUrUrCrCrUrUrCrUrGrUrCrArUrCrUrGrUrUrUrG 1395 TUrCrUrGrUrCrArUrCrUrGrUrUrUrGrUrGrGrArU 1396 TArUrCrCrArCrArArArCrArGrArUrGrArCrArGrA 1397 TGrUrUrUrGrUrGrGrArUrUrGrGrArCrUrCrCrCrC 1398 TGrGrArUrUrGrGrArCrUrCrCrCrCrArGrGrArGrA 1399 TGrGrGrArArGrArCrUrCrCrCrArGrArArCrUrCrC 1400 TGrUrGrUrArUrGrArArUrGrGrArUrGrArGrCrArG 1401 TArGrUrGrUrArUrGrArArUrGrGrArUrGrArGrCrA 1402 TArUrArUrUrUrGrUrCrGrArGrUrGrUrArUrGrArA 1403 TCrArCrArArGrCrCrUrArCrGrGrCrArGrArGrCrA 1404 TGrUrArGrGrCrUrUrGrUrGrGrGrArCrArCrUrArC 1405 TUrArGrGrCrUrUrGrUrGrGrGrArCrArCrUrArCrA 1406 TUrGrUrArGrUrGrUrCrCrCrArCrArArGrCrCrUrA 1407 TArGrGrCrUrUrGrUrGrGrGrArCrArCrUrArCrArG 1408 TArArArGrArArArUrGrArArGrUrCrUrUrGrArUrA 1409 TGrArArArUrGrArArGrUrCrUrUrGrArUrArCrGrG 1410 rArArUrCrUrCrCrCrUrGrUrCrUrGrUrCrCrUrUrA 1411 TUrCrArGrGrUrCrCrUrUrArArGrGrArCrArGrArC 1412 TArArArArUrGrArUrGrUrCrArGrGrUrCrCrUrUrA 1413 rCrCrUrCrArArGrUrCrGrUrCrUrArCrCrGrCrCrU 1414 TCrCrGrArGrGrCrGrGrUrArGrArCrGrArCrUrUrG 1415 TUrUrCrUrCrUrArArUrGrArArGrUrArArCrArUrU 1416 TCrArGrArArArArGrCrArArCrUrGrGrCrArGrGrA 1417 TGrGrGrArCrArGrArArArArGrCrArArCrUrGrGrC 1418 TUrArUrGrGrGrGrArCrArGrArArArArGrCrArArC 1419 TUrUrUrUrGrUrArUrCrUrArUrUrCrUrArArArArA 1420 TUrGrUrArUrCrUrArUrUrCrUrArArArArArUrGrG 1455 mC*mA*mG*rUrGrCrCrCrArUrGrCrArArUrGrGrCrUrU 1456 mA*mU*mG*rArGrUrGrGrCrUrUrCrUrGrGrGrArGrArU 1462 mG*mA*mG*rCrArArCrArCrArGrArGrCrCrCrArGrGrG 1464 mU*mU*mG*rArArArUrUrCrGrArArUrUrCrUrGrCrCrU 1466 mG*mU*mG*rCrGrUrUrGrCrUrUrUrGrArGrUrGrGrGrU 1468 mC*mA*mG*rTrGrCrCrCrArTrGrCrArArTrGrGrCrTrT 1470 mA*mT*mG*rArGrTrGrGrCrTrTrCrTrGrGrGrArGrArT 1451 mA*mG*mC*rUrCrUrGrGrCrArGrGrCrArArUrGrCrUrU 1452 mA*mU*mA*rUrArArArArCrArArArGrCrUrCrUrGrGrC 1453 mA*mA*mA*rUrArUrArUrArArArArCrArArArGrCrUrC1454 mA*mC*mG*rUrGrGrCrGrUrGrArArCrArUrCrUrGrCrC67ME1\59622102.v1Attorney Docket No. 126454-030201474 mA*mA*mU*rCrUrUrUrGrUrArCrArUrArArArGrArArG1475 mU*mA*mC*rArArArArArCrCrUrUrCrUrArUrArCrArG All bases are in upper case Lowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bonds except as noted:represents phosphorothioate modificationTable 6: gRNA sequences for targeting LDLR with SpCas9SEQ gRNA sequenceID NO900 TArCrGrUrGrGrCrGrUrGrArArCrArUrCrUrGrCrCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU901 TCrUrGrGrGrCrArGrGrGrArCrGrGrGrArCrUrCrCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU902 TGrArGrGrUrCrUrCrArGrGrArArGrGrGrUrUrCrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU903 TCrGrArGrGrUrCrUrCrArGrGrArArGrGrGrUrUrCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU904 TArArCrCrCrUrUrCrCrUrGrArGrArCrCrUrCrGrCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU905 TGrGrCrCrGrGrCrGrArGrGrUrCrUrCrArGrGrArArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU906 TArGrGrCrCrGrGrCrGrArGrGrUrCrUrCrArGrGrArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU907 TArArCrArArGrGrCrCrGrGrCrGrArGrGrUrCrUrCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU908 TUrGrArArUrArArArArCrArArGrGrCrCrGrGrCrGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU909 TGrUrCrUrUrUrGrArArUrArArArArCrArArGrGrCrGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC68ME1\59622102.v1Attorney Docket No. 126454-03020TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU910 TCrUrCrUrGrUrCrUrUrUrGrArArUrArArArArCrArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU911 TArGrCrUrCrUrGrGrCrArGrGrCrArArUrGrCrUrUrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU912 TArUrArUrArArArArCrArArArGrCrUrCrUrGrGrCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU913 TArArArUrArUrArUrArArArArCrArArArGrCrUrCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU914 TArUrUrCrArUrCrUrGrGrGrArGrGrCrArGrArArCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU915 TCrUrGrGrGrArGrGrCrArGrArArCrArGrGrCrUrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU916 TUrUrCrGrGrArCrArGrUrGrCrCrCrArUrGrCrArArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU917 TArCrArGrUrGrCrCrCrArUrGrCrArArUrGrGrCrUrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU918 TCrArGrUrGrCrCrCrArUrGrCrArArUrGrGrCrUrUrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU919 TGrCrCrCrArUrGrCrArArUrGrGrCrUrUrGrGrGrUrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU920 TCrCrCrArUrGrCrArArUrGrGrCrUrUrGrGrGrUrUrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU921 rCrCrCrArArCrCrCrArArGrCrCrArUrUrGrCrArUrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrUrUrU69ME1\59622102.v1Attorney Docket No. 126454-03020922 TUrCrCrCrArArCrCrCrArArGrCrCrArUrUrGrCrArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU923 TGrUrUrUrCrUrUrCrCrUrUrUrCrCrUrCrGrUrGrArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU924 TCrUrCrUrUrArUrCrCrUrUrCrArCrGrArGrGrArArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 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TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1127 TArArUrCrUrUrUrGrUrArCrArUrArArArGrArArGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1128 TCrArArArGrArUrUrArUrUrUrGrCrArCrGrArArCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1129 TGrGrArCrUrGrUrGrUrGrCrArArCrGrCrUrUrUrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1130 TGrArCrUrGrUrGrUrGrCrArArCrGrCrUrUrUrUrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1131 TGrUrCrCrCrCrGrUrUrGrUrArUrGrUrArUrGrArGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1132 TArGrCrCrArCrUrCrArUrArCrArUrArCrArArCrGrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU1133 rArArGrCrCrArCrUrCrArUrArCrArUrArCrArArCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU1134 TGrArArGrCrCrArCrUrCrArUrArCrArUrArCrArArGrUrUrUrUrArGrArGrCrUTArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr 86ME1\59622102.v1Attorney Docket No. 126454-03020GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU1135 TUrUrGrUrArUrGrUrArUrGrArGrUrGrGrCrUrUrCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1136 TUrGrUrArUrGrUrArUrGrArGrUrGrGrCrUrUrCrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1137 TUrArUrGrArGrUrGrGrCrUrUrCrUrGrGrGrArGrArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1138 TArUrGrArGrUrGrGrCrUrUrCrUrGrGrGrArGrArUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1139 TUrArCrArArArArArCrCrUrUrCrUrArUrArCrArGrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU1140 TUrUrGrArUrCrArCrArGrUrArArGrArCrArUrUrCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1141 TUrGrCrArUrCrGrArGrUrUrUrArGrArCrArArArUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1142 TUrUrArGrCrGrUrUrGrGrCrCrArCrCrCrGrCrGrGrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU1143 TArArArUrUrArGrCrGrUrUrGrGrCrCrArCrCrCrGrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU1331 TUrUrGrArArArUrUrCrGrArArUrUrCrUrGrCrCrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU1332 TGrUrGrCrGrUrUrGrCrUrUrUrGrArGrUrGrGrGrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG rGrUrGrCrUrUrUrU TArCrArGrArUrGrArCrArGrArArGrGrArArGrUrGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG1421 rGrUrGrCrUrUrUrU87ME1\59622102.v1Attorney Docket No. 126454-03020rUrUrCrCrUrUrCrUrGrUrCrArUrCrUrGrUrUrUrGrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1422 GrUrGrCrUrUrUrU TUrCrUrGrUrCrArUrCrUrGrUrUrUrGrUrGrGrArUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1423 rGrUrGrCrUrUrUrU TArUrCrCrArCrArArArCrArGrArUrGrArCrArGrArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1424 GrUrGrCrUrUrUrU TGrUrUrUrGrUrGrGrArUrUrGrGrArCrUrCrCrCrCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1425 GrUrGrCrUrUrUrU TGrGrArUrUrGrGrArCrUrCrCrCrCrArGrGrArGrArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1426 GrUrGrCrUrUrUrU TGrGrGrArArGrArCrUrCrCrCrArGrArArCrUrCrCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1427 GrUrGrCrUrUrUrU TGrUrGrUrArUrGrArArUrGrGrArUrGrArGrCrArGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1428 rGrUrGrCrUrUrUrU TArGrUrGrUrArUrGrArArUrGrGrArUrGrArGrCrArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1429 rGrUrGrCrUrUrUrU TArUrArUrUrUrGrUrCrGrArGrUrGrUrArUrGrArArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1430 rGrUrGrCrUrUrUrU TCrArCrArArGrCrCrUrArCrGrGrCrArGrArGrCrArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1431 GrUrGrCrUrUrUrU TGrUrArGrGrCrUrUrGrUrGrGrGrArCrArCrUrArCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1432 rGrUrGrCrUrUrUrU TUrArGrGrCrUrUrGrUrGrGrGrArCrArCrUrArCrArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1433 rGrUrGrCrUrUrUrUTUrGrUrArGrUrGrUrCrCrCrArCrArArGrCrCrUrArGrUrUrUrUrArGrArGrCrU1434 TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr 88ME1\59622102.v1Attorney Docket No. 126454-03020GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrUrU TArGrGrCrUrUrGrUrGrGrGrArCrArCrUrArCrArGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1435 rGrUrGrCrUrUrUrU TArArArGrArArArUrGrArArGrUrCrUrUrGrArUrArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1436 rGrUrGrCrUrUrUrU TGrArArArUrGrArArGrUrCrUrUrGrArUrArCrGrGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1437 rGrUrGrCrUrUrUrU TArArUrCrUrCrCrCrUrGrUrCrUrGrUrCrCrUrUrArGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1438 GrUrGrCrUrUrUrU TUrCrArGrGrUrCrCrUrUrArArGrGrArCrArGrArCrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1439 GrUrGrCrUrUrUrU TArArArArUrGrArUrGrUrCrArGrGrUrCrCrUrUrArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1440 rGrUrGrCrUrUrUrU TCrCrUrCrArArGrUrCrGrUrCrUrArCrCrGrCrCrUrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1441 GrUrGrCrUrUrUrU TCrCrGrArGrGrCrGrGrUrArGrArCrGrArCrUrUrGrGrUrUrUrUrArGrArGrCrU TArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCr GrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGr 1442 GrUrGrCrUrUrUrU TUrUrCrUrCrUrArArUrGrArArGrUrArArCrArUrUrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1443 rGrUrGrCrUrUrUrU TCrArGrArArArArGrCrArArCrUrGrGrCrArGrGrArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1444 rGrUrGrCrUrUrUrU TGrGrGrArCrArGrArArArArGrCrArArCrUrGrGrCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1445 rGrUrGrCrUrUrUrU TUrArUrGrGrGrGrArCrArGrArArArArGrCrArArCrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG1446 rGrUrGrCrUrUrUrU89ME1\59622102.v1Attorney Docket No. 126454-03020TUrUrUrUrGrUrArUrCrUrArUrUrCrUrArArArArArGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1447 rGrUrGrCrUrUrUrU TUrGrUrArUrCrUrArUrUrCrUrArArArArArUrGrGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1448 rGrUrGrCrUrUrUrU TArCrArGrArUrGrArCrArGrArArGrGrArArGrUrGrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrC TGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrG 1421 rGrUrGrCrUrUrUrU mC*mA*mG*rUrGrCrCrCrArUrGrCrArArUrGrGrCrUrUrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1459 U mC*mA*mG*rUrGrCrCrCrArUrGrCrArArUrGrGrCrUrUrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1460 U mG*mA*mG*rCrArArCrArCrArGrArGrCrCrCrArGrGrGrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1463 U mU*mU*mG*rArArArUrUrCrGrArArUrUrCrUrGrCrCrUrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1465 U mG*mU*mG*rCrGrUrUrGrCrUrUrUrGrArGrUrGrGrGrUrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1467 U mC*mA*mG*rTrGrCrCrCrArTrGrCrArArTrGrGrCrTrTrGrUrUrUrUrArGrAm GmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGr CrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmGmU 1469 mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU mA*mT*mG*rArGrTrGrGrCrTrTrCrTrGrGrGrArGrArTrGrUrUrUrUrArGrAm GmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGr CrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmGmU 1471 mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU mU*mU*mG*rArArArUrUrCrGrArArUrUrCrUrGrCrCrUrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m1472 U90ME1\59622102.v1Attorney Docket No. 126454-03020mG*mU*mG*rCrGrUrUrGrCrUrUrUrGrArGrUrGrGrGrUrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1473 U mA*mG*mC*rUrCrUrGrGrCrArGrGrCrArArUrGrCrUrUrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1476 U mA*mU*mA*rUrArArArArCrArArArGrCrUrCrUrGrGrCrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1477 U mA*mA*mA*rUrArUrArUrArArArArCrArArArGrCrUrCrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1478 U mA*mC*mG*rUrGrGrCrGrUrGrArArCrArUrCrUrGrCrCrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1479 U mA*mA*mU*rCrUrUrUrGrUrArCrArUrArArArGrArArGrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m 1449 U mU*mA*mC*rArArArArArCrCrUrUrCrUrArUrArCrArGrGrUrUrUrUrArGrA mGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGr GrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m1450 UAll bases are in upper case Lowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bonds except as noted:represents phosphorothioate modificationTable 7: LDLR Target Sequences for AsCas12a WTSEQ ID NO Target Sequence (DNA)549 AATAAAACAAGGCCGGCGAGG550 TTCATCTGGGAGGCAGAACAG551 GTTTCTTCCTTTCCTCGTGAA552 TTCCTTTCCTCGTGAAGGATA553 TCTTATCCTTCACGAGGAAAG554 AGTTTCTCTCCACCGTGACAC91ME1\59622102.v1Attorney Docket No. 126454-03020555 AGGATTGTGTCACGGTGGAGA 556 TCTCCACCGTGACACAATCCT 557 ATCTTCCATGTTTGAGGATTG 558 AACACACAACAGCAGATGGCA 559 CCTCTTCTATGCAAGCCTTGC 560 GAGGTGGGTTTGTACCTTCCT 561 CTGGCTTAAGGAAGGTACAAA 562 CGACGCCATGAATCCCTTTCC 563 GTCTCGGTGCCACCACGGATT 564 GTGAATGAGTTTGGTCTCGGT 565 TTAAGTGCCTGAGACACCCGG 566 TCAGTTCAGAGTTGTACACTG 567 CACTGTTTTCTGTCGTGTGTG 568 AAGTCCCGGTCAACCTGCCCC 569 TGTCCGCCCACCTAGTGCTTC 570 CATAGAAGTGGAAGCACTAGG 571 AACACATACCCATCAACGACA 572 AAACATGCACGGTGAGGCCGG 573 ACGGGACTTCAGGTTCTTTCT 574 AGAAAGAACCTGAAGTCCCGT 575 TGAAATCGCCGTGTTACTGTT 576 TCCCTGGGGACAATTGCCAAT 577 CGTGCTCCGAAACCAGAAAGG 578 TGGTTTCGGAGCACGTAAATG 579 GGAGCACGTAAATGCGTCCCT 580 AACCAATATACAAAGTGCAAA 581 CACTTTGTATATTGGTTGAAA 582 TATATTGGTTGAAACTGTTAT 583 TTTTTGCAAACCCTGGTTGCT 584 CAAACCCTGGTTGCTGTATTT 585 AGAGGCAATAACCCCCTACAC 586 TACATAAAGAAGAGGCATTTC 587 TGTACAAAGATTATTTGCACG 588 CACGAACTGGACTGTGTGCAA 589 GGAGAATGATGTCCCCGTTGT 590 AACCACTGTATAGAAGGTTTT 591 TAGCCTGAATGTCTTACTGTG 592 ATTGATCACAGTAAGACATTC 593 AGAAATTTAATTGATCACAGT 594 GACAAATTGGTTCATTTAAGA 595 TTAAATGAACCAATTTGTCTA596 TCTAAACTCGATGCACGTTCT92ME1\59622102.v1Attorney Docket No. 126454-03020597 TGGATTGGACTCCCCAGGAGA 598 TCAAGTCCCCTGCTCATCCAT599 TCGAGTGTATGAATGGATGAG 600 TTATATCCCCTGTAGTGTCCC601 ATTTCTTATATCCCCTGTAGT602 TTCAGGGGGAGTTTCTCTAAT 603 TGGTGCAGTCACCTGCCAGCA 604 TGGTGGTGGCGCTTTCTGCTG605 CCTCCTATGAGTTTCTGGCTC606 TGGACCTAACCACCCCAGTGT 1319 TTGTCTGGGGACAGAAAAGGC 1320 GCTTCCAAGGAAATGAAGATG 1321 AATGGGCTGATCTCAGGCCTG 1322 AAAGCAGTTCCCACCCACTCA1323 AGTGGGTGGGAACTGCTTTGATable 8: LDLR Targeting Sequences for AsCas12a WTSEQ ID NO gDNA targeting Domain (RNA) 607 TArArUrArArArArCrArArGrGrCrCrGrGrCrGrArGrG 608 TUrUrCrArUrCrUrGrGrGrArGrGrCrArGrArArCrArG 609 rGrUrUrUrCrUrUrCrCrUrUrUrCrCrUrCrGrUrGrArA 610 TUrUrCrCrUrUrUrCrCrUrCrGrUrGrArArGrGrArUrA 611 TUrCrUrUrArUrCrCrUrUrCrArCrGrArGrGrArArArG 612 TArGrUrUrUrCrUrCrUrCrCrArCrCrGrUrGrArCrArC 613 TArGrGrArUrUrGrUrGrUrCrArCrGrGrUrGrGrArGrA 614 TUrCrUrCrCrArCrCrGrUrGrArCrArCrArArUrCrCrU 615 TArUrCrUrUrCrCrArUrGrUrUrUrGrArGrGrArUrUrG 616 TArArCrArCrArCrArArCrArGrCrArGrArUrGrGrCrA 617 rCrCrUrCrUrUrCrUrArUrGrCrArArGrCrCrUrUrGrC 618 TGrArGrGrUrGrGrGrUrUrUrGrUrArCrCrUrUrCrCrU 619 TCrUrGrGrCrUrUrArArGrGrArArGrGrUrArCrArArA 620 TCrGrArCrGrCrCrArUrGrArArUrCrCrCrUrUrUrCrC 621 TGrUrCrUrCrGrGrUrGrCrCrArCrCrArCrGrGrArUrU 622 TGrUrGrArArUrGrArGrUrUrUrGrGrUrCrUrCrGrGrU 623 TUrUrArArGrUrGrCrCrUrGrArGrArCrArCrCrCrGrG 624 TUrCrArGrUrUrCrArGrArGrUrUrGrUrArCrArCrUrG 625 TCrArCrUrGrUrUrUrUrCrUrGrUrCrGrUrGrUrGrUrG 626 TArArGrUrCrCrCrGrGrUrCrArArCrCrUrGrCrCrCrC 627 rUrGrUrCrCrGrCrCrCrArCrCrUrArGrUrGrCrUrUrC 628 TCrArUrArGrArArGrUrGrGrArArGrCrArCrUrArGrG 629 TArArCrArCrArUrArCrCrCrArUrCrArArCrGrArCrA630 TArArArCrArUrGrCrArCrGrGrUrGrArGrGrCrCrGrG93ME1\59622102.v1Attorney Docket No. 126454-03020631 rArCrGrGrGrArCrUrUrCrArGrGrUrUrCrUrUrUrCrU 632 TArGrArArArGrArArCrCrUrGrArArGrUrCrCrCrGrU 633 TUrGrArArArUrCrGrCrCrGrUrGrUrUrArCrUrGrUrU 634 TUrCrCrCrUrGrGrGrGrArCrArArUrUrGrCrCrArArU 635 TCrGrUrGrCrUrCrCrGrArArArCrCrArGrArArArGrG 636 TUrGrGrUrUrUrCrGrGrArGrCrArCrGrUrArArArUrG 637 TGrGrArGrCrArCrGrUrArArArUrGrCrGrUrCrCrCrU 638 TArArCrCrArArUrArUrArCrArArArGrUrGrCrArArA 639 TCrArCrUrUrUrGrUrArUrArUrUrGrGrUrUrGrArArA 640 TUrArUrArUrUrGrGrUrUrGrArArArCrUrGrUrUrArU 641 TUrUrUrUrUrGrCrArArArCrCrCrUrGrGrUrUrGrCrU 642 TCrArArArCrCrCrUrGrGrUrUrGrCrUrGrUrArUrUrU 643 TArGrArGrGrCrArArUrArArCrCrCrCrCrUrArCrArC 644 TUrArCrArUrArArArGrArArGrArGrGrCrArUrUrUrC 645 TUrGrUrArCrArArArGrArUrUrArUrUrUrGrCrArCrG 646 TCrArCrGrArArCrUrGrGrArCrUrGrUrGrUrGrCrArA 647 TGrGrArGrArArUrGrArUrGrUrCrCrCrCrGrUrUrGrU 648 TArArCrCrArCrUrGrUrArUrArGrArArGrGrUrUrUrU 649 TUrArGrCrCrUrGrArArUrGrUrCrUrUrArCrUrGrUrG 650 TArUrUrGrArUrCrArCrArGrUrArArGrArCrArUrUrC 651 TArGrArArArUrUrUrArArUrUrGrArUrCrArCrArGrU 652 TGrArCrArArArUrUrGrGrUrUrCrArUrUrUrArArGrA 653 TUrUrArArArUrGrArArCrCrArArUrUrUrGrUrCrUrA 654 TUrCrUrArArArCrUrCrGrArUrGrCrArCrGrUrUrCrU 655 TUrGrGrArUrUrGrGrArCrUrCrCrCrCrArGrGrArGrA 656 rUrCrArArGrUrCrCrCrCrUrGrCrUrCrArUrCrCrArU 657 TUrCrGrArGrUrGrUrArUrGrArArUrGrGrArUrGrArG 658 rUrUrArUrArUrCrCrCrCrUrGrUrArGrUrGrUrCrCrC 659 rArUrUrUrCrUrUrArUrArUrCrCrCrCrUrGrUrArGrU 660 TUrUrCrArGrGrGrGrGrArGrUrUrUrCrUrCrUrArArU 661 TUrGrGrUrGrCrArGrUrCrArCrCrUrGrCrCrArGrCrA 662 TUrGrGrUrGrGrUrGrGrCrGrCrUrUrUrCrUrGrCrUrG 663 rCrCrUrCrCrUrArUrGrArGrUrUrUrCrUrGrGrCrUrC 664 TUrGrGrArCrCrUrArArCrCrArCrCrCrCrArGrUrGrU 1326 TUrUrGrUrCrUrGrGrGrGrArCrArGrArArArArGrGrC 1327 TGrCrUrUrCrCrArArGrGrArArArUrGrArArGrArUrG 1328 TArArUrGrGrGrCrUrGrArUrCrUrCrArGrGrCrCrUrG 1329 TArArArGrCrArGrUrUrCrCrCrArCrCrCrArCrUrCrA1330 TArGrUrGrGrGrUrGrGrGrArArCrUrGrCrUrUrUrGrA All bases are in upper case Lowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bonds94ME1\59622102.v1Attorney Docket No. 126454-03020Table 9: gRNA sequences for targeting LDLR with AsCas12a (WT)SEQ ID NO gRNA Sequence TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArUrArArArArCrArArG 1144 rGrCrCrGrGrCrGrArGrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrCrArUrCrUrGrGrGrA 1145 rGrGrCrArGrArArCrArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrUrUrCrUrUrCrCrUrU 1146 rUrCrCrUrCrGrUrGrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrCrCrUrUrUrCrCrUrC 1147 rGrUrGrArArGrGrArUrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrUrUrArUrCrCrUrUrC 1148 rArCrGrArGrGrArArArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrUrUrUrCrUrCrUrCrC 1149 rArCrCrGrUrGrArCrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrGrArUrUrGrUrGrUrC 1150 rArCrGrGrUrGrGrArGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrUrCrCrArCrCrGrUrG 1151 rArCrArCrArArUrCrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArUrCrUrUrCrCrArUrGrU 1152 rUrUrGrArGrGrArUrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArCrArCrArCrArArCrA 1153 rGrCrArGrArUrGrGrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrUrCrUrUrCrUrArUrG 1154 rCrArArGrCrCrUrUrGrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrGrUrGrGrGrUrUrU 1155 rGrUrArCrCrUrUrCrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrGrGrCrUrUrArArGrG 1156 rArArGrGrUrArCrArArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrArCrGrCrCrArUrGrA 1157 rArUrCrCrCrUrUrUrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrCrUrCrGrGrUrGrCrC 1158 rArCrCrArCrGrGrArUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrGrArArUrGrArGrUrU 1159 rUrGrGrUrCrUrCrGrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrArArGrUrGrCrCrUrG 1160 rArGrArCrArCrCrCrGrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrArGrUrUrCrArGrArG 1161 rUrUrGrUrArCrArCrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArCrUrGrUrUrUrUrCrU 1162 rGrUrCrGrUrGrUrGrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArGrUrCrCrCrGrGrUrC 1163 rArArCrCrUrGrCrCrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrCrCrGrCrCrCrArC 1164 rCrUrArGrUrGrCrUrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArUrArGrArArGrUrGrG 1165 rArArGrCrArCrUrArGrGTUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArCrArCrArUrArCrCrC1166 rArUrCrArArCrGrArCrA95ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArArCrArUrGrCrArCrG 1167 rGrUrGrArGrGrCrCrGrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrGrGrGrArCrUrUrCrA 1168 rGrGrUrUrCrUrUrUrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrArArArGrArArCrCrU 1169 rGrArArGrUrCrCrCrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrArArArUrCrGrCrCrG 1170 rUrGrUrUrArCrUrGrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrCrCrUrGrGrGrGrArC 1171 rArArUrUrGrCrCrArArU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrUrGrCrUrCrCrGrArA 1172 rArCrCrArGrArArArGrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrUrUrUrCrGrGrArG 1173 rCrArCrGrUrArArArUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrArGrCrArCrGrUrArA 1174 rArUrGrCrGrUrCrCrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArCrCrArArUrArUrArC 1175 rArArArGrUrGrCrArArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArCrUrUrUrGrUrArUrA 1176 rUrUrGrGrUrUrGrArArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArUrArUrUrGrGrUrUrG 1177 rArArArCrUrGrUrUrArU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrUrUrUrGrCrArArArC 1178 rCrCrUrGrGrUrUrGrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArArArCrCrCrUrGrGrU 1179 rUrGrCrUrGrUrArUrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrArGrGrCrArArUrArA 1180 rCrCrCrCrCrUrArCrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArCrArUrArArArGrArA 1181 rGrArGrGrCrArUrUrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrArCrArArArGrArU 1182 rUrArUrUrUrGrCrArCrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArCrGrArArCrUrGrGrA 1183 rCrUrGrUrGrUrGrCrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrArGrArArUrGrArUrG 1184 rUrCrCrCrCrGrUrUrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArCrCrArCrUrGrUrArU 1185 rArGrArArGrGrUrUrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArGrCrCrUrGrArArUrG 1186 rUrCrUrUrArCrUrGrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArUrUrGrArUrCrArCrArG 1187 rUrArArGrArCrArUrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrArArArUrUrUrArArU 1188 rUrGrArUrCrArCrArGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArCrArArArUrUrGrGrU 1189 rUrCrArUrUrUrArArGrATUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrArArArUrGrArArCrC1190 rArArUrUrUrGrUrCrUrA96ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrUrArArArCrUrCrGrA 1191 rUrGrCrArCrGrUrUrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrArUrUrGrGrArCrU 1192 rCrCrCrCrArGrGrArGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrArArGrUrCrCrCrCrU 1193 rGrCrUrCrArUrCrCrArU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrGrArGrUrGrUrArUrG 1194 rArArUrGrGrArUrGrArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrArUrArUrCrCrCrCrU 1195 rGrUrArGrUrGrUrCrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArUrUrUrCrUrUrArUrArU 1196 rCrCrCrCrUrGrUrArGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrCrArGrGrGrGrGrArG 1197 rUrUrUrCrUrCrUrArArU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrUrGrCrArGrUrCrA 1198 rCrCrUrGrCrCrArGrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrUrGrGrUrGrGrCrG 1199 rCrUrUrUrCrUrGrCrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrUrCrCrUrArUrGrArG 1200 rUrUrUrCrUrGrGrCrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrArCrCrUrArArCrC 1201 rArCrCrCrCrArGrUrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrGrUrCrUrGrGrGrGrA 1333 rCrArGrArArArArGrGrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrCrUrUrCrCrArArGrGrA 1334 rArArUrGrArArGrArUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArUrGrGrGrCrUrGrArU 1335 rCrUrCrArGrGrCrCrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArArGrCrArGrUrUrCrC 1336 rCrArCrCrCrArCrUrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrUrGrGrGrUrGrGrGrA 1337 rArCrUrGrCrUrUrUrGrA / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCr U / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUrUrCrUrUrA 1457 rUrCrCrUrUrCrArCrGrArGrGrArArArG / 3InvdT / / 5InvdT / ATGTGTTTTTGTCAAAAGACCTTTT / i2FU / rArArU / i2FU / / i2FU / rCr U / i2FA / / i2FC / rU / i2FC / / i2FU / / i2FU / rG / i2FU / / i2FA / / i2FG / / i2FA / rUrGrGrArGr1458 ArArUrGrArUrGrUrCrCrCrCrGrUrUrGrU / 3InvdT / All bases are in upper case Lowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bondsTable 10: LDLR Target Sequences for AsCas12a RRSEQ ID NO Target Sequence (DNA)665 GGCAGATGTTCACGCCACGTC666 TGAGACCTCGCCGGCCTTGTT667 TCTGGGAGGCAGAACAGGCTT668 AAGCCTGTTCTGCCTCCCAGA97ME1\59622102.v1Attorney Docket No. 126454-03020669 CGAGGAAAGGAAGAAACCAAA 670 TTTCCTCGTGAAGGATAAGAG 671 CCCGGGCCTGTTTCTCTTATC 672 GGAAGTTTTGAGTTTCTCTCC 673 CCGTGACACAATCCTCAAACA 674 TGTTTGAGGATTGTGTCACGG 675 ATGTTTGAGGATTGTGTCACG 676 TCTTCCATGTTTGAGGATTGT 677 CTTTCATCTTCCATGTTTGAG 678 ACACACAACAGCAGATGGCAC 679 GTTGGTGCCATCTGCTGTTGT 680 TGGCTTAAGGAAGGTACAAAC 681 TTAAGCCAGGAAAGGGATTCA 682 ACGCCATGAATCCCTTTCCTG 683 GACGCCATGAATCCCTTTCCT 684 TGGCGTCGGAAATGATCTGGC 685 GCCAGATCATTTCCGACGCCA 686 TGGTGGCACCGAGACCAAACT 687 CCAAATGATGCCACTTCCCAG 688 ATTCCCGTGGTCTCCTTGCAC 689 TTCCCGTGGTCTCCTTGCACT 690 CGTGGTCTCCTTGCACTTTCT 691 GAGTTGTACACTGTGTACATT 692 ATCCCAACACACACGACAGAA 693 TGGCCTGGGATCCCATCCCAA 694 AGGCCAGGGAAAGCCCGTGTC 695 TTGACACGGGCTTTCCCTGGC 696 CGGCATTCATTGACACGGGCT 697 GGTCAACCTGCCCCTCTCTGT 698 CGATCACGGCTTTGAAGTCCC 699 AAGCCGTGATCGTGAATATCG 700 CCCACCTAGTGCTTCCACTTC 701 ACTTCTATGCAAATGCCTCCA 702 CTTCTATGCAAATGCCTCCAA 703 CTTCCCCAATCTTGTCGTTGA 704 GTCTGGATCGTTTGACGGGAC 705 GAAAGAACCTGAAGTCCCGTC 706 GGTTCTTTCTGAAATCGCCGT 707 GAGAGACAGTGACAGCCTCCG 708 CGCGGGAGTCTGACGGAGGCT 709 TCAGACTCCCGCGTGAAGATG710 TTGTGACATCTTCACGCGGGA98ME1\59622102.v1Attorney Docket No. 126454-03020711 GCGTGAAGATGTCACAAGGGA 712 TGGGGACAATTGCCAATCCCT 713 CAGGGACAAAACACTGTGTCC 714 TGCACTGGGGGGGACACAGTG 715 CTGCACTGGGGGGGACACAGT 716 CCCCAGTGCAGGGAACCGTGA 717 AAACCAGAAAGGCTTATCACG 718 GAGCACGTAAATGCGTCCCTG 719 CACTATCTGTACAGGGACGCA 720 TGTACAGATAGTGGGGATTTT 721 GTGACTATTCTCGGGGCCCTG722 GAGGCAATAACCCCCTACACATable 11: LDLR Targeting Sequences for AsCas12a RRSEQ ID NO gDNA targeting Domain (RNA) 751 TGrGrCrArGrArUrGrUrUrCrArCrGrCrCrArCrGrUrC 752 TUrGrArGrArCrCrUrCrGrCrCrGrGrCrCrUrUrGrUrU 753 TUrCrUrGrGrGrArGrGrCrArGrArArCrArGrGrCrUrU 754 TArArGrCrCrUrGrUrUrCrUrGrCrCrUrCrCrCrArGrA 755 TCrGrArGrGrArArArGrGrArArGrArArArCrCrArArA 756 TUrUrUrCrCrUrCrGrUrGrArArGrGrArUrArArGrArG 757 rCrCrCrGrGrGrCrCrUrGrUrUrUrCrUrCrUrUrArUrC 758 TGrGrArArGrUrUrUrUrGrArGrUrUrUrCrUrCrUrCrC 759 TCrCrGrUrGrArCrArCrArArUrCrCrUrCrArArArCrA 760 TUrGrUrUrUrGrArGrGrArUrUrGrUrGrUrCrArCrGrG 761 TArUrGrUrUrUrGrArGrGrArUrUrGrUrGrUrCrArCrG 762 TUrCrUrUrCrCrArUrGrUrUrUrGrArGrGrArUrUrGrU 763 rCrUrUrUrCrArUrCrUrUrCrCrArUrGrUrUrUrGrArG 764 TArCrArCrArCrArArCrArGrCrArGrArUrGrGrCrArC 765 TGrUrUrGrGrUrGrCrCrArUrCrUrGrCrUrGrUrUrGrU 766 TUrGrGrCrUrUrArArGrGrArArGrGrUrArCrArArArC 767 TUrUrArArGrCrCrArGrGrArArArGrGrGrArUrUrCrA 768 TArCrGrCrCrArUrGrArArUrCrCrCrUrUrUrCrCrUrG 769 TGrArCrGrCrCrArUrGrArArUrCrCrCrUrUrUrCrCrU 770 TUrGrGrCrGrUrCrGrGrArArArUrGrArUrCrUrGrGrC 771 TGrCrCrArGrArUrCrArUrUrUrCrCrGrArCrGrCrCrA 772 TUrGrGrUrGrGrCrArCrCrGrArGrArCrCrArArArCrU 773 TCrCrArArArUrGrArUrGrCrCrArCrUrUrCrCrCrArG 774 rArUrUrCrCrCrGrUrGrGrUrCrUrCrCrUrUrGrCrArC 775 rUrUrCrCrCrGrUrGrGrUrCrUrCrCrUrUrGrCrArCrU 776 rCrGrUrGrGrUrCrUrCrCrUrUrGrCrArCrUrUrUrCrU777 TGrArGrUrUrGrUrArCrArCrUrGrUrGrUrArCrArUrU99ME1\59622102.v1Attorney Docket No. 126454-03020778 TArUrCrCrCrArArCrArCrArCrArCrGrArCrArGrArA 779 TUrGrGrCrCrUrGrGrGrArUrCrCrCrArUrCrCrCrArA 780 TArGrGrCrCrArGrGrGrArArArGrCrCrCrGrUrGrUrC 781 TUrUrGrArCrArCrGrGrGrCrUrUrUrCrCrCrUrGrGrC 782 TCrGrGrCrArUrUrCrArUrUrGrArCrArCrGrGrGrCrU 783 rGrGrUrCrArArCrCrUrGrCrCrCrCrUrCrUrCrUrGrU 784 TCrGrArUrCrArCrGrGrCrUrUrUrGrArArGrUrCrCrC 785 TArArGrCrCrGrUrGrArUrCrGrUrGrArArUrArUrCrG 786 rCrCrCrArCrCrUrArGrUrGrCrUrUrCrCrArCrUrUrC 787 TArCrUrUrCrUrArUrGrCrArArArUrGrCrCrUrCrCrA 788 TCrUrUrCrUrArUrGrCrArArArUrGrCrCrUrCrCrArA 789 rCrUrUrCrCrCrCrArArUrCrUrUrGrUrCrGrUrUrGrA 790 TGrUrCrUrGrGrArUrCrGrUrUrUrGrArCrGrGrGrArC 791 TGrArArArGrArArCrCrUrGrArArGrUrCrCrCrGrUrC 792 TGrGrUrUrCrUrUrUrCrUrGrArArArUrCrGrCrCrGrU 793 TGrArGrArGrArCrArGrUrGrArCrArGrCrCrUrCrCrG 794 TCrGrCrGrGrGrArGrUrCrUrGrArCrGrGrArGrGrCrU 795 TUrCrArGrArCrUrCrCrCrGrCrGrUrGrArArGrArUrG 796 TUrUrGrUrGrArCrArUrCrUrUrCrArCrGrCrGrGrGrA 797 TGrCrGrUrGrArArGrArUrGrUrCrArCrArArGrGrGrA 798 TUrGrGrGrGrArCrArArUrUrGrCrCrArArUrCrCrCrU 799 TCrArGrGrGrArCrArArArArCrArCrUrGrUrGrUrCrC 800 TUrGrCrArCrUrGrGrGrGrGrGrGrArCrArCrArGrUrG 801 TCrUrGrCrArCrUrGrGrGrGrGrGrGrArCrArCrArGrU 802 TCrCrCrCrArGrUrGrCrArGrGrGrArArCrCrGrUrGrA 803 TArArArCrCrArGrArArArGrGrCrUrUrArUrCrArCrG 804 TGrArGrCrArCrGrUrArArArUrGrCrGrUrCrCrCrUrG 805 TCrArCrUrArUrCrUrGrUrArCrArGrGrGrArCrGrCrA 806 TUrGrUrArCrArGrArUrArGrUrGrGrGrGrArUrUrUrU 807 TGrUrGrArCrUrArUrUrCrUrCrGrGrGrGrCrCrCrUrG 808 TGrArGrGrCrArArUrArArCrCrCrCrCrUrArCrArCrA 809 TGrUrUrCrGrUrGrCrArArArUrArArUrCrUrUrUrGrU 810 TArArArArArGrCrGrUrUrGrCrArCrArCrArGrUrCrC 811 TCrGrUrUrGrUrArUrGrUrArUrGrArGrUrGrGrCrUrU 812 TGrGrCrUrArCrArArArArArCrCrUrUrCrUrArUrArC 813 TUrUrUrArArGrArArArUrUrUrArArUrUrGrArUrCrA 814 TArGrArArCrUrCrCrGrGrGrCrArArGrArUrGrCrArA 815 TGrGrCrArArGrArUrGrCrArArUrCrUrCrCrGrUrGrG 816 TArCrUrCrArGrArGrArArGrCrGrArCrArGrCrArGrA 817 rCrUrGrCrUrCrArUrCrCrArUrUrCrArUrArCrArCrU 818 TUrUrCrArUrArCrArCrUrCrGrArCrArArArUrArUrC819 TUrArCrArCrUrCrGrArCrArArArUrArUrCrUrGrUrU100ME1\59622102.v1Attorney Docket No. 126454-03020820 rArCrArArGrCrCrUrArCrGrGrCrArGrArGrCrArUrG821 rCrUrGrUrArGrUrGrUrCrCrCrArCrArArGrCrCrUrA822 rGrGrGrGrGrArGrUrUrUrCrUrCrUrArArUrGrArArG823 rUrUrArGrArGrArArArCrUrCrCrCrCrCrUrGrArArG824 rUrGrCrCrArGrUrUrGrCrUrUrUrUrCrUrGrUrCrCrC825 rArGrArUrUrArArArCrArGrGrGrGrGrArCrGrGrUrG826 rUrCrUrCrCrUrGrGrUrUrUrUrGrUrGrGrUrGrGrUrG827 rArGrGrCrUrCrCrArGrArArArCrCrArCrArUrCrUrU828 rCrUrCrCrUrArUrGrArGrUrUrUrCrUrGrGrCrUrCrU829 rCrGrCrCrUrCrGrCrUrCrUrGrCrUrGrGrGrGrArCrC830 rCrCrGrCrCrUrCrGrCrUrCrUrGrCrUrGrGrGrGrArC831 rCrCrGrCrUrCrCrArGrGrCrUrCrUrArUrCrUrCrCrC832 rGrGrCrUrCrUrArUrCrUrCrCrCrArCrCrArGrUrGrC833 rUrGrUrUrGrGrGrCrUrCrArGrCrCrCrGrUrGrGrGrA834 rArCrGrGrGrCrUrGrArGrCrCrCrArArCrArGrGrGrA835 rUrCrArGrCrCrUrGrUrCrCrCrUrGrUrUrGrGrGrCrU836 rArUrCrCrArCrCrCrArGrUrArArGrGrCrArCrCrArUAll bases are in upper case Lowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bondsTable 12: gRNA sequences for targeting LDLR with AsCaslla (RR)SEQ ID NO gRNA sequence TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrArGrArUrGrUrUrCr 1202 ArCrGrCrCrArCrGrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrArGrArCrCrUrCrGrCr 1203 CrGrGrCrCrUrUrGrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrUrGrGrGrArGrGrCrAr 1204 GrArArCrArGrGrCrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArGrCrCrUrGrUrUrCrUr 1205 GrCrCrUrCrCrCrArGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrArGrGrArArArGrGrAr 1206 ArGrArArArCrCrArArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrUrCrCrUrCrGrUrGrAr 1207 ArGrGrArUrArArGrArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrCrGrGrGrCrCrUrGrUr 1208 UrUrCrUrCrUrUrArUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrArArGrUrUrUrUrGrAr 1209 GrUrUrUrCrUrCrUrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrGrUrGrArCrArCrArAr 1210 UrCrCrUrCrArArArCrATUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrUrUrGrArGrGrArUr1211 UrGrUrGrUrCrArCrGrG101ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArUrGrUrUrUrGrArGrGrAr 1212 UrUrGrUrGrUrCrArCrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrUrUrCrCrArUrGrUrUr 1213 UrGrArGrGrArUrUrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrUrUrCrArUrCrUrUrCr 1214 CrArUrGrUrUrUrGrArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrArCrArCrArArCrArGr 1215 CrArGrArUrGrGrCrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrUrGrGrUrGrCrCrArUr 1216 CrUrGrCrUrGrUrUrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrCrUrUrArArGrGrAr 1217 ArGrGrUrArCrArArArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrArArGrCrCrArGrGrAr 1218 ArArGrGrGrArUrUrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrGrCrCrArUrGrArArUr 1219 CrCrCrUrUrUrCrCrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArCrGrCrCrArUrGrArAr 1220 UrCrCrCrUrUrUrCrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrCrGrUrCrGrGrArAr 1221 ArUrGrArUrCrUrGrGrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrCrCrArGrArUrCrArUrUr 1222 UrCrCrGrArCrGrCrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrUrGrGrCrArCrCrGr 1223 ArGrArCrCrArArArCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrArArArUrGrArUrGrCr 1224 CrArCrUrUrCrCrCrArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArUrUrCrCrCrGrUrGrGrUr 1225 CrUrCrCrUrUrGrCrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrCrCrCrGrUrGrGrUrCr 1226 UrCrCrUrUrGrCrArCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrUrGrGrUrCrUrCrCrUr 1227 UrGrCrArCrUrUrUrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrUrUrGrUrArCrArCr 1228 UrGrUrGrUrArCrArUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArUrCrCrCrArArCrArCrAr 1229 CrArCrGrArCrArGrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrCrCrUrGrGrGrArUr 1230 CrCrCrArUrCrCrCrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrGrCrCrArGrGrGrArAr 1231 ArGrCrCrCrGrUrGrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrGrArCrArCrGrGrGrCr 1232 UrUrUrCrCrCrUrGrGrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrGrCrArUrUrCrArUrUr 1233 GrArCrArCrGrGrGrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrUrCrArArCrCrUrGrCr 1234 CrCrCrUrCrUrCrUrGrUTUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrArUrCrArCrGrGrCrUr1235 UrUrGrArArGrUrCrCrC102ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArGrCrCrGrUrGrArUrCr 1236 GrUrGrArArUrArUrCrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrCrArCrCrUrArGrUrGr 1237 CrUrUrCrCrArCrUrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrUrUrCrUrArUrGrCrAr 1238 ArArUrGrCrCrUrCrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrUrCrUrArUrGrCrArAr 1239 ArUrGrCrCrUrCrCrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrUrCrCrCrCrArArUrCr 1240 UrUrGrUrCrGrUrUrGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrCrUrGrGrArUrCrGrUr 1241 UrUrGrArCrGrGrGrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArArArGrArArCrCrUrGr 1242 ArArGrUrCrCrCrGrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrUrUrCrUrUrUrCrUrGr 1243 ArArArUrCrGrCrCrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrArGrArCrArGrUrGr 1244 ArCrArGrCrCrUrCrCrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrCrGrGrGrArGrUrCrUr 1245 GrArCrGrGrArGrGrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrArGrArCrUrCrCrCrGr 1246 CrGrUrGrArArGrArUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrGrUrGrArCrArUrCrUr 1247 UrCrArCrGrCrGrGrGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrCrGrUrGrArArGrArUrGr 1248 UrCrArCrArArGrGrGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrGrGrGrArCrArArUrUr 1249 GrCrCrArArUrCrCrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArGrGrGrArCrArArArAr 1250 CrArCrUrGrUrGrUrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrCrArCrUrGrGrGrGrGr 1251 GrGrArCrArCrArGrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrGrCrArCrUrGrGrGrGr 1252 GrGrGrArCrArCrArGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrCrCrArGrUrGrCrArGr 1253 GrGrArArCrCrGrUrGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArArCrCrArGrArArArGr 1254 GrCrUrUrArUrCrArCrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrCrArCrGrUrArArAr 1255 UrGrCrGrUrCrCrCrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArCrUrArUrCrUrGrUrAr 1256 CrArGrGrGrArCrGrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrArCrArGrArUrArGr 1257 UrGrGrGrGrArUrUrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrGrArCrUrArUrUrCrUr 1258 CrGrGrGrGrCrCrCrUrGTUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrGrCrArArUrArArCr1259 CrCrCrCrUrArCrArCrA103ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrUrCrGrUrGrCrArArAr 1260 UrArArUrCrUrUrUrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArArArArGrCrGrUrUrGr 1261 CrArCrArCrArGrUrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrUrUrGrUrArUrGrUrAr 1262 UrGrArGrUrGrGrCrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrUrArCrArArArArAr 1263 CrCrUrUrCrUrArUrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrUrArArGrArArArUrUr 1264 UrArArUrUrGrArUrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrArArCrUrCrCrGrGrGr 1265 CrArArGrArUrGrCrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrArArGrArUrGrCrAr 1266 ArUrCrUrCrCrGrUrGrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrUrCrArGrArGrArArGr 1267 CrGrArCrArGrCrArGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrGrCrUrCrArUrCrCrAr 1268 UrUrCrArUrArCrArCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrCrArUrArCrArCrUrCr 1269 GrArCrArArArUrArUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArCrArCrUrCrGrArCrAr 1270 ArArUrArUrCrUrGrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrArArGrCrCrUrArCrGr 1271 GrCrArGrArGrCrArUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrGrUrArGrUrGrUrCrCr 1272 CrArCrArArGrCrCrUrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrGrGrGrArGrUrUrUrCr 1273 UrCrUrArArUrGrArArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrArGrArGrArArArCrUr 1274 CrCrCrCrCrUrGrArArG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrCrCrArGrUrUrGrCrUr 1275 UrUrUrCrUrGrUrCrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrArUrUrArArArCrArGr 1276 GrGrGrGrArCrGrGrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrUrCrCrUrGrGrUrUrUr 1277 UrGrUrGrGrUrGrGrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrGrCrUrCrCrArGrArAr 1278 ArCrCrArCrArUrCrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrCrCrUrArUrGrArGrUr 1279 UrUrCrUrGrGrCrUrCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrGrCrCrUrCrGrCrUrCrUr 1280 GrCrUrGrGrGrGrArCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrGrCrCrUrCrGrCrUrCrU 1281 rGrCrUrGrGrGrGrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrGrCrUrCrCrArGrGrCr 1282 UrCrUrArUrCrUrCrCrCTUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrCrUrCrUrArUrCrUrCr1283 CrCrArCrCrArGrUrGrC104ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrUrGrGrGrCrUrCrAr 1284 GrCrCrCrGrUrGrGrGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrGrGrGrCrUrGrArGrCr 1285 CrCrArArCrArGrGrGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrArGrCrCrUrGrUrCrCr 1286 CrUrGrUrUrGrGrGrCrUTUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArUrCrCrArCrCrCrArGrUr1287 ArArGrGrCrArCrCrArUAll bases are in upper case Lowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bondsTable 13: LDLR Target Sequences for AsCaslla RVRSEQ ID NO Target Sequence (DNA)837 TAAAACAAAGCTCTGGCAGGC838 CAAGCCTTGCTAGACAGCCAG839 GAGAACTGCCATTGTCGTCTT840 TCCGCCCACCTAGTGCTTCCA841 CAAATGCCTCCAAGCCATTCA842 TGTTTAAAACATGCACGGTGA843 GTTGTATTTCTGAAATAGGGT844 ACGGTTCCCTGCACTGGGGGG845 TGTACAGGGACGCATTTACGT846 TTTGCACTTTGTATATTGGTT847 TTGGTTGAAACTGTTATCACT848 AGTGATAACAGTTTCAACCAA849 TAAGTGATAACAGTTTCAACC850 TATAAGTGATAACAGTTTCAA851 TATATAAGTGATAACAGTTTC852 TATATATAAGTGATAACAGTT853 TACAAAGATTATTTGCACGAA854 TATGAGTGGCTTCTGGGAGAT855 AGTGGCTTCTGGGAGATGGGT856 CAGTGGTTTAAAAAGTGACAC857 GAAGGTTTTTGTAGCCTGAAT858 AATGGATGAGCAGGGGACTTG859 TGTTGAGTGCTAAGTGCGAAC860 CCCTGTAGTGTCCCACAAGCC861 TCCCCTGTAGTGTCCCACAAG862 GGGACAGAAAAGCAACTGGCA863 GTGCAGTCACCTGCCAGCATA864 CTGGCAGGTGACTGCACCATA865 AGTTTCTGGCTCTCACCAGCT105ME1\59622102.v1Attorney Docket No. 126454-03020Table 14: LDLR Targeting Sequences for AsCaslla RVRSEQ ID NO gDNA targeting Domain (RNA)866 rUrArArArArCrArArArGrCrUrCrUrGrGrCrArGrGrC867 rCrArArGrCrCrUrUrGrCrUrArGrArCrArGrCrCrArG868 rGrArGrArArCrUrGrCrCrArUrUrGrUrCrGrUrCrUrU869 rUrCrCrGrCrCrCrArCrCrUrArGrUrGrCrUrUrCrCrA870 rCrArArArUrGrCrCrUrCrCrArArGrCrCrArUrUrCrA871 rUrGrUrUrUrArArArArCrArUrGrCrArCrGrGrUrGrA872 rGrUrUrGrUrArUrUrUrCrUrGrArArArUrArGrGrGrU873 rArCrGrGrUrUrCrCrCrUrGrCrArCrUrGrGrGrGrGrG874 rUrGrUrArCrArGrGrGrArCrGrCrArUrUrUrArCrGrU875 rUrUrUrGrCrArCrUrUrUrGrUrArUrArUrUrGrGrUrU876 rUrUrGrGrUrUrGrArArArCrUrGrUrUrArUrCrArCrU877 rArGrUrGrArUrArArCrArGrUrUrUrCrArArCrCrArA878 rUrArArGrUrGrArUrArArCrArGrUrUrUrCrArArCrC879 rUrArUrArArGrUrGrArUrArArCrArGrUrUrUrCrArA880 rUrArUrArUrArArGrUrGrArUrArArCrArGrUrUrUrC881 rUrArUrArUrArUrArArGrUrGrArUrArArCrArGrUrU882 rUrArCrArArArGrArUrUrArUrUrUrGrCrArCrGrArA883 rUrArUrGrArGrUrGrGrCrUrUrCrUrGrGrGrArGrArU884 rArGrUrGrGrCrUrUrCrUrGrGrGrArGrArUrGrGrGrU885 rCrArGrUrGrGrUrUrUrArArArArArGrUrGrArCrArC886 rGrArArGrGrUrUrUrUrUrGrUrArGrCrCrUrGrArArU887 rArArUrGrGrArUrGrArGrCrArGrGrGrGrArCrUrUrG888 rUrGrUrUrGrArGrUrGrCrUrArArGrUrGrCrGrArArC889 rCrCrCrUrGrUrArGrUrGrUrCrCrCrArCrArArGrCrC890 rUrCrCrCrCrUrGrUrArGrUrGrUrCrCrCrArCrArArG891 rGrGrGrArCrArGrArArArArGrCrArArCrUrGrGrCrA892 rGrUrGrCrArGrUrCrArCrCrUrGrCrCrArGrCrArUrA893 rCrUrGrGrCrArGrGrUrGrArCrUrGrCrArCrCrArUrA894 rArGrUrUrUrCrUrGrGrCrUrCrUrCrArCrCrArGrCrUAll bases are in upper case Lowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bondsTable 15: gRNA sequences for targeting LDLR with AsCaslla (RVR)SEQ ID NO gRNA Sequence TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArArArArCrArArArGrCr 1288 UrCrUrGrGrCrArGrGrCTUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArArGrCrCrUrUrGrCrUr1289 ArGrArCrArGrCrCrArG106ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArGrArArCrUrGrCrCrAr 1290 UrUrGrUrCrGrUrCrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrCrGrCrCrCrArCrCrUr 1291 ArGrUrGrCrUrUrCrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArArArUrGrCrCrUrCrCr 1292 ArArGrCrCrArUrUrCrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrUrUrArArArArCrAr 1293 UrGrCrArCrGrGrUrGrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrUrGrUrArUrUrUrCrUr 1294 GrArArArUrArGrGrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArCrGrGrUrUrCrCrCrUrGr 1295 CrArCrUrGrGrGrGrGrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrArCrArGrGrGrArCr 1296 GrCrArUrUrUrArCrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrUrGrCrArCrUrUrUrGr 1297 UrArUrArUrUrGrGrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrUrGrGrUrUrGrArArArCr 1298 UrGrUrUrArUrCrArCrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrUrGrArUrArArCrArGr 1299 UrUrUrCrArArCrCrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArArGrUrGrArUrArArCr 1300 ArGrUrUrUrCrArArCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArUrArArGrUrGrArUrAr 1301 ArCrArGrUrUrUrCrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArUrArUrArArGrUrGrAr 1302 UrArArCrArGrUrUrUrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArUrArUrArUrArArGrUr 1303 GrArUrArArCrArGrUrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArCrArArArGrArUrUrAr 1304 UrUrUrGrCrArCrGrArA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrArUrGrArGrUrGrGrCrUr 1305 UrCrUrGrGrGrArGrArU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrUrGrGrCrUrUrCrUrGr 1306 GrGrArGrArUrGrGrGrU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrArGrUrGrGrUrUrUrArAr 1307 ArArArGrUrGrArCrArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrArArGrGrUrUrUrUrUrGr 1308 UrArGrCrCrUrGrArArU TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArArUrGrGrArUrGrArGrCr 1309 ArGrGrGrGrArCrUrUrG TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrGrUrUrGrArGrUrGrCrUr 1310 ArArGrUrGrCrGrArArC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrCrCrUrGrUrArGrUrGrUr 1311 CrCrCrArCrArArGrCrC TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrUrCrCrCrCrUrGrUrArGrUr 1312 GrUrCrCrCrArCrArArGTUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrGrGrArCrArGrArArArAr1313 GrCrArArCrUrGrGrCrA107ME1\59622102.v1Attorney Docket No. 126454-03020TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrGrUrGrCrArGrUrCrArCrCr 1314 UrGrCrCrArGrCrArUrA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrCrUrGrGrCrArGrGrUrGrAr 1315 CrUrGrCrArCrCrArUrATUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArUrArGrUrUrUrCrUrGrGrCrUr1316 CrUrCrArCrCrArGrCrUAll bases are in upper case Lowercase “r” represents RNA, 2’ -hydroxy; bases not modified by an “r” are DNAAll bases are linked via standard phosphodiester bondsTable 16A: Exemplary AsCasll (WT) guide RNA (DNA / RNA oligonucleotide) SEQ ID NO Component Details552 Target sequence TTCCTTTCCTCGTGAAGGATA610 Targeting domain TUrUrCrCrUrUrUrCrCrUrCrGrUrGrArArGrGrArU (corresponding RNA rAsequence)27 AsCasl2a stem loop TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrAr (scaffold sequence) U28 crRNA / gRNA TUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrAr UrrUrUrCrCrUrUrUrCrCrUrCrGrUrGrArArGrGrA rUrA29 gRNA with DNA ATGTGTTTTTGTCAAAAGACCTTTTrUrArArU extension rUrUrCrUrArCrUrCrUrUrGrUrArGrArUrrUrUrCrCrUrUrUrCrCrUrCrGrUrGrArArGrGrArUrA All bases are in upper caseLowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNA All linkages between the nucleotides are standard phosphodiester groups.Table 16B: Exemplary SpCas9 guide RNA (DNA / RNA oligonucleotide)SEQ ID NO Component Details61 Target sequence ACGTGGCGTGAACATCTGCC305 Targeting domain rArCrGrUrGrGrCrGrUrGrArArCrArUrCrUrGrCrC (corresponding RNAsequence)30 SpCas9 stem loop TGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGr (scaffold sequence) CrArArGrUrUrArArArArUrArArGrGrCrUrArGrU TCrCrGrUrUrArUrCrArArCrUrUrGrArArArArAr GrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrUr UrU31 crRNA / gRNA rArCrGrUrGrGrCrGrUrGrArArCrArUrCrUrGrCrC TGrUrUrUrUrArGrArGrCrUrArGrArArArUrArGr CrArArGrUrUrArArArArUrArArGrGrCrUrArGrU TCrCrGrUrUrArUrCrArArCrUrUrGrArArArArAr GrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrUrUrU108ME1\59622102.v1Attorney Docket No. 126454-03020All bases are in upper caseLowercase “r” represents RNA, 2’-hydroxy; bases not modified by an “r” are DNAAll linkages between the nucleotides are standard phosphodiester groups.

[0223] In some embodiments, the targeting domain of the gRNA molecule has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a nucleotide sequence set forth in SEQ ID NOs: 305-548; 607-664; 751-836; or 866-894. In some embodiments, the targeting domain of the gRNA molecule has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a nucleotide sequence set forth in SEQ ID NOs: 305-548; 607-664; 751-836; or 866-894. In some embodiments, the targeting domain of the gRNA molecule has less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations relative to a nucleotide sequence set forth in SEQ ID NOs: 305-548; 607-664; 751-836; or 866-894. In some embodiments, the first gRNA molecule targeting domain and the second gRNA molecule targeting domain comprise, a first 5’ targeting domain, and a second 3’ targeting domain as set forth in Table 17A-17B.Table 17A: 5’ and 3’ targeting domain sequences for targeting LDLR with SpCas95' Targeting Domain 3' Targeting DomainSEQ ID NO: SEQ ID NO:305 119306 123307 188308 275314 277315 280316 282317 283284285286287288291292293296299300301109ME1\59622102.v1Attorney Docket No. 126454-03020Table 17B: 5’ and 3’ targeting domain sequences for targeting LDLR with AsCaslla 5' Targeting Domain 3' Targeting DomainSEQ ID NO: SEQ ID NO:610 643611 6476346374.1 Guide RNA modi fications

[0224] The activity, stability, or other characteristics of gRNAs can be altered through the incorporation of certain modifications. As one example, transiently expressed or delivered nucleic acids can be prone to degradation by, e.g., cellular nucleases. Accordingly, the gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not wishing to be bound by theory it is also believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into cells. Those of skill in the art will be aware of certain cellular responses commonly observed in cells, e.g., mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which can comprise induction of cytokine expression and release and cell death, can be reduced or eliminated altogether by the modifications presented herein.

[0225] Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5’ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5’ end) and / or at or near the 3’ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3’ end). In some cases, modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Casl2a gRNA, and / or a targeting domain of a gRNA. In addition, the entire contents of WO2017136794, W02017004279, W02018107028, Yin, et al. Nat Biotechnol 35(12): 1179-1187 (2017) and Finn, et al. Cell Rep 22(9):2227-2235 (2018) are expressly incorporated in their entirety.

[0226] As one example, the 5’ end of a gRNA can comprise a eukaryotic mRNA cap structure or cap analog (e.g., a G(5 )ppp(5 )G cap analog, a.m7G(5 )ppp(5 )G cap analog, or a 3 ’-O-Me-m7G(5 )ppp(5 )G anti reverse cap analog (ARC A)), as shown below:110ME1\59622102.v1Attorney Docket No. 126454-03020The cap or cap analog can be included during either chemical synthesis or in vitro transcription of the gRNA.

[0227] Along similar lines, the 5’ end of the gRNA can lack a 5’ triphosphate group. For instance, in vitro transcribed gRNAs can be phosphatase-treated (e.g., using calf intestinal alkaline phosphatase) to remove a 5’ triphosphate group.

[0228] Another modification involves the addition, at the 3’ end of a gRNA, of a plurality (e.g., 1-10, 10-20, or 25-200) of adenine (A) residues referred to as a polyA tract or sequence (PAS). The polyA tract can be added to a gRNA during chemical synthesis, following in vitro transcription using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase), or in vivo by means of a polyadenylation sequence, as described in Maeder.

[0229] It should be noted that the modifications described herein can be combined in any suitable manner, e.g., a gRNA, whether transcribed in vivo from a DNA vector, or in vitro transcribed gRNA, can comprise either or both of a 5’ cap structure or cap analog and a 3’ polyA tract.

[0230] Guide RNAs can be modified at a 3’ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:Uo owherein “U” can be an unmodified or modified uridine.

[0231] The 3’ terminal U ribose can be modified with a 2’3’ cyclic phosphate as shown below:111ME1\59622102.v1Attorney Docket No. 126454-03020°x / °wherein “U” can be an unmodified or modified uridine.

[0232] Guide RNAs can contain 3’ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.

[0233] In certain embodiments, sugar-modified ribonucleotides can be incorporated into the gRNA, e.g., wherein the 2’ OH-group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate (PhTx) group. In certain embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-O-methyl, 2’-O-methoxyethyl, or 2’-Fluoro modified including, e.g., 2’-F (as illustrated in FIG. 3) or 2’-O-methyl, adenosine (A), 2’-F or 2’-O-methyl, cytidine (C), 2’-F or 2’-O-methyl, uridine (U), 2’-F or 2’-O-methyl, thymidine (T), 2’-F or 2’-O-methyl, guanosine (G), 2’-O-methoxyethyl-5-methyluridine (Teo), 2’ -O-methoxy ethyladenosine (Aeo), 2’ -O-methoxy ethyl-5-methylcyti dine (m5Ceo), and any combinations thereof.

[0234] Guide RNAs can also comprise “locked” nucleic acids (LNA) in which the 2’ OH-group can be connected, e.g., by a C 1-6 alkylene or Cl -6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar. Any suitable moiety can be used to provide such bridges, which may comprise without limitation methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH2)n-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino,112ME1\59622102.v1Attorney Docket No. 126454-03020heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0235] In certain embodiments, a gRNA can comprise a modified nucleotide which is multi cyclic (e.g., tri cyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3’— >2’)).

[0236] Generally, gRNAs comprise the sugar group ribose, which is a 5-membered ring with an oxygen atom. Exemplary modified gRNAs can comprise, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). Although the majority of sugar analog alterations are localized to the 2’ position, other sites are amenable to modification, including the 4’ position. In certain embodiments, a gRNA comprises a 4’-S, 4’-Se or a 4’-C-aminomethyl-2’-O-Me modification.

[0237] In certain embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In certain embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl adenosine, can be incorporated into the gRNA. In certain embodiments, one or more or all of the nucleotides in a gRNA are deoxynucleotides.

[0238] In certain embodiments, a gRNA comprises one or more 2’F modifications. In certain embodiments, a 2’F modification is positioned on a nucleotide internal to the gRNA (which may be represented herein as “i2F”). In certain embodiments, a gRNA comprises multiple 2’F modifications in an aggressive pattern or a conservative pattern. In certain embodiments, a gRNA hairpin region has the same sequence as the gRNA scaffold sequence. For example, a Casl2a gRNA can have hairpin sequence of SEQ ID NO: 33 and a scaffold sequence of SEQ ID NO: 33 (Table 18B). Herein a Casl2a gRNA with an “aggressive” pattern of hairpin 2’F modifications comprises a hairpin comprising 2’F modifications at each of nucleotide positions 7 and 8 of the hairpin. In certain embodiments, a gRNA comprising an aggressive pattern of hairpin 2’F modifications comprises a hairpin of SEQ ID NO: 37. Herein a Casl2a gRNA with a “conservative” pattern of hairpin 2’F modifications comprises a hairpin lacking 2’F modifications at each of nucleotide positions 7 and 8 of the hairpin. In certain113ME1\59622102.v1Attorney Docket No. 126454-03020embodiments, a gRNA comprising a conservative pattern of hairpin 2’F modifications comprises a hairpin of SEQ ID NO: 34.

[0239] In certain embodiments, gRNAs as used herein may be modified or unmodified gRNAs. In certain embodiments, a gRNA may include one or more modifications. In certain embodiments, the one or more modifications may include a phosphorothioate linkage modification, a phosphorodithioate (PS2) linkage modification, a 2’-O-methyl modification, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof. In general, any combination of modifications may be used. In non-limiting examples, patterns of modification comprising, a combination of 5’ extension; 5’3’ idT; hairpin 2’F in an aggressive pattern (see, e.g., the relevant gRNAs in Table 18A-18B for illustration of an exemplary hairpin_2’F aggressive pattern); a combination of 5’3’ idT and hairpin 2’F in an aggressive pattern; or a combination of 5’3’ idT and 5’ extension combination can be used. The pattern of modification of the gRNA, e.g., the modifications illustrated in Fig. 3 or described elsewhere herein, can be applied to gRNAs with any targeting domain (e.g., targeting LDLK) and / or for targeting genes in a tissue specific manner.5.1 Cas9

[0240] Crystal structures have been determined for S. pyogenes Cas9 (Jinek 2014), and for S. aureus Cas9 in complex with a unimolecular guide RNA and a target DNA (Nishimasu 2014; Anders 2014; and Nishimasu 2015).

[0241] A naturally occurring Cas9 protein comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which comprise particular structural and / or functional domains. The REC lobe comprises an arginine-rich bridge helix (BH) domain, and at least one REC domain (e.g., a RECI domain and, optionally, a REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain. While not wishing to be bound by any theory, mutational analyses suggest specific functional roles for the BH and REC domains: the BH domain appears to play a role in gRNA: DNA recognition, whereas the REC domain is thought to interact with the repeatantirepeat duplex of the gRNA and to mediate the formation of the Cas9 / gRNA complex.

[0242] The NUC lobe comprises a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves the non-complementary (i.e., bottom) strand of the target114ME1\59622102.v1Attorney Docket No. 126454-03020nucleic acid. It can be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in S. pyogenes and S. aureus). The HNH domain, meanwhile, is structurally similar to HNN endonuclease motifs, and cleaves the complementary (i.e., top) strand of the target nucleic acid. The PI domain, as its name suggests, contributes to PAM specificity.

[0243] While certain functions of Cas9 are linked to (but not necessarily fully determined by) the specific domains set forth above, these and other functions can be mediated or influenced by other Cas9 domains, or by multiple domains on either lobe. For instance, in S. pyogenes Cas9, as described in Nishimasu 2014, the repeat: antirepeat duplex of the gRNA falls into a groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem loop structure also interact with amino acids in multiple domains (PI, BH and RECI), as do some nucleotides in the second and third stem loops (RuvC and PI domains).Table 18A. Exemplary Cas9 gRNA modification pattern*Modification gRNA sequence GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAHairpin region 1 GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG CUUUU [SEQ ID NO: 30]Exemplary Targeting12345678901234567890regionExemplary conservative GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA modified pattern GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU (hairpin) GCU*U*U*U [SEQ ID NO: 1360]Exemplary conservative 12345678901234567890GUUUUAGAGCUAGAAAUAGCAAG modified patternUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU(targeting region +GGCACCGAGUCGGUGCU*U*U*U [SEQ ID NO: 1361] hairpin)Exemplary conservative l*2*3*45678901234567890GUUUUAGAGCUAGAAAUAGCA modified patternAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA(targeting region +GUGGCACCGAGUCGGUGCU*U*U*U [SEQ ID NO: 1362] hairpin)Exemplary aggressive GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA modified pattern GUCCGUUAUCAAC*U*U*G*A*A*A*A*A*GUGG*CA*C*C (hairpin) *G*A*G*U*CG*G*U*G*C*U*U*U*U*U [SEQ ID NO: 1363]12345678901234567890GUUUUAGAGCUAGAAAUAGCAAG Exemplary aggressive UUAAAAUAAGGCUAGUCCGUUAUCAAC*U*U*G*A*A*A modified pattern*A*A*GUGG*CA*C*C*G*A*G*U*CG*G*U*G*C*U*U*U* (targeting region +U*Uhairpin)[SEQ ID NO: 1364]Exemplary aggressive1*2*3*45*6*7*8*9*01234567890GUUUUAGAGCUAGAAAUA 2’F pattern (targetingGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC*U*U*G*region + hairpin)115ME1\59622102.v1Attorney Docket No. 126454-03020A*A*A*A*A*GUGG*CA*C*C*G*A*G*U*CG*G*U*G*C*U*U*U*U*U [SEQ ID NO: 32]*Underlined = 2’ F modified nucleotide; Bolded = 2’ OMe modified nucleotide; No underline or bold = unmodified nucleotide; Phosphorothioate bonds are indicated by * Table 18B. Exemplary Cas12a gRNA modification pattern*Modification gRNA sequenceHairpin region UAAUUUCUACUCUUGUAGAU [SEQ ID NO: 33] Exemplary Targeting123456789012345678901regionExemplary conservative UAAUUUCUACUCUUGUAGAU2’F pattern (hairpin) [SEQ ID NO: 34]Exemplary conservativeUAAUUUCUACUCUUGUAGAU123456789012345678901 2’F pattern (hairpin[SEQ ID NO: 35]+ targeting regionExemplary conservativeUAAUUUCUACUCUUGUAGAU123456789012345678901 2’F pattern (targeting[SEQ ID NO: 36]region)Exemplary aggressive UAAUUUCUACUCUUGUAGAU2’F pattern (hairpin) [SEQ ID NO: 37]Exemplary aggressiveUAAUUUCUACUCUUGUAGAU123456789012345678901 2’F pattern (hairpin +[SEQ ID NO: 38]targeting region)Exemplary aggressive UAAUUUCUACUCUUGUAGAU123456789012345678901 2’F pattern (targetingregion) [SEQ ID NO: 39]*Underlined = 2’ F modified nucleotide; No underline = unmodified nucleotide

[0244] In certain embodiments, a modified gRNA as described herein comprises one or more modifications of the gRNA in the hairpin region, the targeting domain, or both. For example, but not by way of limitation, the hairpin region of such a modified gRNA can comprise SEQ ID NO: 33. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 2’Fluorine modifications (e.g., see SEQ ID NOs: 34 or 37 in Table 18B) In certain embodiments, the hairpin region of a modified gRNA comprises a DNA extension at the 5’ end of the hairpin region. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 2’0-methyl modifications. For example, in certain embodiments, a modified gRNA comprises a IxPSOMe modification on a 5’ terminus and / or a 3’ terminus; in certain embodiments, a modified gRNA comprises a 3xPSOMe modification on a 5’ terminus and / or a 3’ terminus. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 5’ inverted dT modifications. In certain embodiments, the hairpin region of a modified gRNA comprises one or more 3’ inverted dT modifications. In certain embodiments, the hairpin region of a modified gRNA comprises a 3’ or 5’ pseudoknot.116ME1\59622102.v1Attorney Docket No. 126454-03020In certain embodiments, the hairpin region of a modified gRNA comprises a 3’ pseudoknot. In certain embodiments, the hairpin region of a modified gRNA comprises a locked nucleic acid (LNA). In certain embodiments, the hairpin region of a modified gRNA comprises a LNA with a 5’ extension.

[0245] In certain embodiments, the hairpin region of a modified gRNA can comprise one or more of 5’ extensions, 2’Fluorine modifications, 2’0-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications, a pseudoknot, or an LNA. For example, in certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a IxPSOMe modification on 5’ and 3’ ends. In certain embodiments, the hairpin region of a modified gRNA comprises a IxPSOMe modification on the 5’ end and a 3’ pseudoknot. In certain embodiments, the hairpin region of the modified gRNAs comprises a 5’ extension and IxPSOMe modification on the 3’ end only. In certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a conservative pattern of 2’F modifications. In certain embodiments, the hairpin region of the modified gRNAs comprises a 5’ extension and an aggressive pattern of 2’F modifications. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and inverted dT modifications at the 5’ and 3’ termini. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension, inverted dT modifications at the 5’ and 3’ termini, and an LNA. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and an aggressive pattern of 2’ modifications, and IxPSOMe modifications on 5’ and 3’ termini. In certain embodiments, a modified gRNA comprises a hairpin with a 5’ extension and an aggressive pattern of 2’F modifications, and inverted dT modifications at the 5’ and 3’ termini. In certain embodiments, the hairpin region of a modified gRNA comprises a 5’ extension and a 2’OMe modification.

[0246] In certain embodiments, the targeting domain of the gRNA can comprise one or more of 5’ extensions, 2’Fluorine modifications, 2’0-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications, a pseudoknot, or an LNA. In certain embodiments the targeting domain of a gRNA comprises 2’F modifications at nucleotide positions 1, 8, 9, 10, 11, 12, 17, 19 and optionally 20 and 21. In certain embodiments the targeting domain of a gRNA comprises 2’F modifications at nucleotide positions 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 and optionally 20 and 21. In certain embodiments, the targeting domain of a gRNA comprises one or more of 2’Fluorine modifications, 2’O-methyl modifications, 5’ inverted dT modifications, or 3’ inverted dT modifications.

[0247] In any of the above embodiments, a gRNA can comprise any pattern of 2’F modifications in the hairpin (e.g., a conservative pattern or an aggressive pattern) and any 117ME1\59622102.v1Attorney Docket No. 126454-03020pattern of 2’F modifications in the targeting domain e.g., 2’F modifications at nucleotide positions 1, 8, 9, 10, 11, 12, 17, 19 of the targeting domain; 2’F modifications at nucleotide positions 1, 2, 3, 7, 8, 9, 10, 11, 12, 14, 15, 17, 19 of the targeting domain; or no 2’F modifications in the targeting domain).

[0248] In certain embodiments, a genome editing system described herein comprises a gRNA comprising an RNA portion comprising a 2’F modification. In certain embodiments, the RNA portion comprises a 5’ hairpin and a 3’ targeting domain. In certain embodiments, a gRNA further comprises an extension region at the 5’ end of the hairpin (e.g., a DNA extension). In certain embodiments, a gRNA comprises a hairpin comprising a 2’F modification at one or more of nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin (i.e., counting from the 5’ end of the hairpin, e.g., having SEQ ID NO: 33). In certain embodiments, a gRNA comprises a hairpin comprising multiple 2’F modifications. In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 or 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin with a pattern of 2’F modifications that consists of 2’F modifications at nucleotide positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin (exemplifying an aggressive pattern of 2’F modifications). In certain embodiments, a gRNA comprises a hairpin that does not include a 2’F modification on at least one of nucleotide positions 7 or 8 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at each of nucleotide positions 7 and 8 of the hairpin. In certain embodiments, a gRNA comprises a hairpin comprising 2’F modifications at nucleotide positions 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin. In certain embodiments, a gRNA comprises a hairpin with a pattern of 2’F modifications that consists of 2’F modifications at nucleotide positions 1, 5, 6, 9, 10, 12, 13, 14, 16, 17, 18 and 19 of the hairpin (exemplifying a conservative pattern of 2’F modifications). In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at one or more of nucleotide positions 2, 3, 4, 11 and 15 of the hairpin. In certain embodiments, a gRNA comprises a hairpin that lacks a 2’F modification at each of nucleotide positions 2, 3, 4, 11 and 15 of the hairpin.118ME1\59622102.v1Attorney Docket No. 126454-030205. RNA-guided nucleases

[0249] RNA-guided nucleases according to the present disclosure comprise, but are not limited to, naturally-occurring Class 2 CRISPR nucleases such as Cas9, and Cas12a, as well as other nucleases derived or obtained therefrom. In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g. complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that comprises (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations can exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease having a certain PAM specificity and / or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs. Casl2a), species (e.g., S. pyogenes vs. S. aureus) or variation (e.g., full-length vs. truncated or split; naturally occurring PAM specificity vs. engineered PAM specificity, etc.) of RNA-guided nuclease.

[0250] The PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary to gRNA targeting domains (or “spacers”). Together with protospacer sequences, PAM sequences define target regions or sequences for specific RNA-guided nuclease / gRNA combinations.

[0251] Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. For example, Cas9 nucleases recognize PAM sequences that are 3’ of the protospacer, while Casl2a, on the other hand, generally recognizes PAM sequences that are 5’ of the protospacer.

[0252] In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3’ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. And F. novicida Cas12a recognizes a TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described by Shmakov et al.,119ME1\59622102.v1Attorney Docket No. 126454-030202015, Molecular Cell 60, 385–397, November 5, 2015. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of reference molecules (for instance, in the case of an engineered RNA-guided nuclease, the reference molecule can be the naturally occurring variant from which the RNA-guided nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to the engineered RNA-guided nuclease).

[0253] In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally-occurring RNA-guided nucleases typically form DSBs in target nucleic acids but engineered variants have been produced that generate only SSBs (discussed above) Ran & Hsu, et al., Cell 154(6), 1380–1389, September 12, 2013 (Ran), incorporated by reference herein), or that that do not cut at all.5.2 Casl2a (formerly known as Cpfl)

[0254] The crystal structure of Acidaminococcus sp. Cas12a in complex with crRNA and a double-stranded (ds) DNA target including a TTTN PAM sequence has been solved by Yamano et al. (Cell. 2016 May 5; 165(4): 949–962 (Yamano), incorporated by reference herein). Casl2a, like Cas9, has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe. The REC lobe comprises RECI and REC2 domains, which lack similarity to any known protein structures. The NUC lobe, meanwhile, comprises three RuvC domains (RuvC-I, -II and -III) and a BH domain. However, in contrast to Cas9, the Cas12a REC lobe lacks an HNH domain, and comprises other domains that also lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I, -II and -III), and a nuclease (Nuc) domain.

[0255] While Cas9 and Cas12a share similarities in structure and function, it should be appreciated that certain Cas12a activities are mediated by structural domains that are not analogous to any Cas9 domains. For instance, cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which differs sequentially and spatially from the HNH domain of Cas9. Additionally, the non-targeting portion of Casl2a gRNA (the handle) adopts a pseudoknot structure, rather than a stem loop structure formed by the repeat: antirepeat duplex in Cas9 gRNAs.

[0256] Non-limiting examples of RNA-guided nucleases include, Cas9 (e.g., SpCas9, SaCas9, (KKH) SaCas9, eSpCas9, Cas9-HF1, HypaCas9, dCas9-Fokl, Sniper-Cas9, xCas9, evoCas9, SpCas9-NG, VRQR, VRER, NmeCas9, CjCas9), Casl2a (also known as Cpfl; e.g.,120ME1\59622102.v1Attorney Docket No. 126454-03020AsCasl2a, LbCasl2a), Cas12b (e.g., AaCas12b, BhCas12b, BhCas12bV4), Cas12c (e.g., Cas12cl, Cas12c2), Cas12h (e.g., Cas12hl), Cas12i (e.g., Cas12il), CasX, CasY, and CasΦ5.3 Modifications of RNA-guided nucleases

[0257] The RNA-guided nucleases described above have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that RNA-guided nucleases can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.

[0258] Turning first to modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe have been described above. Exemplary mutations that can be made in the RuvC domains, in the Cas9 HNH domain, or in the Cas12a Nuc domain are described in Ran and Yamano, as well as in Cotta-Ramusino. In general, mutations that reduce or eliminate activity in one of the two nuclease domains result in RNA-guided nucleases with nickase activity, but it should be noted that the type of nickase activity varies depending on which domain is inactivated. As one example, inactivation of a RuvC domain or of a Cas9 HNH domain results in a nickase.

[0259] Modifications of PAM specificity relative to naturally-occurring Cas9 reference molecules have been described by Kleinstiver et al. for both S. pyogenes (Kleinstiver et al., Nature. 2015 Jul 23;523(7561):481-5 (Kleinstiver I)) and S. aureus (Kleinstiver et al., Nat Biotechnol. 2015 Dec; 33(12): 1293-1298 (Klienstiver II)). Kleinstiver et al. have also described modifications that improve the targeting fidelity of Cas9 (Nature, 2016 January 28; 529, 490-495 (Kleinstiver III)). Each of these references is incorporated by reference herein.

[0260] RNA-guided nucleases have been split into two or more parts, as described by Zetsche et al. (Nat Biotechnol. 2015 Feb;33(2): 139-42 (Zetsche II), incorporated by reference), and by Fine et al. (Sci Rep. 2015 Jul 1;5: 10777 (Fine), incorporated by reference).

[0261] RNA-guided nucleases can be, in certain embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the nuclease while still retaining gRNA association, target and PAM recognition, and cleavage activities. In certain embodiments, RNA guided nucleases are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary bound nucleases and linkers are described by Guilinger et al., Nature Biotechnology 32, 577-582 (2014), which is incorporated by reference for all purposes herein.121ME1\59622102.v1Attorney Docket No. 126454-03020

[0262] RNA-guided nucleases also optionally comprise a tag, such as, but not limited to, a nuclear localization signal (NLS) to facilitate movement of RNA-guided nuclease protein into the nucleus. In certain embodiments, the RNA-guided nuclease can incorporate C- and / or N-terminal nuclear localization signals, e.g., SEQ ID NO: 40, presented herein. Nuclear localization sequences are known in the art and are described in Maeder and elsewhere.

[0263] The foregoing list of modifications is intended to be exemplary in nature, and the skilled artisan will appreciate, in view of the instant disclosure, that other modifications can be possible or desirable in certain applications. For brevity, therefore, exemplary systems, methods and compositions of the present disclosure are presented with reference to particular RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used can be modified in ways that do not alter their operating principles. Such modifications are within the scope of the present disclosure.

[0264] Exemplary suitable nuclease variants comprise, but are not limited to, AsCasl2a variants comprising an M537R substitution, an H800A substitution, and / or an F870L substitution, or any combination thereof (numbering scheme according to AsCasl2a wild-type sequence). Exemplary suitable nuclease variants comprise, but are not limited to, SpCas9 variants comprising an D10A substitution, and / or an H840A substitution, (numbering scheme according to SpCas9 wild-type sequence). The entire contents of WO2019 / 067872 are expressly incorporated by reference herein in their entirety. Other suitable modifications of the AsCasl2a amino acid sequence are known to those of ordinary skill in the art. Some nonlimiting exemplary sequences of wild-type Cas9, Cas9 variants, wild-type AsCasl2a, and AsCasl2a variants are as follows:5.3.1 Cas9 amino acid sequencesSpCas9 amino acid sequence MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLI< RTARRRYTRRI< NRICYLQEIFSNEMAI< VDDSFFHRLEESFLVEEDI< I< HE RHPIFGNIVDEVAYHEI< YPTIYHLRI< I< LVDSTDI< ADLRLIYLALAHMII< FRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT122ME1\59622102.v1Attorney Docket No. 126454-03020VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYI< VREINNYHHAHDAYLNAVVGTALII< I< YPI< LESEFVYGDYI< VYDVRI< MIAI< SEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD [SEQ ID NO: 1338]sNLS-SpCas9-NLS-HA variant 1 amino acid sequence:MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAI KKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSF LKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT123ME1\59622102.v1Attorney Docket No. 126454-03020KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITL KSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGDSGGKRPAATKKAGQAKKKKGSYPYDVPDYA [SEQ ID NO: 1339]SpCas9-sNLS variant 2 amino acid sequence:MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRL1< RTARRRYTRRI< NRICYLQEIFSNEMAI< VDDSFFHRLEESFLVEEDI< I< HE RHPIFGNIVDEVAYHEI< YPTIYHLRI< I< LVDSTDI< ADLRLIYLALAHMII< FRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYI< VREINNYHHAHDAYLNAVVGTALII< I< YPI< LESEFVYGDYI< VYDVRI< MIAI< SEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN124ME1\59622102.v1Attorney Docket No. 126454-03020LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKK KRKV [SEQ ID NO: 1340]SpCas9-sNLS D10A variant 3 amino acid sequence:MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRL1< RTARRRYTRRI< NRICYLQEIFSNEMAI< VDDSFFHRLEESFLVEEDI< I< HE RHPIFGNIVDEVAYHEI< YPTIYHLRI< I< LVDSTDI< ADLRLIYLALAHMII< FRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYI< VREINNYHHAHDAYLNAVVGTALII< I< YPI< LESEFVYGDYI< VYDVRI< MIAI< SEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVL SMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKK KRKV [SEQ ID NO: 1341]SpCas9-sNLS D10A H840A variant 4 amino acid sequence:MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLI< RTARRRYTRRI< NRICYLQEIFSNEMAI< VDDSFFHRLEESFLVEEDI< I< HE RHPIFGNIVDEVAYHEI< YPTIYHLRI< I< LVDSTDI< ADLRLIYLALAHMII< FRGHFLIEG 125ME1\59622102.v1Attorney Docket No. 126454-03020DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS...

Claims

Attorney Docket No. 126454-03020WHAT IS CLAIMED IS:

1. A guide RNA (gRNA) molecule comprising a targeting domain that targets a target sequence of a low-density lipoprotein receptor (LDLR) gene,(i) wherein the target sequence comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 73, 300, 61-72, 74-299, 301-304, 549-606, 665-750, 837-865, 1317-1323, 1365-1392, and 1461; and / or(ii) wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 1452, 1475, 305-322, 324-542, 544-548, 607-664, 751-836, 866-894, 1324-1330, 1393-1420, 1451, 1453-1456, 1462, 1464, 1466, 1468, 1470, and 1474.

2. The gRNA molecule of claim 1,(i) wherein the target sequence comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 73, 300, 61-72, 74, 275-299, 301, 302, 553, 589, 1317, 1318, and 1461;(ii) wherein the targeting domain comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 1452, 1475, 305-318, 519-542, 544-546, 611, 647, 1324, 1325, 1451, 1453-1454, 1456, 1462, 1464, 1466, 1470, and 1474; and / or(iii) wherein the gRNA molecule comprises a nucleotide sequence selected from the group consisting ofany one of SEQ IDNOs: 1477, 1450, 900-913, 1114-1138, 1139-1141, 1148, 1184, 1331, 1332, 1449, 1457, 1458, 1460, 1463, 1465, 1467, 1471-1473, 1476, and 1478-1479.

3. A guide RNA (gRNA) molecule comprising a targeting domain that targets a target sequence of a low-density lipoprotein receptor (LDLR) gene, wherein the target sequence comprises a nucleotide sequence located within SEQ ID NO: 1482.

4. The gRNA molecule of claim 3, wherein the target sequence comprises SEQ ID NO: ATTTA.

5. The gRNA molecule of any one of claims 1-4, wherein the gRNA molecule comprises one or more modifications.257ME1\59622102.v1Attorney Docket No. 126454-030206. The gRNA molecule of any one of claims 1-5, wherein the gRNA molecule comprises one or more modifications selected from the group consisting of a 5’ inverted thymidine (idT) modification, a 3’ idT modification, a 2’ fluoro modification, a 2’ O-m ethyl modification, a phosphorothioate linkage, a 3’ pseudoknot, a locked nucleic acid (LNA), and any combination thereof.

7. The gRNA molecule of claim 6, wherein the gRNA molecule comprises one or more modifications selected from the group consisting of a 2’ O-methyl modification, a phosphorothioate linkage, and any combination thereof.

8. A composition comprising:a first gRNA molecule comprising a first targeting domain that binds to a first target sequence of an LDLR gene, anda second gRNA molecule comprising a second targeting domain that binds to a second target sequence of an LDLR gene,wherein the first and second target sequences of the LDLR gene flank the first AU-rich element (ARE1 element) of the 3’ untranslated region (UTR) of the LDLR gene.

9. The composition of claim 8, wherein the first targeting domain comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1452, 305-318, 1451, and 1454, and wherein the second targeting domain comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1475, 519-542, 544-546, 1456, 1470, and 1474.

10. The composition of claim 8 or claim 9, wherein the first gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1477, 900-913, 1476, 1478, and 1479 and wherein the second gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1450, 1114-1137, 1139-1141, 1449, 1460, and 1471.

11. A genome editing system comprising:at least one gRNA molecule according to any one of claims 1-7, or a nucleic acid molecule encoding the gRNA molecule of any one of claims 1-7; andan RNA-guided nuclease, or a nucleic acid encoding the RNA-guided nuclease.258ME1\59622102.v1Attorney Docket No. 126454-0302012. The genome editing system of claim 11, comprisinga first gRNA molecule according to any one of claims 1-7,a second gRNA molecule according to any one of claims 1-7; anda RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease, wherein the first gRNA molecule and the second gRNA molecule do not have the same nucleotide sequence.

13. A genome editing system comprising:the composition of any one of claims 8-10, or a nucleic acid molecule encoding the first gRNA molecule and the second gRNA molecule of the composition of any one of claims 8-10; andan RNA-guided nuclease, or a nucleic acid encoding the RNA-guided nuclease.

14. The genome editing system of any one of claims 11-13, wherein the nucleic acid encoding the RNA-guided nuclease is an RNA.

15. The genome editing system of any one of claims 11-14, wherein the RNA-guided nuclease is selected from the group consisting of Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Cas12h, Cas12i, CasX, CasY, and CasΦ16. The genome editing system of any one of claims 11-15, wherein the RNA-guided nuclease is a Cas9 nuclease.

17. The genome editing system of claim 16, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9 (SpCas9) nuclease.

18. The genome editing system of any one of claims 11-15, wherein the RNA-guided nuclease is a Casl2a nuclease.

19. The genome editing system of claim 18, wherein the Casl2a nuclease is a modified Cas12a nuclease.

20. The genome editing system of claim 18 or claim 19, wherein the modified Casl2a nuclease is a modified Acidaminococcus sp. Casl2a (AsCasl2a) nuclease.259ME1\59622102.v1Attorney Docket No. 126454-0302021. The genome editing system of any one of claims 11-20, wherein the RNA-guided nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1338-1347.

22. The genome editing system of any one of claims 11-21, wherein the nucleic acid encoding the RNA-guided nuclease comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1350-1359 and 1480.

23. A genome editing system comprising a first gRNA molecule comprisinga first targeting domain selected from the group consisting of SEQ ID NOs: 1452, 305-318, 1451, 1453, and 1454,a second gRNA molecule comprising a second targeting domain selected form the group consisting of SEQ ID NOs: 1475, 519-542, 544-546, 1456, 1470, and 1474, and an RNA encoding an RNA-guided nuclease.

24. The genome editing system of claim 23, wherein the first gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1477, 900-913, 1476, 1478, and 1479; and wherein the second gRNA molecule comprises a sequence selected from the group consisting of any one of SEQ ID NOs: 1450, 1114-1137, 1139-1141, 1449, 1460, and 1471.

25. The genome editing system of claim 23 or claim 24, wherein the first targeting domain and the second targeting domain comprise, respectively,SEQ ID NO: 1452 and SEQ ID NO: 1475;SEQ ID NO: 305 and SEQ ID NO: 532;SEQ ID NO: 316 and SEQ ID NO: 531;SEQ ID NO: 317 and SEQ ID NO: 532;SEQ ID NO: 318 and SEQ ID NO: 532;SEQ ID NO: 305 and SEQ ID NO: 544;SEQ ID NO: 316 and SEQ ID NO: 544;SEQ ID NO: 317 and SEQ ID NO: 544; orSEQ ID NO: 318 and SEQ ID NO: 544.260ME1\59622102.v1Attorney Docket No. 126454-0302026. The genome editing system of any one of claims 23-25, wherein the first gRNA molecule and the second gRNA molecule comprise, respectively,SEQ ID NO: 1477 and SEQ ID NO: 1450;SEQ ID NO: 900 and SEQ ID NO: 1127;SEQ ID NO: 911 and SEQ ID NO: 1126;SEQ ID NO: 912 and SEQ ID NO: 1127;SEQ ID NO: 913 and SEQ ID NO: 1127;SEQ ID NO: 900 and SEQ ID NO: 1139;SEQ ID NO: 911 and SEQ ID NO: 1139;SEQ ID NO: 912 and SEQ ID NO: 1139; orSEQ ID NO: 913 and SEQ ID NO: 1139.

27. The genome editing system of any one of claims 23-26, wherein the first targeting domain comprises SEQ ID NO: 1452, and wherein the second targeting domain comprises SEQ ID NO: 1475.

28. The genome editing system of any one of claims 23-27, wherein the first gRNA molecule comprises a sequence of SEQ ID NO: 1477, and wherein the second gRNA molecule comprises a sequence of SEQ ID NO: 1450.

29. The genome editing system of any one of claims 23-28, wherein the first gRNA molecule comprises a first targeting domain that targets a first target sequence, and wherein the second gRNA molecule comprises a second targeting domain that targets a second target sequence, wherein the first target sequence and the second target sequence comprise a first and second nucleotide sequence, respectively, selected from the group consisting of SEQ ID NO: 73 and SEQ ID NO: 300;SEQ ID NO: 79 and SEQ ID NO: 299;SEQ ID NO: 553 and SEQ ID NO: 589;SEQ ID NO: 64 and SEQ ID NO: 277;SEQ ID NO: 64 and SEQ ID NO: 280;SEQ ID NO: 64 and SEQ ID NO: 284;SEQ ID NO: 64 and SEQ ID NO: 285;SEQ ID NO: 64 and SEQ ID NO: 300;SEQ ID NO: 70 and SEQ ID NO: 277;261ME1\59622102.v1Attorney Docket No. 126454-03020SEQ ID NO: 70 and SEQ ID NO: 280;SEQ ID NO: 70 and SEQ ID NO: 284;SEQ ID NO: 70 and SEQ ID NO: 285;SEQ ID NO: 70 and SEQ ID NO: 300;SEQ ID NO: 72 and SEQ ID NO: 277;SEQ ID NO: 72 and SEQ ID NO: 280;SEQ ID NO: 72 and SEQ ID NO: 284;SEQ ID NO: 72 and SEQ ID NO: 285;SEQ ID NO: 72 and SEQ ID NO: 300;SEQ ID NO: 73 and SEQ ID NO: 284;SEQ ID NO: 73 and SEQ ID NO: 285; andSEQ ID NO: 74 and SEQ ID NO: 300.

30. The genome editing system of any one of claims 23-29, wherein the first targeting domain and the second targeting domain, comprising a first and second nucleotide sequence, respectively, selected from the group consisting ofSEQ ID NO: 1452 and SEQ ID NO: 1475;SEQ ID NO: 317 and SEQ ID NO: 544;SEQ ID NO: 1455 and SEQ ID NO: 1456;SEQ ID NO 1468 and SEQ ID NO: 1470;SEQ ID NO: 611 and SEQ ID NO: 647;SEQ ID NO: 308 and SEQ ID NO: 521;SEQ ID NO: 308 and SEQ ID NO: 524;SEQ ID NO: 308 and SEQ ID NO: 528;SEQ ID NO: 308 and SEQ ID NO: 529;SEQ ID NO: 308 and SEQ ID NO: 544;SEQ ID NO: 314 and SEQ ID NO: 521;SEQ ID NO: 314 and SEQ ID NO: 524;SEQ ID NO: 314 and SEQ ID NO: 528;SEQ ID NO: 314 and SEQ ID NO: 529;SEQ ID NO: 314 and SEQ ID NO: 544;SEQ ID NO: 316 and SEQ ID NO: 521;SEQ ID NO: 316 and SEQ ID NO: 521;SEQ ID NO: 316 and SEQ ID NO: 528;262ME1\59622102.v1Attorney Docket No. 126454-03020SEQ ID NO: 316 and SEQ ID NO: 529;SEQ ID NO: 316 and SEQ ID NO: 544;SEQ ID NO: 317 and SEQ ID NO: 528;SEQ ID NO: 317 and SEQ ID NO: 529; andSEQ ID NO: 318 and SEQ ID NO: 544.

31. The genome editing system of any one of claims 11-30, wherein the first and second gRNA molecules comprise a first and second nucleotide sequence, respectively, selected from the group consisting ofSEQ ID NO: 1477 and SEQ ID NO: 1450;SEQ ID NO: 900 and SEQ ID NO: 1139;SEQ ID NO: 1459 and SEQ ID NO: 1460;SEQ ID NO: 1469 and SEQ ID NO: 1471;SEQ ID NO: 1148 and SEQ ID NO: 1184;SEQ ID NO: 903 and SEQ ID NO: 1116;SEQ ID NO: 903 and SEQ ID NO: 1119;SEQ ID NO: 903 and SEQ ID NO: 1123;SEQ ID NO: 903 and SEQ ID NO: 1124;SEQ ID NO: 903 and SEQ ID NO: 1139;SEQ ID NO: 909 and SEQ ID NO: 1116;SEQ ID NO: 909 and SEQ ID NO: 1119;SEQ ID NO: 909 and SEQ ID NO: 1123;SEQ ID NO: 909 and SEQ ID NO: 1124;SEQ ID NO: 909 and SEQ ID NO: 1139;SEQ ID NO: 911 and SEQ ID NO: 1116;SEQ ID NO: 911 and SEQ ID NO: 1123;SEQ ID NO: 911 and SEQ ID NO: 1124;SEQ ID NO: 911 and SEQ ID NO: 1139;SEQ ID NO: 912 and SEQ ID NO: 1123;SEQ ID NO: 912 and SEQ ID NO: 1124; andSEQ ID NO: 913 and SEQ ID NO: 1139.263ME1\59622102.v1Attorney Docket No. 126454-0302032. The genome editing system of claim 30 or claim 31, wherein the first targeting domain comprises a sequence of SEQ ID NO: 1452, and wherein the second targeting domain comprises a sequence of SEQ ID NO: 1475.

33. The genome editing system of any one of claims 30-32, wherein the first gRNA molecule comprises a sequence comprising SEQ ID NO: 1477, and wherein the second gRNA molecule comprises a sequence comprising SEQ ID NO: 1450.

34. The genome editing system of any one of claims 11-33, wherein the RNA guided nuclease is a Streptococcus pyogenes Cas9 (SpCas9) nuclease.

35. The genome editing system of any one of claims 11-34, wherein the genome editing system increases LDLR expression in a target cell by at least about 7-fold as compared to LDLR expression in a control cell or in the target cell prior to contacting with the genome editing system, optionally wherein the LDLR expression is increased in the target cell by at least about 8-fold, at least about 9-fold, or at least about 10-fold, further optionally wherein the LDLR expression is increased by about 7-fold to about 11 -fold, about 8-fold to about 10-fold, or about 9-fold to about 10-fold.

36. A ribonucleoprotein (RNP) complex comprising the genome editing system of any one of claims 11-35.

37. A delivery system for delivering the genome editing system of any one of claims 11-35, wherein the delivery system comprises the at least one gRNA molecule and an RNA molecule encoding the RNA-guided nuclease.

38. The delivery system of claim 37, wherein the delivery system comprises a lipid nanoparticle (LNP) encapsulating the at least one gRNA molecule and the RNA molecule encoding the RNA-guided nuclease.

39. The delivery system of claim 38, wherein the LNP comprises a targeting moiety.

40. The delivery system of claim 39, wherein the targeting moiety is one or more N-acetylgalactosamine (GalNAc) or GalNAc derivatives.264ME1\59622102.v1Attorney Docket No. 126454-0302041. The delivery system of any one of claims 37-40, wherein the LNP comprises an ionizable lipid, a polyethylene glycol (PEG) lipid, a helper lipid, a sterol, or any combination thereof.

42. A method of editing an LDLR gene in a target cell, the method comprising contacting the target cell with the composition of any one of claims 8-10, the genome editing system of any one of claims 11-35, the RNP complex of claim 36, or the delivery system of any one of claims 37-41.

43. The method of claim 42, wherein the target cell is in vivo, ex vivo, or in vitro.

44. The method of claim 43, wherein the target cell is in vivo.

45. The method of any one of claims 42-44, wherein the target cell is from a subject with heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH).

46. The method of any one of claims 42-45, wherein the target cell is a hepatocyte.

47. The method of any one of claims 42-46, wherein contacting the target cell with the genome editing system, the RNP complex, or the delivery system increases an LDLR protein level in the target cell by at least about 1.5-fold to at least about 30-fold, relative to the LDLR protein level in the target cell prior to contacting the target cell with the genome editing system, the RNP complex, or the delivery system, or relative to a control cell.

48. The method of claim 47, wherein the increase in LDLR protein level is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 18.5-fold, at least about 20-fold, at least about 25-fold, at least about 30 fold.

49. A method of treating a disease or a disorder in a subject, the method comprising administering to the subject the genome editing system of any one of claims 11-35, the RNP 265ME1\59622102.v1Attorney Docket No. 126454-03020complex of claim 36, or the delivery system of any one of claims 37-41, thereby treating the disease or disorder.

50. The method of claim 49, wherein the subject has heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH).

51. The method of claim 50, wherein the disease or disorder is heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH).

52. The method of any one of claims 49-51, wherein administering the genome editing system, the RNP complex, or the delivery system increases an LDLR protein level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by at least about 5-fold to at least about 30-fold, relative to the LDLR protein level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject.

53. The method of any one of claims 49-52, wherein administering the genome editing system, the RNP complex, or the delivery system decreases an LDL-cholesterol (LDL-C) level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by at least about 50% to at least about 95%, relative to the LDL-C level in the cell, tissue, fluid, or subject prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell, tissue, fluid, or subject.

54. The method of any one of claims 49-53, wherein administering the genome editing system, the RNP complex, or the delivery system decreases an Apolipoprotein B (ApoB) level in the subject, or in a cell, tissue, or fluid of the subject, e.g., in a liver cell or the liver of the subject, by about at least about 30% to at least about 98% relative to the ApoB level prior to administering the genome editing system, the RNP complex, or the delivery system, or relative to a control cell.

55. The method of any one of claims 49-54, wherein administering the genome editing system, the RNP complex, or the delivery system to the subject decreases a Lipoprotein(a) (Lp(a)) level in the subject, or in a cell, tissue, or fluid of the subject.266ME1\59622102.v1Attorney Docket No. 126454-0302056. The method of claim 55, wherein the Lp(a) level in the subject is decreased by about 50% to about 95% relative to an Lp(a) level in the subject prior to administering the genome editing system, the RNP complex, or the delivery system to the subject, or relative to a control Lp(a) level.

57. The method of any one of claims 55 or 56, wherein the Lp(a) level in the subject is a serum or plasma Lp(a) level.

58. The method of any one of claims 49-57, wherein administering the genome editing system, the RNP complex, or the delivery system reduces a serum or plasma Lp(a) level in the subject by at least about 80%, 85%, or 90% relative to a serum or plasma Lp(a) level in the subject prior to administration.

59. The method of any one of claims 49-58, wherein administering the genome editing system, the RNP complex, or the delivery system reduces a serum or plasma Lp(a) level in the subject to 100 mg / dL or lower.

60. The method of any one of claims 49-59, wherein the subject has a serum or plasma Lp(a) level of 150 mg / dL or higher prior to administering of the genome editing system, the RNP complex, or the delivery system.

61. The method of any one of claims 42-60, wherein the first gRNA molecule comprises a first targeting domain that targets a first target sequence, wherein the first target sequence is a first nucleotide sequence that is located 5’ to SEQ ID NO: 74 in the LDLR gene, and does not comprise SEQ ID NO: 74.

62. The method of claim 61, wherein the first nucleotide sequence is SEQ ID NO: 73.

63. The method of claim 61 or claim 62, wherein the second gRNA molecule comprises a second targeting domain that targets a second target sequence, wherein the second target sequence comprises a second nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 284, 285, 288, 289, or 300.

64. The method of claim 63, wherein the second nucleotide sequence is SEQ ID NO: 300.267ME1\59622102.v1Attorney Docket No. 126454-0302065. The method of any one of claims 61-64, wherein contacting the target cell with, or administering to the subject, the genome editing system, the RNP complex, or the delivery system increases an LDLR protein level in the target cell, or in a cell, tissue, or fluid of the subject, by at least about 2-fold,relative to a LDLR protein level in a target cell, or in a cell, tissue, or fluid of a subject, contacted with or administered a second genome editing system, RNP complex or delivery system which comprises a third gRNA molecule that comprises a third targeting domain that targets a third sequence, wherein the third target sequence is a third nucleotide sequence that comprises SEQ ID NO: 74, or that is located 3’ to SEQ ID NO: 74 in the LDLR gene.268ME1\59622102.v1

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