Targeting system and use thereof

Abstract

Disclosed in the present invention are a targeting system and a use thereof. The system targets and modulates the expression of any one, two or more genes selected from the following: ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and EDNRB. The targeting system of the present invention can be used for prevention or treatment of diseases such as obesity, fatty liver, hypercholesterolemia, hyperlipidemia, diabetes, hypertension, hyperuricemia, and gout.

Description

Targeting system and use thereof

[0001] This application claims priority to Chinese patent application 2024113025922 with a filing date of 2024 / 9 / 18 and Chinese patent application 2025103287232 with a filing date of 2025 / 3 / 19. This application incorporates the entire text of the above-mentioned Chinese patent applications. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to a targeting system and use thereof. BACKGROUND

[0003] There is still an urgent need in the clinic for the prevention and treatment of diseases and conditions such as hypertension, hyperlipidemia, amyloidosis, obesity, etc. SUMMARY

[0004] The first aspect of the present disclosure provides a targeting system, which targets the expression of any one, two or more genes selected from the group consisting of:

[0005] ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA and EDNRB.

[0006] In some embodiments of the present disclosure, the system targets the expression of any one, two or more genes selected from the group consisting of:

[0007] TTR, APOC3, PCSK9, INHBE, ASGR1, LPA, AGT, ANGPTL3, PNPLA3, HSD17B13 and APOB.

[0008] In some embodiments of the present disclosure, the targeting system is a DNA targeting system, an RNA targeting system or a protein targeting system. In some embodiments of the present disclosure, the targeting system is a DNA targeting system.

[0009] In some embodiments of the present disclosure, the targeting system is a gene editing system.

[0010] In some embodiments of the disclosure, the system targets modulating the expression of the ANGPTL3 gene. In some embodiments of the disclosure, the system targets modulating the expression of the LPA gene. In some embodiments of the disclosure, the system targets modulating the expression of the ASGR1 gene. In some embodiments of the disclosure, the system targets modulating the expression of the PNPLA3 gene. In some embodiments of the disclosure, the system targets modulating the expression of the DLG4 gene. In some embodiments of the disclosure, the system targets modulating the expression of the SLC30A8 gene. In some embodiments of the disclosure, the system targets modulating the expression of the RALA gene. In some embodiments of the disclosure, the system targets modulating the expression of the GPR75 gene. In some embodiments of the disclosure, the system targets modulating the expression of the INHBE gene. In some embodiments of the disclosure, the system targets modulating the expression of the AGT gene. In some embodiments of the disclosure, the system targets modulating the expression of the AGTR1 gene. In some embodiments of the disclosure, the system targets modulating the expression of the XDH gene. In some embodiments of the disclosure, the system targets modulating the expression of the SLC2A9 gene. In some embodiments of the disclosure, the system targets modulating the expression of the ALAS1 gene. In some embodiments of the disclosure, the system targets modulating the expression of the EDNRA gene. In some embodiments of the disclosure, the system targets modulating the expression of the EDNRB gene.

[0011] In some embodiments of the disclosure, the targeting system knocks out the target gene, alters the nucleotide sequence of the target gene, inhibits the transcription of the target gene, or activates the transcription of the target gene.

[0012] In some embodiments of the disclosure, the targeting system alters the nucleotide sequence of the target gene. In some embodiments of the disclosure, the targeting system knocks out the target gene. In some embodiments of the disclosure, the targeting system introduces an indel (insertion / deletion) on the nucleotide sequence of the target gene. In some embodiments of the disclosure, the targeting system introduces a base transition on the nucleotide sequence of the target gene. In some embodiments of the disclosure, the targeting system inhibits the transcription of the target gene. In some embodiments of the disclosure, the targeting system activates the transcription of the target gene. In some embodiments of the disclosure, the targeting system increases the transcription level of the target gene. In some embodiments of the disclosure, the targeting system decreases the transcription level of the target gene.

[0013] In some embodiments of the disclosure, the targeting system includes, but is not limited to, a ZFN gene editing system, a TALEN gene editing system, a CRISPR gene editing system, and other artificial nucleases or natural endonuclease systems capable of achieving targeted action at a specific nucleotide sequence.

[0014] For example, RNA-guided nucleases such as Cas9, Cas12, TnpB, IscB, IsrB, Fanzor, Tas (TIGR-Tas system) can be used to target and recognize the DNA strand of the target gene under the guidance of their respective specific guide polynucleotides (e.g., gRNA, ωRNA, tigRNA, etc.), and to cut or edit the target gene as needed.

[0015] In some embodiments of the present disclosure, the alteration of the nucleotide sequence of the target gene includes, but is not limited to, nucleotide base substitution (including but not limited to single base editing), addition of at least one nucleotide, or deletion of at least one nucleotide on the nucleotide sequence.

[0016] Non-limiting examples include, but are not limited to:

[0017] Single base editing: nickase Cas9, nickase Cas12, nickase IscB, or nickase TnpB are fused with base conversion enzymes (such as cytosine deaminase or adenine deaminase), and can be further fused with 0-2 UGI domains, thereby achieving single base precise substitution of C→T or A→G;

[0018] Introducing substitution, insertion or deletion: using Prime editor (such as Cas9 nickase fused with reverse transcriptase) to achieve base substitution, insertion or deletion under the guidance of gRNA or pegRNA;

[0019] Large segment nucleotide modification: using double nicking system (such as Cas9 or Cas12) combined with exogenous donor DNA to achieve fragment insertion, large fragment deletion or gene knock-in by homologous directed repair (HDR) or non-homologous end joining (NHEJ).

[0020] In some embodiments of the present disclosure, the inhibition of the transcription of the target gene can be achieved by fusing a gene editing system with an epigenetic regulation domain, then targeting the regulatory region of the target gene, thereby inducing chromatin state changes to achieve transcription inhibition. Non-limiting examples include, for example, dead Cas12, dead Cas9 or dead TnpB nucleases with inactivated nuclease activity are fused with DNMT1, NMT3A, DNMT3B, DNMT3L, SETDB1, SUV39H1, Ezh2 and / or HDAC, etc. Epigenetic regulation domain fusion can mediate DNA methylation, histone methylation or deacetylation modification, etc., thereby inducing chromatin compression or blocking transcription initiation complex binding, etc., to achieve epigenetic silencing of the target gene.

[0021] In some embodiments of the disclosure, the inhibition of the transcription of the target gene can be achieved by fusing a gene editing system with a transcriptional regulatory domain, and then targeting the promoter or enhancer region of the target gene, thereby directly inhibiting the transcription initiation or transcription elongation process. Non-limiting examples include, for example, dead Cas12, dead Cas9 or dead TnpB nuclease with inactivated nuclease activity fused with KRAB, SID (mSin3 interaction domain), Mxil or other transcriptional repression domains, which can recruit co-repressors and block the activity of RNA polymerase II, thereby achieving transcriptional repression.

[0022] In some embodiments of the disclosure, the inhibition of the transcription of the target gene can be achieved by fusing a gene editing system with a transcriptional regulatory domain, and then targeting the target gene, thereby causing transcriptional repression of the target gene. Non-limiting examples include, for example, dead Cas12, dead Cas9 or dead TnpB nuclease with inactivated nuclease activity fused with KRAB, DNMT1, DNMT3A, DNMT3B, DNMT3L, SETDB1 and / or Ezh2 domain, thereby used to down-regulate the transcription level of the target gene.

[0023] In some embodiments of the disclosure, the target gene is ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA and / or EDNRB gene. In some embodiments of the disclosure, the target nucleic acid is a DNA sequence of ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA and / or EDNRB gene.

[0024] In some embodiments of the disclosure, the target nucleic acid is selected from any one, two or more of the following:

[0025] TTR gene DNA sequence, APOC3 gene DNA sequence, PCSK9 gene DNA sequence, INHBE gene DNA sequence, ASGR1 gene DNA sequence, LPA gene DNA sequence, AGT gene DNA sequence, ANGPTL3 gene DNA sequence, PNPLA3 gene DNA sequence, HSD17B13 gene DNA sequence, APOB gene DNA sequence.

[0026] In some embodiments of the disclosure, the target gene is a mammalian gene. In some embodiments of the disclosure, the target gene is a human gene.

[0027] In some embodiments of the disclosure, the sequence of the gene includes, but is not limited to, exons, introns, and promoter regions located upstream of the transcription initiation site. In some embodiments of the disclosure, the sequence of the gene includes, but is not limited to, enhancers, silencers, and other cis-acting regulatory elements, as well as 5' untranslated regions (5' UTR), 3' untranslated regions (3' UTR), polyadenylation signals, and regulatory elements related to mRNA splicing, stability, and translation efficiency. In some embodiments of the disclosure, the sequence of the gene includes, but is not limited to, variants with a certain proportion of homology to the natural sequence, such as homologous sequences with a sequence homology of no less than 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, synonymous mutant sequences, or functionally equivalent derivative sequences. In some embodiments of the disclosure, the gene also includes the reverse complementary strands of the above-mentioned sequences, as well as nucleic acid molecules after methylation, sulfuration, phosphorylation, or other artificial modifications. In terms of obtaining methods, the sequence of the gene can be derived from natural genomic cloning, cDNA synthesis, in vitro transcription, chemical synthesis, or vector construction, etc.

[0028] In some embodiments of the disclosure, the targeting system knocks down the level of a target RNA, changes the nucleotide sequence of a target RNA, activates the translation of a target RNA, or inhibits the translation of a target RNA, the target RNA being selected from any one, two or more of the following:

[0029] ANGPTL3 RNA, LPA RNA, ASGR1 RNA, PNPLA3 RNA, TTR RNA, APOC3 RNA, PCSK9 RNA, INHBE RNA, AGT RNA, HSD17B13 RNA, APOB RNA, DLG4 RNA, SLC30A8 RNA, RALA RNA, GPR75 RNA, AGTR1 RNA, XDH RNA, SLC2A9 RNA, ALAS1 RNA, EDNRA RNA, and EDNRB RNA. Alternatively, the RNA is mRNA and / or pre-mRNA.

[0030] In some embodiments of the disclosure, the targeting system is: a CRISPR-Cas system (including but not limited to Cas13 + gRNA), siRNA, shRNA, ASO, snRNA, or snoRNA.

[0031] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in any one of SEQ ID NOs: 28-37, or the reverse complement thereof.

[0032] In some embodiments of the disclosure, the targeting system targets an ASGR1 gene sequence, or the reverse complement thereof. In some specific embodiments of the disclosure, the nucleotide sequence of the ASGR1 gene sequence is set forth in SEQ ID NO: 38.

[0033] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 28-38, or the reverse complement thereof.

[0034] The sequence set forth in any one of genes ASGR1, TTR, APOC3, PCSK9, INHBE, APOB, LPA, AGT, ANGPTL3, and HSD17B13, and the reverse complement thereof, can refer to a DNA sequence, or can refer to an RNA sequence (i.e., T and U are interchangeable in the sequence).

[0035] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the guide sequences in Table 17 and Table 18 herein, or the reverse complement thereof.

[0036] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 28, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, such as nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splicing variants, mutants, or conservatively substituted derivative sequences of the natural sequence.

[0037] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 29, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, such as nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splicing variants, mutants, or conservatively substituted derivative sequences of the natural sequence.

[0038] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 30, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0039] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 31, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0040] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 32, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0041] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 33, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0042] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 34, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0043] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 35, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0044] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 36, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0045] In some embodiments of the disclosure, the targeting system targets a sequence as set forth in SEQ ID NO: 38, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0046] In some embodiments of the disclosure, the targeting system targets a GPR75 gene sequence as set forth in SEQ ID NO: 25, or the reverse complement thereof; in some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0047] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of human chromosome 22 (RefSeq: NC_000022.11) at positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, and / or 43928819-43928876 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, such as nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0048] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, or 43928819-43928876 of the NCBI Reference Genome assembly GRCh38.p14.

[0049] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 human chromosome 8 (RefSeq: NC_000008.11) positions 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, and / or 117172598-117172624; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a proportion of homology to the sequence, for example nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0050] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of NCBI Reference Genome assembly GRCh38.p14 human chromosome 8 (RefSeq: NC_000008.11) positions 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, or 117172598-117172624.

[0051] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of human chromosome 7 (RefSeq: NC_000007.14) from position 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, and / or 39696801-39696877 of NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0052] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 7 (RefSeq: NC_000007.14) from position 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, or 39696801-39696877 of NCBI Reference Genome assembly GRCh38.p14.

[0053] In some embodiments of the disclosure, the targeting system targets the nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14, human chromosome 3 (RefSeq: NC_000003.12) positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, and / or 148741430-148741469; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a proportion of sequence homology to the sequence, for example nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0054] In some embodiments of the disclosure, the targeting system comprises the nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of NCBI Reference Genome assembly GRCh38.p14, human chromosome 3 (RefSeq: NC_000003.12) positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, or 148741430-148741469.

[0055] In some embodiments of the disclosure, the targeting system targets the nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14, human chromosome 2 (RefSeq: NC_000002.12) positions 31414656-31414686, 31386496-31386559, 31375408-31375460, and / or 31370393-31370424; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a proportion of sequence homology to the sequence, for example nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0056] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of NCBI Reference Genome assembly GRCh38.p14 human chromosome 2 (RefSeq: NC_000002.12) positions 31414656-31414686, 31386496-31386559, 31375408-31375460, or 31370393-31370424.

[0057] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 human chromosome 4 (RefSeq: NC_000004.12) positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, and / or 10018991-10019028; optionally, the targeting can occur on the 5’→3’ strand and / or 3’→5’ strand of the segment. In some embodiments, the targeting system can also recognize variants having a proportion of homology to the sequence, for example nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0058] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of NCBI Reference Genome assembly GRCh38.p14 human chromosome 4 (RefSeq: NC_000004.12) positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, or 10018991-10019028.

[0059] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, and / or 52199384-52199419 of NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, such as nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0060] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 3 (RefSeq: NC_000003.12) at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, or 52199384-52199419 of NCBI Reference Genome assembly GRCh38.p14.

[0061] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) at positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, and / or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a proportion of homology to the sequence, for example nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0062] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 4 (RefSeq: NC_000004.12) positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14.

[0063] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of human chromosome 13 (RefSeq: NC_000013.11) positions 77899844-77899928 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, such as nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0064] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 13 (RefSeq: NC_000013.11) positions 77899844-77899928.

[0065] In some embodiments of the disclosure, the targeting system targets a nucleotide sequence of human chromosome 17 (RefSeq: NC_000017.11) at positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, and / or 7174012-7174091 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the targeting system can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0066] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 17 (RefSeq: NC_000017.11) at positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, or 7174012-7174091 of the NCBI Reference Genome assembly GRCh38.p14.

[0067] In some embodiments of the disclosure, the targeting system targets a sequence fragment of SEQ ID NO: 28 at positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, and / or 5464-5487, or the reverse complement thereof.

[0068] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, or 5464-5487 of the sequence of SEQ ID NO: 28.

[0069] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, and / or 4961-4985 of the sequence of SEQ ID NO: 29, or the reverse complement thereof.

[0070] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, or 4961-4985 of the sequence of SEQ ID NO: 29.

[0071] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, and / or 4938-4983 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0072] In some embodiments of the disclosure, the targeting system comprises at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, or 4938-4983 of the sequence set forth in SEQ ID NO: 29.

[0073] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0074] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986 of the sequence set forth in SEQ ID NO: 30.

[0075] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, and / or 23943-23970 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0076] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, or 23943-23970 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0077] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0078] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, or 3372-3395 of the sequence set forth in SEQ ID NO: 31.

[0079] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0080] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, or 3372-3395 of the sequence set forth in SEQ ID NO: 31.

[0081] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, and / or 5195-5222 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0082] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, or 5195-5222 of the sequence of SEQ ID NO: 32.

[0083] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, and / or 5170-5217 of the sequence of SEQ ID NO: 32, or the reverse complement thereof.

[0084] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, or 5170-5217 of the sequence of SEQ ID NO: 32.

[0085] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, and / or 1275-1309 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0086] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, or 1275-1309 of the sequence of SEQ ID NO: 33.

[0087] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, and / or 20759-20810 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0088] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, or 20759-20810 of the sequence of SEQ ID NO: 33.

[0089] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, of the sequence set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0090] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, 10401-10676, 10401-10443, 10420-10676, 10423-10676, 10420-10597, 10498-10576, 10524-10576, 10524-10562, 10401-10489, 10605-10630, 10605-10652, 10605-10654, 10605-10663, 10605-10671, 10605-10672, 10630-10652, 10630-10654, 10630-10663, 10630-10671, or 10630-10672 of a sequence as set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0091] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, and / or 2942-2969 of a sequence as set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0092] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, or 2942-2969 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0093] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, and / or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0094] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0095] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, and / or 6765-6810 of the sequence set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0096] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, or 6765-6810 of the sequence set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0097] In some embodiments of the disclosure, the targeting system targets a sequence fragment of positions 7179-7207, 7364-7405, 7686-7754, 7858-7932, 7858-7900, 7877-7932, 8232-8282, 8837-8993, 8918-8993, 8918-8961, and / or 8938-8993 of the GPR75 gene sequence set forth in SEQ ID NO: 25, or the reverse complement thereof.

[0098] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of the GPR75 gene sequence set forth in SEQ ID NO: 25 at positions 7179-7207, 7364-7405, 7686-7754, 7858-7932, 7858-7900, 7877-7932, 8232-8282, 8837-8993, 8918-8993, 8918-8961, or 8938-8993.

[0099] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0100] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0101] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 17 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0102] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 18 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0103] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 19 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0104] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 20 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0105] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 21 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0106] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 22 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0107] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 23 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0108] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0109] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of 17-24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0110] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at most 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0111] In some embodiments of the disclosure, the targeting system is a gene editing system comprising a guide polynucleotide.

[0112] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0113] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any 2 guide sequences in Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure, respectively. In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of at least 17 contiguous nucleotides of any 2 guide sequences in Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure, respectively. In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence of any 2 guide sequences in Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0114] In some embodiments of the disclosure, the targeting system comprises a guide polynucleotide 1 and a guide polynucleotide 2,

[0115] the guide polynucleotide 1 comprises a guide sequence 1 and the guide polynucleotide 2 comprises a guide sequence 2,

[0116] the guide sequence 1 comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any 1 guide sequence in Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure,

[0117] the guide sequence 2 comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any 1 guide sequence in Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0118] Further, the targeting system comprises an RNA-guided nuclease having 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 15, or 16.

[0119] In some embodiments of the disclosure, the targeting system targets a sequence or sequence fragment of one or more genes selected from the group consisting of genes ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and EDNRB, dependent on a nucleotide sequence in the targeting system that is reverse complementary to the sequence or sequence fragment of the gene, which nucleotide sequence can hybridize or interact with the sequence or sequence fragment of the targeted gene.

[0120] In some embodiments of the disclosure, the targeting system targets a sequence or sequence fragment of one or more genes selected from the group consisting of genes ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and EDNRB, dependent on a nucleotide sequence in the targeting system that is reverse complementary to the sequence or sequence fragment of the gene, which nucleotide sequence can hybridize or interact with the sequence or sequence fragment of the targeted gene; the nucleotide sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the sequence or sequence fragment of the gene.

[0121] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, and / or 5464-5487 of the sequence as set forth in SEQ ID NO: 28, or the reverse complement thereof.

[0122] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, and / or 4961-4985 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0123] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, and / or 4938-4983 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0124] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986, or the reverse complement thereof, of the sequence set forth in SEQ ID NO: 30.

[0125] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, and / or 23943-23970, or the reverse complement thereof, of the sequence set forth in SEQ ID NO: 30.

[0126] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, and / or 3372-3395, or the reverse complement thereof, of the sequence set forth in SEQ ID NO: 31.

[0127] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0128] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, and / or 5195-5222 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0129] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, and / or 5170-5217 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0130] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, and / or 1275-1309 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0131] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, and / or 20759-20810 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0132] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, 10401-10676, 10401-10443, 10420-10676, 10423-10676, 10420-10597, 10498-10576, 10524-10576, 10524-10562, 10401-10489, 10605-10630, 10605-10652, 10605-10654, 10605-10663, 10605-10671, 10605-10672, 10630-10652, 10630-10654, 10630-10663, 10630-10671, and / or 10630-10672 of a sequence as set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0133] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, and / or 2942-2969 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0134] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, and / or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0135] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, and / or 6765-6810 of the sequence set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0136] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 7179-7207, 7364-7405, 7686-7754, 7858-7932, 7858-7900, 7877-7932, 8232-8282, 8837-8993, 8918-8993, 8918-8961, and / or 8938-8993 of the GPR75 gene sequence set forth in SEQ ID NO: 25, or the reverse complement thereof.

[0137] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of NCBI Reference Genome assembly GRCh38.p14 of human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, and / or 43928819-43928876, or the reverse complement thereof.

[0138] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment from nucleotide 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, and / or 117172598-117172624 of human chromosome 8 (RefSeq: NC_000008.11) of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0139] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment from nucleotide 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, and / or 39696801-39696877 of human chromosome 7 (RefSeq: NC_000007.14) of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0140] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 3 (RefSeq: NC_000003.12) positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, and / or 148741430-148741469, or the reverse complement thereof.

[0141] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 2 (RefSeq: NC_000002.12) positions 31414656-31414686, 31386496-31386559, 31375408-31375460, and / or 31370393-31370424, or the reverse complement thereof.

[0142] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 4 (RefSeq: NC_000004.12) positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, and / or 10018991-10019028, or the reverse complement thereof.

[0143] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of the sequence of nucleotides at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, and / or 52199384-52199419 of human chromosome 3 (RefSeq: NC_000003.12) of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0144] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of the sequence of nucleotides at positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, and / or 147486066-147486115 of human chromosome 4 (RefSeq: NC_000004.12) of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0145] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 13 (RefSeq: NC_000013.11) positions 77899844-77899928, or the reverse complement thereof.

[0146] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 17 (RefSeq: NC_000017.11) positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, and / or 7174012-7174091, or the reverse complement thereof.

[0147] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0148] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 90% sequence identity to any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0149] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having 100% sequence identity to any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0150] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of any of the guide sequences in Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0151] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of any of the guide sequences shown in Table 3 or 17, or the reverse complement thereof.

[0152] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of any of the guide sequences shown in Table 4, or the reverse complement thereof.

[0153] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of any of the guide sequences shown in Table 5, or the reverse complement thereof.

[0154] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of any of the guide sequences shown in Table 6 or 28, or the reverse complement thereof.

[0155] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of any of the guide sequences shown in Table 7 or 22, or the reverse complement thereof.

[0156] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of any of the guide sequences shown in Table 8 or 21, or the reverse complement thereof.

[0157] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 9, Table 29, Table 40, or Table 42, or the reverse complement thereof.

[0158] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0159] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0160] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0161] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0162] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0163] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 14 or the reverse complement thereof.

[0164] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 24 or the reverse complement thereof.

[0165] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 25 or the reverse complement thereof.

[0166] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 26 or the reverse complement thereof.

[0167] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 27 or the reverse complement thereof.

[0168] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 30 or the reverse complement thereof.

[0169] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 31 or the reverse complement thereof.

[0170] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 32 or the reverse complement thereof.

[0171] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 33 or the reverse complement thereof.

[0172] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 34 or the reverse complement thereof.

[0173] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 35 or the reverse complement thereof.

[0174] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), it is targeting binding to the sequence or targeting cleaving the sequence (introducing a strand break at least on one nucleotide strand of the sequence).

[0175] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), it is targeting binding to the sequence; further, a specific guide polynucleotide directs RNA-guided nuclease binding to the sequence, the guide polynucleotide comprising a guide sequence that is complementary to (or hybridizes to) the target sequence (or the reverse complement thereof); optionally, the complementarity is perfect complementarity, or imperfect complementarity, for example, including 1, 2, or more base mismatches.

[0176] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), it is targeting binding to the sequence.

[0177] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), it is targeting cleaving the sequence (introducing a strand break at least on one nucleotide strand of the sequence).

[0178] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), it is targeting binding to the sequence, and further inducing base conversion.

[0179] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), it is targeting binding to the sequence, and down-regulating the transcription level of a target gene. In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), it is targeting binding to the sequence, and up-regulating the transcription level of a target gene.

[0180] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), the targeting refers to targeted binding mediated by an endonuclease and its guide polynucleotide (such as gRNA, pegRNA or ωRNA) to the sequence, and further inducing base conversion within or near the target sequence. For example, in some embodiments, Cas proteins fused with deaminases (such as Cas9 or Cas12 fused with deaminases such as APOBEC1, AID or TadA) can be used to achieve C→T, A→G or other programmable base editing within or near the target sequence.

[0181] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), the targeting refers to binding to the sequence and down-regulating the transcription level of a target gene. For example, in some embodiments, Cas proteins carrying transcription inhibition domains (such as dCas9 or dCas12 fused with domains such as KRAB, DNMT3A, DNMT3L or EZH2) can be used to achieve silencing effect of DNA methylation or histone modification.

[0182] In some embodiments of the disclosure, when referring to targeting any particular sequence (target sequence), the targeting refers to binding to the sequence and up-regulating the transcription level of a target gene. For example, in some embodiments, Cas proteins carrying transcription activation domains (such as dCas9 or dCas12 fused with factors such as VP64, p65, Rta (VPR), SAM system or TET1) can be used to induce open conformation of the promoter region and improve the expression level of the target gene.

[0183] Another aspect of the disclosure provides a gene editing system, comprising:

[0184] (a) a guide polynucleotide, or a polynucleotide sequence encoding the guide polynucleotide; and

[0185] (b) an RNA-guided nuclease, or a polynucleotide sequence encoding the RNA-guided nuclease;

[0186] The guide polynucleotide is capable of forming a complex with the RNA-guided nuclease and directing the complex to sequence-specific binding to a target nucleic acid.

[0187] In some embodiments of the disclosure, the guide polynucleotide comprises a guide sequence.

[0188] In some embodiments of the disclosure, the guide polynucleotide comprises a backbone sequence. In some embodiments of the disclosure, the backbone sequence interacts with the RNA-guided nuclease. In some embodiments of the disclosure, the backbone sequence forms a complex with the RNA-guided nuclease. In some embodiments of the disclosure, the backbone sequence forms a complex with the RNA-guided nuclease, and the guide polynucleotide directs the complex to sequence-specific binding to a target nucleic acid.

[0189] In some embodiments of the disclosure, the target nucleic acid is an ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and / or EDNRB gene sequence.

[0190] In some embodiments of the disclosure, the target nucleic acid is a TTR, APOC3, PCSK9, INHBE, ASGR1, LPA, AGT, ANGPTL3, PNPLA3, HSD17B13, and / or APOB gene sequence.

[0191] In some embodiments of the disclosure, the target nucleic acid is a TTR, APOC3, PCSK9, INHBE, ASGR1, LPA, AGT, ANGPTL3, PNPLA3, HSD17B13, and / or APOB gene DNA sequence.

[0192] In some embodiments of the disclosure, the target nucleic acid is selected from the sequences as set forth in any one of SEQ ID NOs: 25, 28-38, or the reverse complement thereof.

[0193] In some embodiments of the disclosure, the target nucleic acid is a sense strand of a target gene.

[0194] In some embodiments of the disclosure, the target nucleic acid is an antisense strand of a target gene.

[0195] In some embodiments of the disclosure, the target nucleic acid is an RNA sequence encoded by an ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and / or EDNRB gene.

[0196] In some embodiments of the disclosure, the target nucleic acid is a pre-mRNA sequence encoded by an ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and / or EDNRB gene.

[0197] In some embodiments of the disclosure, the target nucleic acid is an mRNA sequence encoded by an ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and / or EDNRB gene.

[0198] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOs: 25, 28-38, or the reverse complement thereof.

[0199] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence as set forth in any one of (a)-(j) below, or the reverse complement thereof:

[0200] (a) the nucleotide sequence of human chromosome 22 (RefSeq: NC_000022.11) at positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, or 43928819-43928876 of the NCBI Reference Genome assembly GRCh38.P14;

[0201] (b) the nucleotide sequence of human chromosome 8 (RefSeq: NC_000008.11) at positions 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, or 117172598-117172624 of the NCBI Reference Genome assembly GRCh38.P14;

[0202] (c) the nucleotide sequence of human chromosome 7 (RefSeq: NC_000007.14) at positions 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, or 39696801-39696877 of the NCBI Reference Genome assembly GRCh38.p14;

[0203] (d) the nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, or 148741430-148741469 of the NCBI Reference Genome assembly GRCh38.p14;

[0204] (e) the nucleotide sequence of human chromosome 2 (RefSeq: NC_000002.12) at positions 31414656-31414686, 31386496-31386559, 31375408-31375460, or 31370393-31370424 of the NCBI Reference Genome assembly GRCh38.p14;

[0205] (f) the nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) at positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, or 10018991-10019028 of the NCBI Reference Genome assembly GRCh38.p14;

[0206] (g) the nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, or 52199384-52199419 of the NCBI Reference Genome assembly GRCh38.p14;

[0207] (h) the nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) at positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14;

[0208] (i) the nucleotide sequence of human chromosome 13 (RefSeq: NC_000013.11) at positions 77899844-77899928 of the NCBI Reference Genome assembly GRCh38.p14;

[0209] (j) the nucleotide sequence of human chromosome 17 (RefSeq: NC_000017.11) at positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, or 7174012-7174091 of the NCBI Reference Genome assembly GRCh38.p14.

[0210] In some embodiments of the disclosure, the guide polynucleotide comprises a guide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the guide sequence sequences in Tables 3-14, Table 17, Table 18, Tables 20-38, Table 40, Table 42, Table 47, Table 49, and Table 50 of the disclosure, or the reverse complement thereof.

[0211] In some embodiments of the disclosure, the guide polynucleotide comprises a guide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences set forth in (a)-(j) below, or the reverse complement thereof:

[0212] (a) the nucleotide sequence of human chromosome 22 (RefSeq: NC_000022.11) at positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, or 43928819-43928876 of the NCBI Reference Genome assembly GRCh38.P14;

[0213] (b) the nucleotide sequence of human chromosome 8 (RefSeq: NC_000008.11) at positions 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, or 117172598-117172624 of the NCBI Reference Genome assembly GRCh38.P14;

[0214] (c) the nucleotide sequence of human chromosome 7 (RefSeq: NC_000007.14) at positions 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, or 39696801-39696877 of the NCBI Reference Genome assembly GRCh38.P14;

[0215] (d) the nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, or 148741430-148741469 of the NCBI Reference Genome assembly GRCh38.P14;

[0216] (e) the nucleotide sequence of human chromosome 2 (RefSeq: NC_000002.12) at positions 31414656-31414686, 31386496-31386559, 31375408-31375460, or 31370393-31370424 of the NCBI Reference Genome assembly GRCh38.P14;

[0217] (f) the nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) at positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, or 10018991-10019028 of the NCBI Reference Genome assembly GRCh38.P14;

[0218] (g) the nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, or 52199384-52199419 of the NCBI Reference Genome assembly GRCh38.p14;

[0219] (h) the nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) at positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14;

[0220] (i) the nucleotide sequence of human chromosome 13 (RefSeq: NC_000013.11) at positions 77899844-77899928 of the NCBI Reference Genome assembly GRCh38.p14;

[0221] (j) the nucleotide sequence of human chromosome 17 (RefSeq: NC_000017.11) at positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, or 7174012-7174091 of the NCBI Reference Genome assembly GRCh38.p14.

[0222] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of the guide sequences of Table 17 and Table 18 herein, or the reverse complement thereof.

[0223] In some embodiments of the disclosure, the guide sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of the guide sequences of Table 17 and Table 18 herein, or the reverse complement thereof.

[0224] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, and / or 5464-5487 of the sequence set forth in SEQ ID NO: 28, or the reverse complement thereof.

[0225] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, and / or 4961-4985 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0226] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, and / or 4938-4983 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0227] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0228] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, and / or 23943-23970 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0229] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0230] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0231] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, and / or 5195-5222 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0232] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, and / or 5170-5217 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0233] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, and / or 1275-1309 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0234] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, and / or 20759-20810 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0235] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, 10401-10676, 10401-10443, 10420-10676, 10423-10676, 10420-10597, 10498-10576, 10524-10576, 10524-10562, 10401-10489, 10605-10630, 10605-10652, 10605-10654, 10605-10663, 10605-10671, 10605-10672, 10630-10652, 10630-10654, 10630-10663, 10630-10671, and / or 10630-10672 of a sequence set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0236] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, and / or 2942-2969 of a sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0237] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, and / or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0238] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, and / or 6765-6810 of the sequence set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0239] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 1239-1269, 1243-1269, 1239-1265, 1278-1334, 1279-1334, 1289-1334, 1290-1334, 1278-1312, 1279-1312, 1289-1312, 1278-1311, 1279-1311, 1278-1301, 1390-1475, 1425-1475, 1426-1475, 1430-1475, 1431-1475, 1390-1453, 1425-1453, 1426-1453, 1430-1453, 1390-1452, 1425-1452, 1426-1452, 1390-1448, 1425-1448, 1390-1447, 1469-1568, 1470-1563, 1469-1500, 1470-1500, 1469-1492, 1592-1627, 1596-1627, 1599-1627, 1592-1621, 1596-1621, 1592-1618, 1680-1715, 1726-1749, 5117-5179, 5131-5179, 5142-5179, 5117-5164, 5131-5164, 5117-5153, 5187-5218, 5189-5218, 5187-5211, 5208-5240, 5208-5236, 5214-5240, 5256-5280, 5287-5321, 5293-5321, 5294-5321, 5295-5321, 5287-5317, 5293-5317, 5294-5317, 5287-5316, 5293-5316, 5287-5315, 7011-7038, 7015-7038, 7011-7037, and / or 7041-7072 of a sequence as set forth in SEQ ID NO: 37, or the reverse complement thereof.

[0240] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 1239-1265, 1278-1312, 1278-1301, 1289-1312, 1279-1311, 1390-1448, 1390-1447, 1425-1448, 1430-1475, 1469-1492, 1487-1568, 1487-1563, 1490-1563, 1541-1568, 1541-1567, 1592-1627, 1596-1627, 1599-1627, 1596-1621, 1680-1715, 1726-1749, 5117-5179, 5117-5164, 5117-5153, 5131-5164, 5131-5179, 5208-5236, 5256-5280, 5293-5321, 5294-5321, 7011-7038, 1487-1568, 1490-1568, 1541-1568, 1545-1568, 1487-1567, 1490-1567, 1541-1567, 1487-1563, 1490-1563, and / or 1487-1512 of a sequence as set forth in SEQ ID NO: 37, or the reverse complement thereof.

[0241] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 3 or 17, or the reverse complement thereof.

[0242] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 4, or the reverse complement thereof.

[0243] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 5, or the reverse complement thereof.

[0244] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 6 or Table 28, or the reverse complement thereof.

[0245] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 7 or Table 22, or the reverse complement thereof.

[0246] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 8 or Table 21, or the reverse complement thereof.

[0247] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 9, Table 29, Table 40, or Table 42, or the reverse complement thereof.

[0248] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0249] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in SEQ ID NO: 26, or the reverse complement thereof.

[0250] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 11 or the reverse complement thereof.

[0251] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 12 or Table 23 or the reverse complement thereof.

[0252] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 13 or the reverse complement thereof.

[0253] In some embodiments of the disclosure, the gene editing system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 14 or Table 18 or the reverse complement thereof.

[0254] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 24 or the reverse complement thereof.

[0255] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 25 or the reverse complement thereof.

[0256] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 26 or the reverse complement thereof.

[0257] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 27 or the reverse complement thereof.

[0258] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 30 or the reverse complement thereof.

[0259] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 31 or the reverse complement thereof.

[0260] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 32 or the reverse complement thereof.

[0261] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 33 or the reverse complement thereof.

[0262] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 34 or the reverse complement thereof.

[0263] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 35 or the reverse complement thereof.

[0264] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 3 or 17 or the reverse complement thereof.

[0265] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 4 or the reverse complement thereof.

[0266] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the sequences set forth in Table 50 or the reverse complement thereof.

[0267] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 6 or Table 28 or the reverse complement thereof.

[0268] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 7 or Table 22, or the reverse complement thereof.

[0269] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 8 or Table 21, or the reverse complement thereof.

[0270] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 9, Table 29, Table 40, or Table 42, or the reverse complement thereof.

[0271] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0272] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 9, Table 29, Table 40, or Table 42, or the reverse complement thereof.

[0273] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0274] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 12 or Table 23, or the reverse complement thereof.

[0275] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 13, or the reverse complement thereof.

[0276] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 14 or Table 18, or the reverse complement thereof.

[0277] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 24, or the reverse complement thereof.

[0278] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 25, or the reverse complement thereof.

[0279] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 26, or the reverse complement thereof.

[0280] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 27, or the reverse complement thereof. In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 30, or the reverse complement thereof.

[0281] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 31, or the reverse complement thereof.

[0282] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 32, or the reverse complement thereof.

[0283] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 33, or the reverse complement thereof.

[0284] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 34, or the reverse complement thereof.

[0285] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 35, or the reverse complement thereof.

[0286] In some embodiments, the guide sequence is engineered to hybridize to a target nucleic acid.

[0287] In some embodiments of the disclosure, the RNA-guided nuclease optionally comprises: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12f / CasZ, Cas12g, Cas12h, Cas12i, Cas12j, Cas12l, Cas12m, Cas12n, Cas12o, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, TnpB, IscB, IsrB, Fancor, Tas (TIGR-Tas system), or fragments thereof (non-limiting examples include nucleic acid binding domain fragments).

[0288] In some embodiments of the disclosure, the RNA-guided nuclease is optionally selected from Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor, Tas (TIGR-Tas system) nuclease; or fragments thereof, including but not limited to nucleic acid binding domain fragments.

[0289] In some embodiments of the disclosure, the RNA-guided nuclease is optionally selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, PpnCas9, FrCas9, SauCas9, SauriCas9, ScaCas9, St1Cas9, BlatCas9, CdiCas9, GeoCas9, fragments thereof, and mutants or mutant fragments thereof.

[0290] In some embodiments of the disclosure, the RNA-guided nuclease is optionally selected from AsCpf1, enAsCas12a (addgene plasmid #196724), dFnCas12a (addgene plasmid #136379), ErCas12a, LbCas12a D832A, LbCas12a H759A, LbCas12a E795L, FnCas12a3, FnCas12a D917A, AsCas12a R1226A, AsCas12a D908A, AsCas12a E174R / S542R, AsCas12a (S542R / K548V / N552R), PrCas12a, PxCas12a, PcCas12a, PdCas12a, Mb2Cas12a, Mb3Cas12a, MlCas12a, CMaCas12a, CMtCas12a, HkCas12a, Lb5Cas12a, ErCas12a, TsCas12a, FnCpf1, LbCas12a, ttHsCas12a, AaCas12b, AaCas12b D570A, AaCas12b Q119F / E475R / E758R, BhCas12b, BvCas12b, BrCas12b, AkCas12b, AmCas12b, BsCas12b, OspCas12c, Cas12c2 (addgene plasmid #183072), Cas12c_4 (addgene plasmid #183071), Cas12c1 (addgene plasmid #120872), CasY.1 (from Katanobacteria), CasY.2 (from Vogelbacteria), CasY.3 (from Vogelbacteria), CasY.4 (from Parcubacteria), CasY.5 (from Komeilibacteria), CasY.6 (from Kerfeldbacteria), PlmCasX, DpbCasX, UnlCas12f, CnCas12fl, enRhCas12fl, AsCas12fl, SpaCas12fl, Cas12gl (addgene plasmid #120879), Cas12h of WO2021113522A1 (SEQ ID NO: 1), Cas12il (addgene plasmid #171670), Cas12i2 (addgene plasmid #188275), Cas12il (addgene plasmid #171670), Cas12i2 (addgene plasmid #188275), Cas12j (addgene plasmid #120878), Cas12k (addgene plasmid #120877), Cas12l (addgene plasmid #120876), Cas12m (addgene plasmid #120875), Cas12n (addgene plasmid #120874), Cas12o (addgene plasmid #120873), Cas12p (addgene plasmid #120881), Cas12q (addgene plasmid #120882), Cas12r (addgene plasmid #120883), Cas12s (addgene plasmid #120884), Cas12t (addgene plasmid #120885), Cas12u (addgene plasmid #120886), Cas12v (addgene plasmid #120887), Cas12w (addgene plasmid #120888), Cas12x (addgene plasmid #120889), Cas12y (addgene plasmid #120890), Cas12z (addgene plasmid #120891), or any combination thereof.plasmid #120882), Cas12i2 (addgene plasmid #120883), Cas12i protein named Cas12f.4 / Cas12f.5 / Cas12f.6 in CN111757889B, dSiCas12i (D1049A), SiCas12i, Si2Cas12i, WiCas12i, Wi2Cas12i, Wi3Cas12i, SaCas12i, Sa2Cas12i, Sa3Cas12i, WaCas12i, Wa2Cas12i, xCas12i, hfCas12Max, Cas12i-Max (addgene plasmid #188276), Cas12i1 D647A (addgene plasmid #171671), Cas12i-HiFi (addgene plasmid #188269), Cas12i1 D647A, Cas12j3 (addgene plasmid #188497), Cas12j2 (addgene plasmid #188498), AsCas12j-2 (addgene plasmid #191655), Cas12j-8 (addgene plasmid #194966), ShCas12k, N7Cas12k, AcCas12k, Cas12k-TniQ (addgene plasmid #181787), Cas12k-TnsC (addgene plasmid #181789), Cas12l, MmCas12m, MmCas12mΔZF (H549A, C552A), dCas12m-ΔZF (D485A, H549A, C552A), AcCas12n, dAcCas12n (D240), TnpB Actinomadura cellulosilytica strain DSM 45823, TnpB Actinomadura namibiensis strain DSM 44197, TnpB Actinomadura umbrina strain DSM 43927 $, TnpB Actinoplanes lobatus strain DSM 43150 (TnpB-1 and TnpB-2), TnpB Alicyclobacillus macrosporagiidus strain DSM 17980, TnpB Haloactinospora alba Strain DSM 45015, TnpBLipingzhangella halophila strain DSM 102030, TnpB Meiothermus silvanus DSM 9946, TnpB QNFX01000004, ISDra2 TnpB (PDB: 8H1J), KraIscB-1, AwaIscB, OgeuIscB, GtFz1 (from Guillardia theta), SpuFz1 (from Spizellomyces punctatus), NlovFz2 (from Percolozoa Naegleria lovaniensis), MmeFz2 (from Mercenaria mercenaria), fragments thereof, and mutants or fragments of mutants thereof.

[0291] In some embodiments of the disclosure, the RNA-guided nuclease is a Cas12 protein. Optionally, the Cas12d protein comprises: a wild-type Cas12 protein, a fragment of a wild-type Cas12 protein, a variant of a wild-type Cas12 protein, a fragment of a variant of a wild-type Cas12 protein. The fragment can be a functional fragment.

[0292] In some embodiments of the disclosure, the RNA-guided nuclease is an IscB protein. Optionally, the IscB protein comprises: a wild-type IscB protein, a fragment of a wild-type IscB protein, a variant of a wild-type IscB protein, a fragment of a variant of a wild-type IscB protein. The fragment can be a functional fragment.

[0293] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0294] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0295] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0296] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0297] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of C12-279-V4 protein (SEQ ID NO: 1).

[0298] In some embodiments of the disclosure, the Cas12 protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of Cas12i1-69 protein (SEQ ID NO: 15).

[0299] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2.

[0300] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4.

[0301] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 13.

[0302] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 17.

[0303] In some embodiments of the disclosure, the RNA-guided nuclease includes, but is not limited to, a wild-type RNA-guided nuclease, a functional fragment of a wild-type RNA-guided nuclease, a variant of a wild-type RNA-guided nuclease (e.g., a mutant with complete loss of cleavage activity, a mutant with partial loss of cleavage activity, a mutant with improved cleavage activity, a mutant with reduced off-target effects, a mutant with reduced collateral effects), a functional fragment of a variant of a wild-type RNA-guided nuclease, and a fusion protein thereof with other homologous or heterologous domains.

[0304] In some embodiments of the disclosure, the RNA-guided nuclease comprises any one or more of the following fused domains: a subcellular localization signal, a deaminase domain, a transcription activation domain, a transcription repression domain, a translation activation domain, a translation repression domain, a DNA methylation domain, a DNA demethylation domain, a histone methylation domain, a histone demethylation domain, a histone acetylation domain, a histone deacetylation domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0305] In some embodiments of the disclosure, the RNA-guided nuclease comprises a subcellular localization signal.

[0306] In some embodiments of the disclosure, the RNA-guided nuclease comprises a subcellular localization signal and a deaminase domain.

[0307] In some embodiments of the disclosure, the subcellular localization signal is optionally selected from the group consisting of a nuclear localization signal and a nuclear export signal.

[0308] In some embodiments of the disclosure, the polynucleotide sequence encoding the guide polynucleotide is linked to a first regulatory sequence for regulating expression of the guide polynucleotide; the polynucleotide sequence encoding the RNA-guided nuclease is linked to a second regulatory sequence for regulating expression of the RNA-guided nuclease.

[0309] In some embodiments of the disclosure, the RNA-guided nuclease comprises a nuclease moiety and a fused homologous or heterologous domain.

[0310] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; and

[0311] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in any one of SEQ ID NOs: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416.

[0312] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3; and

[0313] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in any one of SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918.

[0314] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and

[0315] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 133, 119, 166, 211, 127, 167, 72, 212, 236, 273, 214, 239, 309, 304, 326, 213, 283, 237, 287, 248, 272, 505, 395, 397, 628, 402, 513, 654, 691, 482, 501, 752, 797, 838, 726, 1094, 995, 1080, 981, 1259, 1279, 1315, 1317, 1280, 1294, 1303, 1295, 1522, 1507, 1450, 1403, 1396, 1496, 1558, 1491, 1389, 1442, 1478, 1565, 1720, 1682, 1763, 1714, 1796, 1762, 1777, 1838, 1831, 1916, 3136, 3137, 3148, 3149, 3150, 3151, 3152, 3153, 1346, 1441, 1564, 1560, 1520, and 1529.

[0316] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and

[0317] the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2908, 2850, 2871, 2906, 2816, 2894, 2858, 2826, 2966, 2942, 2963, 1217, and 1360.

[0318] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and

[0319] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 188, 63, 69, 62, 2067, 1911, 1910, 1438, 1420, 1023, 1051, 878, 886, 1753, 2154, 1761, 2153, 2139, 2145, 2142, 2144, 1763, 2166, 2191, 2182, 2192, 2180, 2202, 2169, 2204, 2168, 2195, 2248, 2264, 2277, 2265, 2235, 2240, 2274, 2262, 2263, 2267, 2272, 2295, 2310, 2347, 2348, 2289, 2314, 2349, 2328, 2332, 2290, 2380, 2365, 793, 771, 2409, 797, 780, 772, 832, 795, 800, 769, 2414, 1271, 1290, 1317, 2417, 1289, 2433, 2439, 2430, 2464, 2758, 2746, 2735, 2761, 2718, 2675, 2706, 2704, 2708, 2713, 2715, 2709, 2690, 2628, 2627, 2659, 2637, 2635, 2639, 2554, 2551, 2590, 2560, 2553, 2550, 2562, 2606, 2579, 2567, 2558, 2533, 2520, 2517, 2519, and 2544.

[0320] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16; and

[0321] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2134, 2137, 2135, and 2133.

[0322] In some embodiments, the gRNA nucleotides are modified with phosphorothioate (PS) linkages. In some embodiments, the first three nucleotides at the 5' end, and the last three nucleotides at the 3' end are modified. In some embodiments, the first four nucleotides at the 5' end, and the last four nucleotides at the 3' end are linked with phosphorothioate (PS) linkages. In some embodiments, the gRNA can also use modified ribonucleotides, deoxyribonucleotides, other synthetic bases and synthetic backbones linkages (such as peptide nucleic acid (PNA), locked nucleic acid (LNA), etc.). In some embodiments, the nucleic acids that make up the gRNA sequence comprise natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2- thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), inserted bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N- phosphoramidite linkages).

[0323] In some embodiments of the disclosure, the guide RNA comprises modified nucleotides. The modifications include, but are not limited to, 2'-O-methyl, 2'-O-methyl-3'-phosphorothioate, or 2'-O-methyl-3'-thioPACE modifications. In some embodiments of the disclosure, the guide RNA comprises modified nucleotides selected from deoxyribonucleotides, locked nucleic acids (LNAs). In some embodiments, the guide RNA comprises at least one chemically modified nucleotide. In some embodiments, the guide RNA is a hybrid RNA-DNA guide, i.e., some RNA nucleotides in the guide RNA are replaced by DNA nucleotides. In some embodiments, the guide RNA is a hybrid RNA-LNA (locked nucleic acid) guide, i.e., some RNA nucleotides in the guide RNA are replaced by LNA nucleotides. In some embodiments of the disclosure, the guide RNA comprises modifications that are phosphorothioate modifications.

[0324] In some embodiments of the disclosure, the guide RNA is linked to a DNA sequence at the 5' end of the DR sequence.

[0325] In some embodiments of the disclosure, the 1-3 nucleotides at the 3’ end of the guide sequence of the guide RNA are phosphorothioate modified and / or 2’-methoxy modified nucleotides. In some embodiments of the disclosure, the 3 nucleotides at the 3’ end of the guide sequence of the guide RNA are phosphorothioate modified and 2’-methoxy modified nucleotides.

[0326] In some embodiments of the disclosure, the guide RNA is linked to a dG* dT* dT* dGdCdAdAdTdCdCdCdAdAdG sequence at the 5’ end of the DR sequence (* indicates phosphorothioate modification) (SEQ ID NO: 5), and the 3 nucleotides at the 3’ end of the guide sequence are phosphorothioate modified and 2’-methoxy modified.

[0327] In some embodiments of the disclosure, the guide RNA

[0328] Another aspect of the disclosure provides a guide polynucleotide comprising a guide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the guide sequences in Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure, or the reverse complement thereof.

[0329] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, and / or 5464-5487 of the sequence set forth in SEQ ID NO: 28, or the reverse complement thereof.

[0330] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, and / or 4961-4985 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0331] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, and / or 4938-4983 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0332] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986, or the reverse complement thereof, of the sequence set forth in SEQ ID NO: 30.

[0333] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, and / or 23943-23970, or the reverse complement thereof, of the sequence set forth in SEQ ID NO: 30.

[0334] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, and / or 3372-3395, or the reverse complement thereof, of the sequence set forth in SEQ ID NO: 31.

[0335] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0336] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, and / or 5195-5222 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0337] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, and / or 5170-5217 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0338] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, and / or 1275-1309 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0339] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, and / or 20759-20810 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0340] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, 10401-10676, 10401-10443, 10420-10676, 10423-10676, 10420-10597, 10498-10576, 10524-10576, 10524-10562, 10401-10489, 10605-10630, 10605-10652, 10605-10654, 10605-10663, 10605-10671, 10605-10672, 10630-10652, 10630-10654, 10630-10663, 10630-10671, and / or 10630-10672 of a sequence as set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0341] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, and / or 2942-2969 of a sequence as set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0342] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, and / or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0343] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence fragment of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, and / or 6765-6810 of the sequence set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0344] In some embodiments of the disclosure, the guide sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 7179-7207, 7364-7405, 7686-7754, 7858-7932, 7858-7900, 7877-7932, 8232-8282, 8837-8993, 8918-8993, 8918-8961, and / or 8938-8993 of the GPR75 gene sequence as set forth in SEQ ID NO: 25, or the reverse complement thereof.

[0345] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and

[0346] The guide sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416.

[0347] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and

[0348] The guide sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918.

[0349] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and

[0350] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 133, 119, 166, 211, 127, 167, 72, 212, 236, 273, 214, 239, 309, 304, 326, 213, 283, 237, 287, 248, 272, 505, 395, 397, 628, 402, 513, 654, 691, 482, 501, 752, 797, 838, 726, 1094, 995, 1080, 981, 1259, 1279, 1315, 1317, 1280, 1294, 1303, 1295, 1522, 1507, 1450, 1403, 1396, 1496, 1558, 1491, 1389, 1442, 1478, 1565, 1720, 1682, 1763, 1714, 1796, 1762, 1777, 1838, 1831, 1916, 3136, 3137, 3148, 3149, 3150, 3151, 3152, 3153, 1346, 1441, 1564, 1560, 1520, and 1529.

[0351] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and

[0352] the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2908, 2850, 2871, 2906, 2816, 2894, 2858, 2826, 2966, 2942, 2963, 1217, and 1360.

[0353] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and

[0354] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 188, 63, 69, 62, 2067, 1911, 1910, 1438, 1420, 1023, 1051, 878, 886, 1753, 2154, 1761, 2153, 2139, 2145, 2142, 2144, 1763, 2166, 2191, 2182, 2192, 2180, 2202, 2169, 2204, 2168, 2195, 2248, 2264, 2277, 2265, 2235, 2240, 2274, 2262, 2263, 2267, 2272, 2295, 2310, 2347, 2348, 2289, 2314, 2349, 2328, 2332, 2290, 2380, 2365, 793, 771, 2409, 797, 780, 772, 832, 795, 800, 769, 2414, 1271, 1290, 1317, 2417, 1289, 2433, 2439, 2430, 2464, 2758, 2746, 2735, 2761, 2718, 2675, 2706, 2704, 2708, 2713, 2715, 2709, 2690, 2628, 2627, 2659, 2637, 2635, 2639, 2554, 2551, 2590, 2560, 2553, 2550, 2562, 2606, 2579, 2567, 2558, 2533, 2520, 2517, 2519, and 2544.

[0355] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16; and

[0356] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2134, 2137, 2135, and 2133.

[0357] Another aspect of the disclosure provides a guide polynucleotide comprising a guide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any of (a)-(j), or the reverse complement thereof:

[0358] (a) the nucleotide sequence of human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, or 43928819-43928876 of the NCBI Reference Genome assembly GRCh38.p14;

[0359] (b) the nucleotide sequence of human chromosome 8 (RefSeq: NC_000008.11) at positions 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, or 117172598-117172624 of the NCBI Reference Genome assembly GRCh38.P14;

[0360] (c) the nucleotide sequence of human chromosome 7 (RefSeq: NC_000007.14) at positions 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, or 39696801-39696877 of the NCBI Reference Genome assembly GRCh38.P14;

[0361] (d) the nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, or 148741430-148741469 of the NCBI Reference Genome assembly GRCh38.P14;

[0362] (e) the nucleotide sequence of human chromosome 2 (RefSeq: NC_000002.12) at positions 31414656-31414686, 31386496-31386559, 31375408-31375460, or 31370393-31370424 of the NCBI Reference Genome assembly GRCh38.p14;

[0363] (f) the nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) at positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, or 10018991-10019028 of the NCBI Reference Genome assembly GRCh38.p14;

[0364] (g) the nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, or 52199384-52199419 of the NCBI Reference Genome assembly GRCh38.p14;

[0365] (h) the nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) at positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14;

[0366] (i) the nucleotide sequence of human chromosome 13 (RefSeq: NC_000013.11) from position 77899844-77899928 of the NCBI Reference Genome assembly GRCh38.p14;

[0367] (j) the nucleotide sequence of human chromosome 17 (RefSeq: NC_000017.11) from position 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, or 7174012-7174091 of the NCBI Reference Genome assembly GRCh38.p14.

[0368] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the guide sequences shown in Table 3 or 17, or the reverse complement thereof.

[0369] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the guide sequences shown in Table 4, or the reverse complement thereof.

[0370] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the guide sequences shown in Table 5, or the reverse complement thereof.

[0371] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the guide sequences shown in Table 6 or Table 28, or the reverse complement thereof.

[0372] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 7 or Table 22, or the reverse complement thereof.

[0373] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 7 or Table 22, or the reverse complement thereof.

[0374] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 9, Table 29, Table 40, or Table 42, or the reverse complement thereof.

[0375] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0376] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 11, or the reverse complement thereof.

[0377] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 12 or Table 23, or the reverse complement thereof.

[0378] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 13, or the reverse complement thereof.

[0379] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 14 or Table 18, or the reverse complement thereof.

[0380] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 24, or the reverse complement thereof.

[0381] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 25, or the reverse complement thereof.

[0382] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 26, or the reverse complement thereof.

[0383] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 27, or the reverse complement thereof.

[0384] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 30, or the reverse complement thereof.

[0385] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 31 or the reverse complement thereof.

[0386] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 32 or the reverse complement thereof.

[0387] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 33 or the reverse complement thereof.

[0388] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 34 or the reverse complement thereof.

[0389] In some embodiments of the disclosure, the guide sequence is a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 35 or the reverse complement thereof.

[0390] In some embodiments of the disclosure, the guide polynucleotide is for use in a gene editing system.

[0391] In some embodiments of the disclosure, the guide polynucleotide is a guide RNA (gRNA).

[0392] In some embodiments of the disclosure, the guide sequence is engineered to hybridize to a target nucleic acid. In some embodiments of the disclosure, the guide sequence is engineered to hybridize to a target nucleic acid with no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide of mismatch.

[0393] In some embodiments of the disclosure, the guide polynucleotide is capable of forming a complex with an RNA-guided nuclease and directing the complex to bind to a sequence of a target RNA with sequence specificity.

[0394] In some embodiments of the disclosure, the guide polynucleotide is capable of forming a complex with an RNA-guided nuclease and directing the complex to bind to and cleave a target nucleic acid.

[0395] In some embodiments of the disclosure, the RNA-guided nuclease is a CRISPR-Cas protein.

[0396] In some embodiments of the disclosure, the guide RNA is capable of forming a CRISPR complex with a Cas protein and directing the CRISPR complex to bind to a sequence of a target nucleic acid with sequence specificity.

[0397] In some embodiments of the disclosure, the guide RNA is capable of forming a CRISPR complex with a Cas protein and directing the CRISPR complex to bind to and cleave a target nucleic acid.

[0398] In some embodiments of the disclosure, the RNA-guided nuclease includes, but is not limited to, Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor, Tas (TIGR-Tas system) nuclease; and fragments thereof, including but not limited to nucleic acid binding domain fragments.

[0399] In some embodiments of the disclosure, the RNA-guided nuclease is a Cas12 protein or an IscB protein.

[0400] In some embodiments of the disclosure, the RNA-guided nuclease is a Cas12i protein.

[0401] In some embodiments of the disclosure, the RNA-guided nuclease is a Cas12 protein. Alternatively, the Cas12d protein includes: a wild type Cas12 protein, a fragment of a wild type Cas12 protein, a variant of a wild type Cas12 protein, a fragment of a variant of a wild type Cas12 protein. The fragment can be a functional fragment.

[0402] In some embodiments of the disclosure, the RNA-guided nuclease is an IscB protein. Alternatively, the IscB protein includes: a wild type IscB protein, a fragment of a wild type IscB protein, a variant of a wild type IscB protein, a fragment of a variant of a wild type IscB protein. The fragment can be a functional fragment.

[0403] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of C12-279-V4 protein (SEQ ID NO: 1).

[0404] In some embodiments of the disclosure, the Cas12 protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of Cas12i1-69 protein (SEQ ID NO: 15).

[0405] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in SEQ ID NO: 3.

[0406] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in SEQ ID NO: 16.

[0407] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in SEQ ID NO: 2.

[0408] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in SEQ ID NO: 4.

[0409] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 13.

[0410] In some embodiments of the disclosure, the backbone sequence of the guide polynucleotide has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 17.

[0411] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and

[0412] The guide sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOS: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416.

[0413] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and

[0414] the guide sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918.

[0415] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and

[0416] the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 133, 119, 166, 211, 127, 167, 72, 212, 236, 273, 214, 239, 309, 304, 326, 213, 283, 237, 287, 248, 272, 505, 395, 397, 628, 402, 513, 654, 691, 482, 501, 752, 797, 838, 726, 1094, 995, 1080, 981, 1259, 1279, 1315, 1317, 1280, 1294, 1303, 1295, 1522, 1507, 1450, 1403, 1396, 1496, 1558, 1491, 1389, 1442, 1478, 1565, 1720, 1682, 1763, 1714, 1796, 1762, 1777, 1838, 1831, 1916, 3136, 3137, 3148, 3149, 3150, 3151, 3152, 3153, 1346, 1441, 1564, 1560, 1520, and 1529.

[0417] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3; and

[0418] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOS: 2908, 2850, 2871, 2906, 2816, 2894, 2858, 2826, 2966, 2942, 2963, 1217, and 1360.

[0419] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15; and

[0420] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 188, 63, 69, 62, 2067, 1911, 1910, 1438, 1420, 1023, 1051, 878, 886, 1753, 2154, 1761, 2153, 2139, 2145, 2142, 2144, 1763, 2166, 2191, 2182, 2192, 2180, 2202, 2169, 2204, 2168, 2195, 2248, 2264, 2277, 2265, 2235, 2240, 2274, 2262, 2263, 2267, 2272, 2295, 2310, 2347, 2348, 2289, 2314, 2349, 2328, 2332, 2290, 2380, 2365, 793, 771, 2409, 797, 780, 772, 832, 795, 800, 769, 2414, 1271, 1290, 1317, 2417, 1289, 2433, 2439, 2430, 2464, 2758, 2746, 2735, 2761, 2718, 2675, 2706, 2704, 2708, 2713, 2715, 2709, 2690, 2628, 2627, 2659, 2637, 2635, 2639, 2554, 2551, 2590, 2560, 2553, 2550, 2562, 2606, 2579, 2567, 2558, 2533, 2520, 2517, 2519, and 2544.

[0421] In some embodiments of the disclosure, the RNA-guided nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16; and

[0422] The guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2134, 2137, 2135, and 2133.

[0423] In some embodiments of the disclosure, the RNA-guided nuclease comprises a nuclease portion and a fused homologous or heterologous domain.

[0424] In some embodiments of the disclosure, the RNA-guided nuclease comprises any one or more of: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0425] In some embodiments of the disclosure, the Cas protein comprises any one or more of: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0426] In some embodiments of the disclosure, the subcellular localization signal is optionally selected from the group consisting of a nuclear localization signal and a nuclear export signal sequence.

[0427] In some embodiments of the disclosure, the guide RNA comprises a guide sequence and a backbone sequence. In some embodiments of the disclosure, the backbone sequence interacts with the RNA-guided nuclease.

[0428] In some embodiments of the disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence. In some embodiments of the disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence that interacts with the RNA-guided nuclease.

[0429] In some embodiments of the disclosure, the guide RNA comprises a guide sequence, a direct repeat sequence, and a tracrRNA sequence. The direct repeat sequence interacts with the tracrRNA sequence and the RNA-guided nuclease. Typically, the direct repeat sequence and the tracrRNA sequence have a region of complementarity that interact, such as the direct repeat sequence and the tracrRNA sequence of SpCas9.

[0430] In some embodiments of the disclosure, the complex reduces the level of the target nucleic acid in an animal, such as in a human.

[0431] In some embodiments of the disclosure, the complex reduces the level of the target nucleic acid in a cell, such as the level of a target nucleic acid in a cell expressing the target nucleic acid.

[0432] In some embodiments of the disclosure, the complex reduces the level of the target nucleic acid in a cell 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 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction of the level of the target nucleic acid can be tested using methods routine in the art; including but not limited to methods as described in the Examples, using untreated cells or cells treated with a gene editing system targeting non-mammalian genomes as negative controls, the level of knockdown of the target nucleic acid in the experimental group compared to the negative controls can be calculated.

[0433] In some embodiments of the disclosure, the number of off-target genes when the complex binds to and cleaves the target nucleic acid is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by methods routine in the art.

[0434] In some embodiments of the disclosure, the complex reduces the level of the protein encoded by the target nucleic acid in an animal, such as a human.

[0435] In some embodiments of the disclosure, the complex reduces the level of the protein encoded by the target nucleic acid in a cell.

[0436] The reduction of the level of the protein encoded by the target RNA can be tested using methods routine in the art; including but not limited to ELISA, Western Blotting, using untreated cells or cells treated with a gene editing system targeting non-mammalian genomes as negative controls, the level of knockdown of the protein encoded by the target nucleic acid in the experimental group compared to the negative controls can be calculated.

[0437] Another aspect of the disclosure provides an isolated nucleic acid encoding the targeting system according to the disclosure.

[0438] In some embodiments of the disclosure, an isolated nucleic acid encoding the gene editing system according to the disclosure is provided.

[0439] Another aspect of the disclosure provides a vector comprising a nucleic acid encoding the targeting system according to the disclosure, and a regulatory sequence regulating the expression of the targeting system.

[0440] In some embodiments of the disclosure, a vector comprising a nucleic acid encoding a gene editing system according to the disclosure, and a regulatory sequence is provided.

[0441] In some embodiments of the disclosure, the vector comprises a polynucleotide sequence encoding a guide polynucleotide according to the disclosure, and a regulatory sequence for regulating expression of the guide polynucleotide.

[0442] In some embodiments of the disclosure, the vector is an mRNA. In some embodiments of the disclosure, the vector is an mRNA delivered by LNP (lipid nanoparticle).

[0443] In some embodiments of the disclosure, the vector is a viral vector. In some embodiments of the disclosure, the vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an HIV viral vector.

[0444] In some embodiments of the disclosure, the regulatory sequence is a promoter and / or enhancer sequence.

[0445] In another aspect of the disclosure, a vector system comprising

[0446] (a) a polynucleotide sequence encoding a guide polynucleotide according to the disclosure, and a first regulatory sequence regulating expression of the guide polynucleotide; and

[0447] (b) a polynucleotide sequence encoding the RNA-guided nuclease, and a second regulatory sequence regulating expression of the RNA-guided nuclease.

[0448] In some embodiments of the disclosure, the vector system comprises one or more vectors.

[0449] In some embodiments of the disclosure, the vector system comprises only one vector.

[0450] In some embodiments of the disclosure, the vector system comprises a plurality of vectors, the polynucleotide sequence encoding the guide RNA and the first regulatory sequence regulating expression of the guide RNA are located on a first vector, and the polynucleotide sequence encoding the RNA-guided nuclease and the second regulatory sequence regulating expression of the RNA-guided nuclease are located on a second vector.

[0451] In some embodiments of the disclosure, the first regulatory sequence and / or the second regulatory sequence is a promoter and / or enhancer sequence. In some embodiments of the disclosure, the first regulatory sequence or the second regulatory sequence is a promoter sequence. In some embodiments of the disclosure, the first regulatory sequence and the second regulatory sequence are a promoter and an enhancer sequence.

[0452] Another aspect of the disclosure provides an adeno-associated viral vector comprising the targeting system, the gene editing system, the vector, or the vector system of the disclosure, wherein the adeno-associated viral vector comprises DNA encoding the RNA- guided nuclease and the guide RNA of the disclosure.

[0453] Another aspect of the disclosure provides a lipid nanoparticle comprising the targeting system, the gene editing system, the vector, or the vector system of the disclosure, wherein the lipid nanoparticle comprises the guide polynucleotide and the mRNA encoding the RNA-guided nuclease of the disclosure.

[0454] Another aspect of the disclosure provides a lentiviral vector comprising the targeting system, the gene editing system, the vector, or the vector system of the disclosure, wherein the lentiviral vector comprises the guide RNA and the mRNA encoding the RNA-guided nuclease of the disclosure; optionally, the lentiviral vector is pseudotyped with an envelope protein; optionally, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

[0455] Another aspect of the disclosure provides a ribonucleoprotein complex comprising the targeting system, the gene editing system, the vector, or the vector system of the disclosure, wherein the ribonucleoprotein complex is formed by the guide polynucleotide and the RNA-guided nuclease of the disclosure.

[0456] Another aspect of the disclosure provides a virus-like particle comprising the targeting system, the vector, or the vector system of the disclosure, wherein the virus-like particle comprises a ribonucleoprotein complex formed by the guide polynucleotide and the RNA-guided nuclease of the disclosure; optionally, the RNA-guided nuclease is fused to a gag protein.

[0457] Another aspect of the disclosure provides a eukaryotic cell comprising the targeting system, the gene editing system, the guide polynucleotide, the nucleic acid, the vector, the vector system, the adeno-associated viral vector, the lipid nanoparticle, the lentiviral vector, the ribonucleoprotein complex, or the virus-like particle of the disclosure; optionally, the eukaryotic cell is a mammalian cell.

[0458] Another aspect of the disclosure provides a pharmaceutical composition comprising the targeting system of the disclosure, the gene editing system of the disclosure, the guide polynucleotide of the disclosure, the nucleic acid of the disclosure, the vector of the disclosure, the vector system of the disclosure, the adeno-associated viral vector of the disclosure, the lipid nanoparticle of the disclosure, the lentiviral vector of the disclosure, the ribonucleoprotein complex of the disclosure, the virus-like particle of the disclosure, or the eukaryotic cell of the disclosure.

[0459] In some embodiments of the disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0460] Another aspect of the disclosure provides use of a targeting system according to the disclosure, a gene editing system according to the disclosure, a guide polynucleotide according to the disclosure, a nucleic acid according to the disclosure, a vector according to the disclosure, a vector system according to the disclosure, an adeno-associated viral vector according to the disclosure, a lipid nanoparticle according to the disclosure, a lentiviral vector according to the disclosure, a ribonucleoprotein complex according to the disclosure, a virus-like particle according to the disclosure, a cell according to the disclosure, and / or a pharmaceutical composition according to the disclosure in any one of the following or in the preparation of a reagent for effecting any one of the following protocols:

[0461] cleaving, nicking, binding, activating or upregulating transcription of, repressing or downregulating transcription of, activating or upregulating translation of, repressing or downregulating translation of, altering the nucleotide sequence of (base substitution, insertion or deletion), inactivating, visualizing or labeling or detecting, transporting, and masking one or more target nucleic acid molecules.

[0462] In some embodiments of the disclosure, use of a targeting system according to the disclosure, a gene editing system according to the disclosure, a guide polynucleotide according to the disclosure, a nucleic acid according to the disclosure, a vector according to the disclosure, a vector system according to the disclosure, an adeno-associated viral vector according to the disclosure, a lipid nanoparticle according to the disclosure, a lentiviral vector according to the disclosure, a ribonucleoprotein complex according to the disclosure, a virus-like particle according to the disclosure, a cell according to the disclosure, and / or a pharmaceutical composition according to the disclosure is in any one of the following or in the preparation of a reagent for effecting any one of the following protocols:

[0463] cleaving one or more target nucleic acid molecules.

[0464] In some embodiments of the disclosure, use of a targeting system according to the disclosure, a gene editing system according to the disclosure, a guide polynucleotide according to the disclosure, a nucleic acid according to the disclosure, a vector according to the disclosure, a vector system according to the disclosure, an adeno-associated viral vector according to the disclosure, a lipid nanoparticle according to the disclosure, a lentiviral vector according to the disclosure, a ribonucleoprotein complex according to the disclosure, a virus-like particle according to the disclosure, a cell according to the disclosure, and / or a pharmaceutical composition according to the disclosure is in any one of the following or in the preparation of a reagent for effecting any one of the following protocols:

[0465] binding one or more target nucleic acid molecules.

[0466] In some embodiments of the disclosure, the targeting system of the disclosure, the gene editing system of the disclosure, the guide polynucleotide of the disclosure, the nucleic acid of the disclosure, the vector of the disclosure, the vector system of the disclosure, the adeno-associated viral vector of the disclosure, the lipid nanoparticle of the disclosure, the lentiviral vector of the disclosure, the ribonucleoprotein complex of the disclosure, the virus-like particle of the disclosure, the cell of the disclosure, and / or the pharmaceutical composition of the disclosure is used in any one or in the preparation of reagents for achieving any one of the following:

[0467] altering the nucleotide sequence (base substitution, insertion, or deletion) of one or more target nucleic acid molecules.

[0468] In some embodiments of the disclosure, the targeting system of the disclosure, the gene editing system of the disclosure, the guide polynucleotide of the disclosure, the nucleic acid of the disclosure, the vector of the disclosure, the vector system of the disclosure, the adeno-associated viral vector of the disclosure, the lipid nanoparticle of the disclosure, the lentiviral vector of the disclosure, the ribonucleoprotein complex of the disclosure, the virus-like particle of the disclosure, the cell of the disclosure, and / or the pharmaceutical composition of the disclosure is used in any one or in the preparation of reagents for achieving any one of the following:

[0469] activating or upregulating the transcription of one or more target nucleic acid molecules, inhibiting or downregulating the transcription of one or more target nucleic acid molecules.

[0470] In some embodiments of the disclosure, the target nucleic acid is selected from any one, two, or more of the following:

[0471] ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and EDNRB.

[0472] In some embodiments of the disclosure, the cleaving, nicking, binding, activating or upregulating transcription of, inhibiting or downregulating transcription of, activating or upregulating translation of, inhibiting or downregulating translation of, altering the nucleotide sequence of (base substitution, insertion or deletion), inactivating, visualizing or labeling or detecting, transporting, and masking of one or more target nucleic acid molecules can be achieved by the targeted system (e.g., the gene editing system) through indel or exogenous template strand-based HDR repair, single base editing (e.g., CBE, ABE), prime editing, CRISPRi, etc.

[0473] In another aspect of the disclosure, a method of diagnosing, treating or preventing a disease or disorder associated with a target nucleic acid is provided, comprising administering to a sample of a subject in need thereof or to a subject in need thereof an effective amount of the targeted system according to the disclosure, the gene editing system according to the disclosure, the guide polynucleotide according to the disclosure, the nucleic acid according to the disclosure, the vector according to the disclosure, the vector system according to the disclosure, the adeno-associated viral vector according to the disclosure, the lipid nanoparticle according to the disclosure, the lentiviral vector according to the disclosure, the ribonucleoprotein complex according to the disclosure, the virus-like particle according to the disclosure, the cell according to the disclosure, and / or the pharmaceutical composition according to the disclosure.

[0474] In some embodiments of the disclosure, the disease or disorder associated with a target nucleic acid comprises a disease or disorder caused by abnormal expression of the target nucleic acid.

[0475] In some embodiments of the disclosure, the disease or disorder associated with a target nucleic acid comprises, but is not limited to, a series of metabolic-related diseases such as obesity, fatty liver, hypercholesterolemia, hyperlipidemia, diabetes, and the like, and hypertension, hyperuricemia, gout, kidney-related diseases, tumors, cancers, skin diseases, joint diseases, ophthalmic diseases, cardiovascular and cerebrovascular diseases, nervous system diseases, infectious diseases, respiratory system diseases, immune or allergic diseases, and digestive system diseases.

[0476] In some embodiments of the disclosure, the disease or disorder associated with a target nucleic acid comprises, but is not limited to, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, coronary heart disease, stroke, myocardial infarction, non-alcoholic fatty liver disease, alcoholic fatty liver disease, obesity, amyloidosis.

[0477] Another aspect of the disclosure provides use of the targeting system according to the disclosure, the gene editing system according to the disclosure, the guide polynucleotide according to the disclosure, the nucleic acid according to the disclosure, the vector according to the disclosure, the vector system according to the disclosure, the adeno-associated viral vector according to the disclosure, the lipid nanoparticle according to the disclosure, the lentiviral vector according to the disclosure, the ribonucleoprotein complex according to the disclosure, the virus-like particle according to the disclosure, the cell according to the disclosure and / or the pharmaceutical composition according to the disclosure in the preparation of a medicament for diagnosing, treating or preventing a disease or disorder associated with a target nucleic acid.

[0478] In some embodiments of the disclosure, the disease or disorder associated with the target nucleic acid comprises a disease or disorder caused by abnormal expression of the target nucleic acid.

[0479] In some embodiments of the disclosure, the disease or disorder associated with the target nucleic acid comprises, but is not limited to, a series of metabolic-related diseases such as obesity, fatty liver, hypercholesterolemia, hyperlipidemia, diabetes, and the like, as well as hypertension, hyperuricemia, gout, kidney-related diseases, tumors, cancers, skin diseases, joint diseases, ophthalmic diseases, cardiovascular and cerebrovascular diseases, nervous system diseases, infectious diseases, respiratory system diseases, immune or allergic diseases, and digestive system diseases.

[0480] In some embodiments of the disclosure, the disease or disorder associated with the target nucleic acid comprises, but is not limited to, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, coronary heart disease, stroke, myocardial infarction, non-alcoholic fatty liver disease, alcoholic fatty liver disease, obesity, amyloidosis. BRIEF DESCRIPTION OF DRAWINGS

[0481] Figure 1 is a C12-279-V4 J10866 plasmid vector map.

[0482] Figure 2 shows a result graph of TTR levels in serum after editing at different doses.

[0483] Figure 3 shows editing efficiency after editing at different doses.

[0484] Figure 4 shows ANGPTL3 protein levels in cell supernatant after co-transfection of C12-279 V4 mRNA and gRNA in HUH7 cells.

[0485] Figure 5 shows ANGPTL3 editing efficiency after co-transfection of C12-279 V4 mRNA and gRNA in HUH7 cells.

[0486] Figure 6 shows editing efficiency of targeting ANGPTL3 after co-transfection of gRNA targeting the CDS region of ANGPTL3 and C12-279 V4 mRNA in HUH7 cells. * indicates human / cynomolgus homology.

[0487] Figure 7 shows the editing efficiency of three variants of C12-279 targeting ANGPTL3 gene in HUH7 cells, different points represent different gRNAs.

[0488] Figure 8 shows the editing efficiency of gRNAs targeting the promoter region of ANGPTL3 in HUH7 cells. * indicates human / cynomolgus homology.

[0489] Figure 9 shows the editing efficiency of gRNAs targeting PCSK9 after co-transfection of Hela cells with C12-279 V4 mRNA.

[0490] Figure 10 shows the results of quantitative analysis of PCSK9 protein levels in cell supernatant after gRNA editing of Hela cells.

[0491] Figure 11 shows the quantitative real-time PCR assay for PCSK9 mRNA levels after editing.

[0492] Figure 12 shows the editing efficiency of gRNAs targeting exon 1 and the promoter region of the ANGPTL3 gene on ANGPTL3 in 293T cells.

[0493] Figure 13 shows the comparison results of editing efficiency of sgRNAs targeting ANGPTL3 with different PAMs on ANGPTL3 in 293T cells.

[0494] Figure 14 shows the editing efficiency of gRNAs targeting exon 1 and the promoter region of the ANGPTL3 gene on ANGPTL3 in HUH7 cells.

[0495] Figure 15 shows the comparison results of editing efficiency of sgRNAs targeting ANGPTL3 with different PAMs on ANGPTL3 in HUH7 cells.

[0496] Figure 16 shows the editing efficiency of gRNAs with editing efficiency higher than 40% in combination with C12-334 variants in Huh7 cells.

[0497] Figures 17-18 show the editing efficiency of 10 gRNAs targeting the exon region of the ANGPTL3 gene and 2 gRNAs targeting the promoter region in combination with 4 variants (V2, V7, V8, V9 respectively) of C12-334 respectively.

[0498] Figures 19-29 show the results of GUIDE-seq off-target test of C12-279 V4 protein and C12-334-V2 protein 2 respectively in combination with gRNAs.

[0499] FIG. 30 shows the editing efficiency of AGT in 293T cells by gRNAs targeting the promoter region and the exon region of AGT.

[0500] FIG. 31 shows the editing efficiency of C12-334 mutants in 293T cells by targeting different PAM sequences of AGT.

[0501] FIG. 32 shows the editing efficiency of AGT after transfecting Hep3B cells with unsaturated doses of RNA (total amount of 2 μg / mL, mRNA:gRNA = 2:1). * indicates human-chimpanzee homology.

[0502] FIG. 33 shows the level of AGT protein in the cytoplasm after transfecting Hep3B cells with unsaturated doses of RNA (total amount of 2 μg / mL, mRNA:gRNA = 2:1). * indicates human-chimpanzee homology.

[0503] FIG. 34 shows the editing efficiency of AGT after transfecting Hep3B cells with saturated doses of RNA (total amount of 8 μg / mL, mRNA:gRNA = 2:1). * indicates human-chimpanzee homology.

[0504] FIG. 35 shows the level of AGT protein in the cytoplasm after transfecting Hep3B cells with saturated doses of RNA (total amount of 8 μg / mL, mRNA:gRNA = 2:1).

[0505] FIG. 36 shows the relative expression level of mRNA of target genes after transfecting Huh7 cells with RNA (total amount of 8 μg / mL, mRNA:gRNA = 2:1).

[0506] FIG. 37 shows the editing efficiency of target genes after transfecting Huh7 cells with RNA (total amount of 8 μg / mL, mRNA:gRNA = 2:1).

[0507] FIG. 38 shows the editing efficiency and off-target effect of each gRNA plasmid vector to be tested.

[0508] FIG. 39 shows the editing efficiency of INHBE-g33 gRNA and ANGPTL3 m106 gRNA on INHBE in vivo in mice. DETAILED DESCRIPTION

[0509] The term “guide sequence” is used interchangeably with “targeting domain”.

[0510] When referring to “guide polynucleotide”, “t” in the sequence can be used interchangeably with “u”.

[0511] When referring to “guide sequence”, “t” in the sequence can be used interchangeably with “u”.

[0512] When referring to RNA sequences, "t" in the sequence can be used interchangeably with "u".

[0513] When referring to "direct repeat sequence", "t" in the sequence can be used interchangeably with "u".

[0514] Definitions section:

[0515] In some embodiments of the disclosure, the targeting system is a gene editing system.

[0516] The term gene editing system refers to a protein, nucleic acid, or a combination thereof, which is capable of modifying an endogenous target nucleic acid sequence (e.g., target RNA, target DNA) when introduced into a cell, essentially relying on any one of: (A) an RNA and an RNA-guided gene editing protein; (B) a gene editing protein that does not rely on an RNA-guided gene editing protein, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN). Wherein the RNA-guided gene editing protein includes but is not limited to: Cas proteins, and IscB, IsrB, TnpB, Fanzor, and the like proteins, or their modified domains, variants, and the like. The RNA can have various forms of expression, including but not limited to: guide RNA, gRNA, arRNA, ωRNA, guide RNA, and the like. Many gene editing systems suitable for use in the present disclosure are known in the art, such as: CRISPR system, OMEGA system, ZFN, TALEN, LEAPER, REPAIR, RESTORE, CUSPER, RESCUE, PASTE, and the like.

[0517] The terms guide RNA, guide polynucleotide, guide RNA, guide RNA, guide RNA, guide RNA, and gRNA are used interchangeably. The term guide RNA is used to refer to a molecule in a gene editing system that forms a complex with an RNA-guided nuclease and directs the complex to bind sequence specifically to a target sequence. The guide RNA comprises a guide sequence, which can hybridize to the target sequence. When the RNA-guided nuclease is a Cas protein such as Cas9 or Cas12 protein, the guide polynucleotide typically comprises a direct repeat sequence linked to the guide sequence, and in some cases the guide polynucleotide further comprises a tracrRNA sequence.

[0518] The term "guide sequence" is used interchangeably with "targeting domain", "guide sequence", "guide sequence", "guide sequence", "spacer sequence", "spacer sequence", "spacer", "protospacer", and the like, and refers to a contiguous sequence of nucleotides in a gRNA that has partial or complete complementarity (reverse complement) to a target sequence in a target nucleic acid. In some cases, the guide sequence can hybridize to a target sequence in a target nucleic acid through base pairing promoted by an RNA-guided gene editing protein. Complete complementarity of a guide sequence to a target sequence described herein is not required, so long as there is sufficient complementarity to cause hybridization or to promote the formation of a gene editing complex. A guide sequence can be at least 15 nt in length, for example, it can be about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt, about 21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt, about 30 nt, about 31 nt, about 32 nt, about 33 nt, about 34 nt, or about 35 nt.

[0519] Suitable direct repeat (DR) sequences can be found in the CRISPR locus structure of prokaryotes (e.g., bacteria, archaea), obtained by experimental screening; or can be obtained by sequence modification or optimization based thereon, non-limiting examples of which include deletion, substitution or addition of 1, 2, 3, 4 or more complementary base pairs in the double-stranded region complementary to the secondary structure of the DR sequence, deletion, substitution or addition of nucleotides in the loop of the stem-loop structure of the secondary structure of the DR sequence (e.g., an aptamer sequence can be inserted in the loop). The size of the direct repeat sequence is usually tens of nucleotides, part of which are reverse complements of each other, i.e., meaning that a secondary structure, e.g., a stem-loop structure (often referred to as a hairpin structure), is formed within the RNA molecule, and the other part is unstructured. When the RNA-guided nuclease is a Cas protein, the direct repeat sequence is a constant part of the gRNA molecule, which contains a strong secondary structure, which is conducive to the interaction between the Cas protein and the gRNA molecule.

[0520] The term "hybridization" or "hybridizing" refers to the process of polynucleotide strands that are completely or partially complementary coming together under appropriate hybridization conditions to form a double-stranded structure or region, including association between nucleic acids by hydrogen bonding. As used herein, the term hybridization includes instances in which the double-stranded structure or region contains one or more bulges or mismatches. Hybridization and the strength of hybridization (i.e., the strength of association between nucleic acids) are influenced by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the Tmof the hybrid formed. While hydrogen bonds are generally formed between adenine and thymine, adenine and uracil, or cytosine and guanine, other non-canonical base pairs can also form hydrogen bonds. It is contemplated that modified nucleotides can form hydrogen bonds in non-canonical ways that allow or facilitate hybridization.

[0521] As used herein, the term target nucleic acid refers to a polynucleotide containing a target sequence, representing a particular sequence or its reverse complement sequence that one wishes to bind, target, or modify using a targeting system. It can be, for example, a stretch of DNA sequence, a mature mRNA molecule, or a pre-mRNA molecule. In some embodiments of the disclosure, the target nucleic acid is a DNA sequence of the ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and / or EDNRB gene. In some embodiments of the disclosure, the target nucleic acid is a pre-mRNA transcribed from the ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and / or EDNRB gene. In some embodiments of the disclosure, the target nucleic acid is a mature mRNA transcribed from the ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and / or EDNRB gene.

[0522] As used herein, the term target sequence refers to a stretch of sequence in a target nucleic acid molecule that can be reverse-complementary (fully complementary or partially complementary) to the guide sequence of a gRNA molecule. The gene editing complex is sequence-specifically localized to the target sequence by the guide sequence and performs the corresponding function at or near (one- or three-dimensional space) this location. The length of the target sequence is often in the tens of nt (nucleotides), e.g., can be about 15 nt, about 20 nt, about 25 nt, about 30 nt, about 35 nt, about 40 nt, about 50 nt, about 60 nt.

[0523] As used herein, the term cleavage refers to the breaking of a covalent bond (e.g., a covalent phosphodiester bond) in the ribosyl phosphate diester backbone of a polynucleotide. This includes breaking of a DNA duplex, or breaking of one of the single strands in a DNA duplex.

[0524] The ability of a guide RNA to direct sequence-specific binding of a gene editing complex to a target nucleic acid can be assessed by any suitable assay. For example, components of a gene editing system sufficient to form a CRISPR complex, including a guide RNA to be tested, can be provided to a host cell having a corresponding target nucleic acid molecule, e.g., by transfection of a vector encoding components of the CRISPR complex, and then the preferential cleavage within the target sequence is assessed. Similarly, cleavage of a target nucleic acid sequence can be assessed in vitro by providing the target nucleic acid, components of a CRISPR complex, including a guide RNA to be tested and a control guide RNA different from the test guide RNA, and comparing the ability of the test and control guide RNAs to bind the target nucleic acid or the rate of cleavage of the target nucleic acid. The ability of a guide RNA to direct cleavage of a target nucleic acid by a complex can also be assessed by the assays described above.

[0525] The terms guide RNA-guided gene editing protein, RNA-guided nuclease, RNA-guided nucleic acid binding protein are used interchangeably. As used herein, when referring to the term RNA-guided nuclease, it is not intended to limit that it must have nuclease activity; i.e., RNA-guided nucleases include proteins that are nuclease inactive (able to bind but not cleave a target sequence), such as dCas9 proteins, nCas9 proteins, dCas12 proteins, nCas12 proteins, etc. (dead Cas, nickase Cas). In some embodiments of the disclosure, the gene editing protein is an RNA-guided nuclease. The term RNA-guided nuclease refers to a polypeptide that binds to a specific target nucleic acid sequence in a sequence-specific manner, and the polypeptide is guided to the target nucleic acid by a guide RNA that is complexed with the polypeptide and hybridizes to a target sequence on the target nucleic acid. In some cases, cleavage of the target sequence by the RNA-guided nuclease can result in a strand break. The RNA-guided nucleases described in the disclosure include, but are not limited to, wild-type RNA-guided nucleases, variants thereof (e.g., mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off-target effects, mutants with reduced collateral cleavage effects), and functional fragments or fusion proteins thereof; e.g., C12-279 proteins and variants thereof, C12-334 proteins and variants thereof.

[0526] As used herein, the term Cas protein is a CRISPR-associated (Cas) polypeptide or protein that, when complexed or functionally combined with one or more guide RNAs, is capable of being guided to a target sequence in a target nucleic acid, and sometimes can subsequently also bind or cleave the target nucleic acid. The term Cas protein also includes nuclease-inactivated Cas proteins that are able to bind but not cleave a target sequence. The Cas proteins described in the disclosure include, but are not limited to, wild-type Cas proteins, variants thereof (e.g., mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off-target effects, mutants with reduced collateral cleavage effects), and functional fragments or fusion proteins thereof.

[0527] As used herein, when referring to a nucleotide sequence / DNA / RNA encoding a protein, RNA, CRISPR complex, the encoding sequence can be codon-optimized. For example, the encoding sequence is codon-optimized for expression in a eukaryotic cell environment, codon-optimized for expression in a mammalian cell environment, or codon-optimized for expression in a human cell environment.

[0528] As used herein, the term "sequence identity" (identity or percent identity) is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., each position in both of the DNA molecules is occupied by adenine, or each position in both of the polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent sequence identity" between two sequences is a function of the number of matching positions shared by the sequences, divided by the number of positions in the comparison x 100%. For example, if two sequences have 6 of 10 positions that are matched, then the sequences have 60% sequence identity. Generally, the comparison is made over the full length of the two sequences being compared, when the sequences are aligned to produce the maximum sequence identity. Such alignment can be achieved by use of publicly available and commercially available alignment algorithms and programs, such as, but not limited to, Clustal Omega, MAFFT, Probcons, T-Coffee, Probalign, BLAST, as can be reasonably selected by one of ordinary skill in the art. One of skill in the art can determine appropriate parameters for aligning sequences, for example, including any algorithm required to achieve an optimal alignment or best fit over the full length of the sequences being compared, and any algorithm required to achieve an optimal alignment or best fit over a portion of the sequences being compared. When a Cas13 protein described in the present disclosure is fused to other domains, the sequence identity is compared for the portion of the Cas13 protein.

[0529] As used herein, the term "regulatory sequence" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly U sequences). Regulatory sequences include those that direct sequential expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression in a desired tissue of interest, such as muscle, neuronal, skeletal, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., neuronal cells, lymphocytes). Regulatory sequences can also direct expression in a time-dependent manner, such as cell cycle-dependent or developmental stage-dependent, which can or can not be tissue-specific or cell type-specific. The term regulatory sequence also encompasses enhancer elements, such as the WPRE, CMV enhancer, SV40 enhancer, HRE enhancer element, and the intron sequence between exons 2 and 3 of rabbit beta-globin.

[0530] As used herein, the term "promoter" has the meaning generally accepted in the art.

[0531] As used herein, the term enhancer has the meaning generally understood in the art.

[0532] Those skilled in the art appreciate that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, and the like. The vector can be introduced into a host cell to thereby produce the RNA-guided nuclease and / or guide RNA of the present disclosure.

[0533] As used herein, the term pharmaceutically acceptable excipient means a diluent, adjuvant, pharmaceutical carrier, or other excipient with which the effective ingredient is administered. The selection of excipient depends on the use and intended method of administration. The excipient should not be incompatible with the effective ingredient, for example, it should not kill the cells or otherwise interact in a deleterious manner with any other components of the pharmaceutical composition. The pharmaceutical composition can be prepared by known methods in the art of pharmaceutical manufacture.

[0534] Guide RNA

[0535] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with the RNA-guided nuclease (also referred to as a gene editing complex) and directing the complex to bind to the target nucleic acid sequence specifically.

[0536] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with the RNA-guided nuclease and directing the complex to bind to and cleave the target nucleic acid.

[0537] In some embodiments of the present disclosure, the complex reduces the level of the target nucleic acid in an animal, such as in a human.

[0538] In some embodiments of the present disclosure, the complex reduces the level of the target nucleic acid in a cell, such as the level of the target nucleic acid in a cell expressing the target nucleic acid.

[0539] In some embodiments of the present disclosure, the complex reduces the level of the target nucleic acid in a cell 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 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the level of the target nucleic acid can be tested using methods routine in the art; the level of the target nucleic acid in the experimental group can be compared to the level of the target nucleic acid in a negative control, which can be untreated cells or cells treated with a targeting system that targets a non-mammalian genome.

[0540] In some embodiments of the disclosure, the number of off-target genes when the complex binds to and cleaves the target nucleic acid is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by routine methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection of the set of differentially expressed genes determined by RNA sequencing and the set of program predicted off-target genes. Non-limiting examples of the program predicted method are: using the EMBOSS-water program to make predictions in the whole genome and whole cDNA sequences of the target species (Homo sapiens / Mus musculus) with the parameters set to gap_extend=0.5 & gap_extend=10, using both the forward and reverse strands of the gRNA guide sequence for alignment, filtering the results to obtain the predicted potential target genes (including both on-target and off-target genes), and removing the on-target genes from the set of off-target genes.

[0541] In some embodiments of the disclosure, the complex reduces the level of a protein encoded by the target nucleic acid in an animal, such as a human.

[0542] In some embodiments of the disclosure, the complex reduces the level of a protein encoded by the target nucleic acid in a cell.

[0543] In some embodiments of the disclosure, the RNA-guided nuclease is a Cas protein. In some embodiments, the RNA-guided nuclease has complete or partial loss of cleavage activity.

[0544] In some embodiments of the disclosure, the RNA-guided nuclease is a Cas protein, and the guide RNA comprises a guide sequence and a homing repeat sequence.

[0545] In some embodiments of the disclosure, the RNA-guided nuclease comprises a nuclease moiety and a fused homologous or heterologous domain.

[0546] In some embodiments of the disclosure, the RNA-guided nuclease comprises any one or more of: a subcellular localization signal, a deaminase domain, a translation-activating domain, a translation-inhibiting domain, an RNA-methylating domain, an RNA-demethylating domain, a nuclease domain, a splicing factor domain, a reporter domain, a reporter tag, an affinity domain, and an affinity tag.

[0547] In some embodiments of the disclosure, the guide sequence comprises 20-40, 20-35, 20-30, or 25-30 nucleotides.

[0548] In some embodiments of the disclosure, the guide sequence hybridizes to the target nucleic acid with no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide mismatch.

[0549] In some embodiments of the disclosure, the guide sequence is 100% identical, i.e., perfectly complementary, to the target nucleic acid sequence.

[0550] The guide RNA comprises a guide sequence and a scaffold sequence, the guide sequence is reverse complementary to the target sequence, the scaffold sequence is linked to the guide sequence, and the scaffold sequence interacts with (e.g., binds to) the RNA-guided nuclease. The scaffold sequence is the sequence that is usually kept unchanged in the design of the guide RNA molecule. For example, the scaffold sequence can refer to the part of the guide RNA molecule other than the guide sequence. The guide sequence used herein is located at the 3' end or 5' end of the scaffold sequence. In some cases, the scaffold sequence comprises a direct repeat sequence (DR). In some cases, the scaffold sequence comprises a tracrRNA sequence. The direct repeat (DR), tracrRNA sequence can exist in the CRISPR locus structure of prokaryotes (such as bacteria, archaea), which can be screened to obtain; or can be obtained on this basis by sequence modification or optimization, non-limiting examples include deletion, substitution or addition of 1, 2, 3, 4 or more complementary base pairs of the secondary structure complementary double-stranded region of the scaffold sequence, deletion, substitution or addition of nucleotides on the loop of the stem-loop structure of the secondary structure of the scaffold sequence (for example, an aptamer sequence can be inserted on the loop). The size of the scaffold sequence is usually tens to 300 nucleotides, and the size of the direct repeat sequence is usually tens of nucleotides. The scaffold sequence is the constant part of the guide RNA molecule, which contains a strong secondary structure, which is conducive to the interaction between the RNA-guided gene editing protein and the guide RNA molecule. For example, it can be an editing system of Cas9 protein (such as SaCas9, SpCas9, CjCas9, NmeCas9) combined with gRNA (containing crRNA and tracrRNA); it can be an editing system of some Cas12 proteins (such as AsCas12a, dFnCas12a, Cas12h, Cas12i1, Cas12i2, dSiCas12i, hfCas12Max, AsCas12j-2) combined with gRNA (crRNA, comprising a guide sequence and a DR sequence); it can be an editing system of other Cas12 proteins (such as BhCas12b, BvCas12b, OspCas12c, CasY.1, CasY.6, PlmCasX, DpbCasX, SpCas12f, Un1Cas12f, AsCas12f1, SpaCas12f1) combined with gRNA (containing crRNA and tracrRNA).

[0551] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a scaffold sequence. In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence. In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a direct repeat sequence, which interacts with the RNA-guided nuclease.

[0552] In some embodiments of the disclosure, the guide sequence is located at the 3’ end or the 5’ end of the direct repeat sequence. In some embodiments of the disclosure, the guide sequence is located at the 3’ end of the direct repeat sequence. In some embodiments of the disclosure, the guide sequence is located at the 5’ end of the direct repeat sequence.

[0553] The gRNA used herein can be chemically modified gRNA, which can be modified using chemical modification methods well known to those skilled in the art, for example, 2’-O-methyl (2’-O-Me) modification, 2’-fluoro (2’-F) modification. In some embodiments, the modification between the nucleotides of the gRNA comprises a phosphorothioate (PS) bond. In some embodiments, the first three nucleotides at the 5’ end and the last three nucleotides at the 3’ end are modified. In some embodiments, the first four nucleotides at the 5’ end and the last four nucleotides at the 3’ end are connected with a phosphorothioate (PS) bond. In some embodiments, the gRNA can also use modified ribonucleotides, deoxyribonucleotides, other synthetic bases and synthetic backbone connections (such as peptide nucleic acid (PNA), locked nucleic acid (LNA), etc.). In some embodiments, the nucleic acids constituting the gRNA sequence comprise natural nucleosides (such as adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), nucleoside analogs (such as 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine), chemically modified bases, biologically modified bases (such as methylated bases), inserted bases, modified sugars (such as 2’-fluororibose, ribose, 2’-deoxyribose, arabinose and hexose), and / or modified phosphate groups (such as phosphorothioate and 5’-N-phosphoramidite linkage).

[0554] In some embodiments of the disclosure, the guide RNA comprises modified nucleotides. The modifications include, but are not limited to, 2'-O-methyl, 2'-O-methyl-3'-thio phosphate or 2'-O-methyl-3'-thio PACE modification. In some embodiments of the disclosure, the guide RNA comprises modified nucleotides selected from deoxyribonucleotides, locked nucleic acid (LNA). In some embodiments, the guide RNA comprises at least one chemically modified nucleotide. In some embodiments, the guide RNA is a hybrid RNA-DNA guide, i.e., some RNA nucleotides in the guide RNA are replaced by DNA nucleotides. In some embodiments, the guide RNA is a hybrid RNA-LNA (locked nucleic acid) guide, i.e., some RNA nucleotides in the guide RNA are replaced by LNA nucleotides. In some embodiments of the disclosure, the guide RNA comprises a modification, which is a phosphorothioate modification.

[0555] In some embodiments of the disclosure, the guide RNA is linked to a DNA sequence at the 5' end of the DR sequence.

[0556] In some embodiments of the disclosure, the 1-3 nucleotides at the 3' end of the guide sequence of the guide RNA are phosphorothioate modified and / or 2'-methoxy modified nucleotides. In some embodiments of the disclosure, the 3 nucleotides at the 3' end of the guide sequence of the guide RNA are phosphorothioate modified and 2'-methoxy modified nucleotides.

[0557] In some embodiments of the disclosure, the guide RNA is linked to a dG*dT*dT*dGdCdAdAdTdCdCdCdAdAdG sequence at the 5' end of the DR sequence (* indicates phosphorothioate modification) (SEQ ID NO: 5), and the 3 nucleotides at the 3' end of the guide sequence are phosphorothioate modified and 2'-methoxy modified.

[0558] In some embodiments of the disclosure, the target nucleic acid is located in the nucleus and / or cytoplasm of a eukaryotic cell.

[0559] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, and / or 43928819-43928876; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of homology to the sequence, for example nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0560] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, or 43928819-43928876 of the NCBI Reference Genome assembly GRCh38.p14.

[0561] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of human chromosome 8 (RefSeq: NC_000008.11) from position 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, and / or 117172598-117172624 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0562] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 8 (RefSeq: NC_000008.11) from position 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, or 117172598-117172624 of the NCBI Reference Genome assembly GRCh38.p14.

[0563] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of human chromosome 7 (RefSeq: NC_000007.14) from position 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, and / or 39696801-39696877 of NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0564] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 7 (RefSeq: NC_000007.14) from position 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, or 39696801-39696877 of NCBI Reference Genome assembly GRCh38.p14.

[0565] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14, human chromosome 3 (RefSeq: NC_000003.12) at positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, and / or 148741430-148741469; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of sequence homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0566] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14, human chromosome 3 (RefSeq: NC_000003.12) at positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, or 148741430-148741469 of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides.

[0567] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14, human chromosome 2 (RefSeq: NC_000002.12) at positions 31414656-31414686, 31386496-31386559, 31375408-31375460, and / or 31370393-31370424; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of sequence homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0568] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 2 (RefSeq: NC_000002.12) positions 31414656-31414686, 31386496-31386559, 31375408-31375460, or 31370393-31370424 of the NCBI Reference Genome assembly GRCh38.p14.

[0569] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, and / or 10018991-10019028 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to, splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0570] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 4 (RefSeq: NC_000004.12) positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, or 10018991-10019028 of the NCBI Reference Genome assembly GRCh38.p14.

[0571] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of human chromosome 3 (RefSeq: NC_000003.12) at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, and / or 52199384-52199419 of NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur on the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a certain proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0572] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 3 (RefSeq: NC_000003.12) at positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, or 52199384-52199419 of NCBI Reference Genome assembly GRCh38.p14.

[0573] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of human chromosome 4 (RefSeq: NC_000004.12) from the position of 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, and / or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to, splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0574] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 4 (RefSeq: NC_000004.12) positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14.

[0575] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of human chromosome 13 (RefSeq: NC_000013.11) positions 77899844-77899928 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0576] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 13 (RefSeq: NC_000013.11) positions 77899844-77899928 of the NCBI Reference Genome assembly GRCh38.p14.

[0577] In some embodiments of the disclosure, the guide polynucleotide targets a nucleotide sequence of human chromosome 17 (RefSeq: NC_000017.11) at positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, and / or 7174012-7174091 of the NCBI Reference Genome assembly GRCh38.p14; optionally, the targeting can occur to the 5'→3' strand and / or the 3'→5' strand of the segment. In some embodiments, the guide polynucleotide can also recognize variants having a proportion of homology to the sequence, for example, nucleotide sequences having no less than 80%, 90%, 95%, 98%, or 99% sequence homology, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence.

[0578] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of human chromosome 17 (RefSeq: NC_000017.11) at positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, or 7174012-7174091 of the NCBI Reference Genome assembly GRCh38.p14.

[0579] In some embodiments of the disclosure, the guide polynucleotide comprises a sequence fragment targeting positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, and / or 5464-5487 of the sequence set forth in SEQ ID NO: 28, or the reverse complement thereof.

[0580] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, or 5464-5487 of the sequence of SEQ ID NO: 28.

[0581] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, and / or 4961-4985 of the sequence of SEQ ID NO: 29, or the reverse complement thereof.

[0582] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, or 4961-4985 of the sequence of SEQ ID NO: 29.

[0583] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, and / or 4938-4983 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0584] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, or 4938-4983 of the sequence set forth in SEQ ID NO: 29.

[0585] In some embodiments of the disclosure, the guide polynucleotide targets a sequence segment of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0586] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986 of the sequence set forth in SEQ ID NO: 30.

[0587] In some embodiments of the disclosure, the guide polynucleotide targets a sequence segment of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, and / or 23943-23970 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0588] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, or 23943-23970 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0589] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0590] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, or 3372-3395 of the sequence set forth in SEQ ID NO: 31.

[0591] In some embodiments of the disclosure, the guide polynucleotide targets a sequence segment of positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0592] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, or 3372-3395 of the sequence set forth in SEQ ID NO: 31.

[0593] In some embodiments of the disclosure, the guide polynucleotide targets a sequence segment of positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, and / or 5195-5222 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0594] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, or 5195-5222 of the sequence set forth in SEQ ID NO: 32.

[0595] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, and / or 5170-5217 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0596] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, or 5170-5217 of the sequence set forth in SEQ ID NO: 32.

[0597] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, and / or 1275-1309 of the sequence set forth in SEQ ID NO: 33, or the reverse complement thereof.

[0598] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, or 1275-1309 of the sequence set forth in SEQ ID NO: 33.

[0599] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, and / or 20759-20810 of the sequence set forth in SEQ ID NO: 33, or the reverse complement thereof.

[0600] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, or 20759-20810 of the sequence set forth in SEQ ID NO: 33.

[0601] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, 10401-10676, 10401-10443, 10420-10676, 10423-10676, 10420-10597, 10498-10576, 10524-10576, 10524-10562, 10401-10489, 10605-10630, 10605-10652, 10605-10654, 10605-10663, 10605-10671, 10605-10672, 10630-10652, 10630-10654, 10630-10663, 10630-10671, and / or 10630-10672 of a sequence as set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0602] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, 10401-10676, 10401-10443, 10420-10676, 10423-10676, 10420-10597, 10498-10576, 10524-10576, 10524-10562, 10401-10489, 10605-10630, 10605-10652, 10605-10654, 10605-10663, 10605-10671, 10605-10672, 10630-10652, 10630-10654, 10630-10663, 10630-10671, or 10630-10672 of a sequence as set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0603] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, and / or 2942-2969 of a sequence as set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0604] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, or 2942-2969 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0605] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, and / or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0606] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0607] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, and / or 6765-6810 of the sequence set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0608] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, or 6765-6810 of the sequence set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0609] In some embodiments of the disclosure, the guide polynucleotide targets a sequence fragment of positions 7179-7207, 7364-7405, 7686-7754, 7858-7932, 7858-7900, 7877-7932, 8232-8282, 8837-8993, 8918-8993, 8918-8961, and / or 8938-8993 of the GPR75 gene sequence set forth in SEQ ID NO: 25, or the reverse complement thereof.

[0610] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of positions 7179-7207, 7364-7405, 7686-7754, 7858-7932, 7858-7900, 7877-7932, 8232-8282, 8837-8993, 8918-8993, 8918-8961, or 8938-8993 of a GPR75 gene sequence as set forth in SEQ ID NO: 25.

[0611] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0612] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0613] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0614] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0615] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0616] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0617] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 21 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0618] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 22 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0619] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 23 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0620] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at least 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0621] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of 17-24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0622] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of at most 24 contiguous nucleotides of any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0623] In some embodiments of the disclosure, the guide polynucleotide targets one or more gene sequences or sequence fragments selected from the group consisting of genes ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and EDNRB, dependent on a nucleotide sequence in the guide polynucleotide that is reverse complementary to the gene sequence or sequence fragment, which nucleotide sequence can hybridize or interact with the targeted XXXX sequence or sequence fragment.

[0624] In some embodiments of the disclosure, the guide polynucleotide targets one or more gene sequences or sequence fragments selected from the group consisting of the gene ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and EDNRB, dependent on a nucleotide sequence in the guide polynucleotide that is reverse complementary to the gene sequence or sequence fragment, which nucleotide sequence can hybridize or interact with the targeted gene sequence or sequence fragment; the nucleotide sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the gene sequence or sequence fragment.

[0625] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 3109-3133, 3129-3152, 3165-3188, 3188-3211, 5321-5344, 5388-5417, 5406-5432, and / or 5464-5487 of the sequence set forth in SEQ ID NO: 28, or the reverse complement thereof.

[0626] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence fragment of positions 1332-1441, 1332-1440, 1332-1436, 1332-1361, 1850-1900, 1850-1899, 1850-1893, 1850-1873, 2861-2896, 4856-4908, 4856-4907, 4856-4902, 4856-4888, 4866-4908, 4866-4907, 4866-4902, 4938-4985, and / or 4961-4985 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0627] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 1104-1175, 1107-1174, 1107-1139, 1228-1252, 1253-1276, 1258-1304, 1258-1303, 1321-1354, 1339-1441, 1339-1440, 1339-1436, 1519-1550, 1640-1672, 1640-1663, 1710-1754, 1710-1750, 1710-1734, 1710-1733, 1711-1754, 1711-1750, 1711-1734, 1711-1733, 1712-1754, 1712-1750, 1712-1734, 1712-1733, 1728-1754, 1798-1872, 1798-1851, 1851-1899, 1851-1893, 1928-1951, 2032-2055, 2657-2683, 4880-4908, 4933-4983, and / or 4938-4983 of the sequence set forth in SEQ ID NO: 29, or the reverse complement thereof.

[0628] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 1593-1619, 1610-1651, 6273-6300, 8971-9010, 23954-23986 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0629] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 1474-1498, 1475-1498, 1533-1556, 1563-1615, 1596-1619, 1664-1712, 1664-1688, 1688-1712, 6311-6333, 6434-6459, 6444-6477, 8982-9010, 9021-9049, 18435-18458, 18441-18479, 19895-19918, 19925-19948, 20468-20491, 20542-20568, 20563-20598, 20596-20621, 21931-21970, 22072-22096, 23942-23970, and / or 23943-23970 of the sequence set forth in SEQ ID NO: 30, or the reverse complement thereof.

[0630] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 2772-2844, 2772-2820, 2772-2803, 2772-2796, 2910-2942, 2910-2941, 3233-3267, 3371-3395, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0631] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment from positions 1267-1427, 1962-1987, 1962-1985, 2222-2326, 2348-2378, 2395-2422, 2766-2794, 2774-2844, 2774-2803, 2798-2844, 2893-2919, 2906-2932, 2919-3001, 2919-2942, 2952-2993, 3024-3139, 3031-3098, 3031-3074, 3076-3139, 3144-3181, 3233-3267, 3371-3395, 3371-3394, and / or 3372-3395 of the sequence set forth in SEQ ID NO: 31, or the reverse complement thereof.

[0632] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment from positions 3739-3789, 3739-3787, 3739-3783, 3739-3767, 3745-3789, 3745-3787, 3745-3783, 3761-3789, 3761-3787, 5137-5171, 5138-5171, 5170-5222, and / or 5195-5222 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0633] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment from positions 2579-2608, 2616-2642, 3761-3787, 5137-5160, 5149-5171, 5170-5231, and / or 5170-5217 of the sequence set forth in SEQ ID NO: 32, or the reverse complement thereof.

[0634] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 851-874, 985-1059, 985-1034, 1012-1059, 1007-1059, 1007-1041, 1245-1269, and / or 1275-1309 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0635] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 985-1034, 1007-1034, 1019-1059, 1124-1153, 1203-1227, 1456-1480, 1461-1485, 15136-15160, 15265-15291, 15286-15320, 15295-15320, and / or 20759-20810 of the sequence of SEQ ID NO: 33, or the reverse complement thereof.

[0636] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment of positions 2187-2229, 2284-2408, 2284-2322, 2334-2408, 2450-2473, 5887-5936, 5896-5936, 5887-5935, 5896-5935, 5990-6014, 6059-6139, 6059-6098, 6112-6139, 6193-6292, 6226-6292, 6247-6292, 6253-6292, 6256-6292, 6266-6292, 6226-6278, 6247-6278, 6253-6278, 6442-6465, 6476-6554, 6496-6554, 6512-6554, 6583-6647, 6602-6635, 6602-6647, 10401-10676, 10401-10443, 10420-10676, 10423-10676, 10420-10597, 10498-10576, 10524-10576, 10524-10562, 10401-10489, 10605-10630, 10605-10652, 10605-10654, 10605-10663, 10605-10671, 10605-10672, 10630-10652, 10630-10654, 10630-10663, 10630-10671, and / or 10630-10672 of the sequence set forth in SEQ ID NO: 34, or the reverse complement thereof.

[0637] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment from positions 2373-2415, 2434-2465, 2434-2463, 2558-2594, 2558-2593, 2558-2590, 2560-2594, 2560-2593, 2560-2590, 2567-2594, 2567-2593, 2603-2630, 2628-2652, 2634-2659, 2691-2722, 2699-2722, 2942-2970, and / or 2942-2969 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0638] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment from positions 2280-2306, 2292-2323, 2296-2323, 2325-2352, 2327-2352, 2334-2366, 2335-2366, 2340-2366, 2334-2362, 2335-2364, 2335-2362, 2366-2390, 2441-2465, 2558-2583, 2597-2621, 2634-2658, 2678-2706, 2691-2719, 2859-2944, 2859-2887, 2879-2944, 2942-2995, and / or 2946-2970 of the sequence set forth in SEQ ID NO: 35, or the reverse complement thereof.

[0639] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 755-778, 766-804, 785-863, 785-821, 800-859, 800-855, 800-851, 800-841, 800-828, 800-823, 801-863, 801-859, 801-855, 801-851, 801-841, 801-828, 801-823, 806-863, 806-859, 806-855, 806-851, 806-841, 819-863, 819-859, 819-855, 819-851, 866-889, 876-911, 876-910, 876-909, 876-908, 876-899, 877-911, 877-910, 877-909, 877-908, 886-911, 886-910, 886-909, 887-991, 887-910, 888-911, 917-946, 917-942, 920-946, 941-984, 942-984, 962-1043, 1188-1212, 1189-1212, 1230-1253, and / or 6765-6810 of the sequence as set forth in SEQ ID NO: 36, or the reverse complement thereof.

[0640] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of positions 7179-7207, 7364-7405, 7686-7754, 7858-7932, 7858-7900, 7877-7932, 8232-8282, 8837-8993, 8918-8993, 8918-8961, and / or 8938-8993 of the GPR75 gene sequence as set forth in SEQ ID NO: 25, or the reverse complement thereof.

[0641] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, and / or 43928819-43928876, or the reverse complement thereof.

[0642] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment from nucleotides 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, and / or 117172598-117172624 of human chromosome 8 (RefSeq: NC_000008.11) of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0643] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence fragment from nucleotides 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, and / or 39696801-39696877 of human chromosome 7 (RefSeq: NC_000007.14) of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0644] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 3 (RefSeq: NC_000003.12) positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, and / or 148741430-148741469, or the reverse complement thereof.

[0645] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 2 (RefSeq: NC_000002.12) positions 31414656-31414686, 31386496-31386559, 31375408-31375460, and / or 31370393-31370424, or the reverse complement thereof.

[0646] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of NCBI Reference Genome assembly GRCh38.p14 human chromosome 4 (RefSeq: NC_000004.12) positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, and / or 10018991-10019028, or the reverse complement thereof.

[0647] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of chromosome 3 (RefSeq: NC_000003.12) positions 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, and / or 52199384-52199419 of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0648] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of chromosome 4 (RefSeq: NC_000004.12) positions 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, and / or 147486066-147486115 of the NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0649] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of human chromosome 13 (RefSeq: NC_000013.11) positions 77899844-77899928 of NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0650] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence fragment of human chromosome 17 (RefSeq: NC_000017.11) positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, and / or 7174012-7174091 of NCBI Reference Genome assembly GRCh38.p14, or the reverse complement thereof.

[0651] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0652] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0653] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having 100% sequence identity to any of the guide sequences of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0654] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence of any of Tables 3-14, 17, 18, 20-38, 40, 42, 47, 49, and 50 of the disclosure.

[0655] In some embodiments of the disclosure, the guide polynucleotide is a nucleotide sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences set forth in SEQ ID NOs: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416.

[0656] In some embodiments of the disclosure, the guide polynucleotide is a nucleotide sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences set forth in SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918.

[0657] In some embodiments of the disclosure, the guide polynucleotide is comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to 62, 63, 69, 72, 119, 127, 133, 166, 167, 188, 211, 212, 213, 214, 236, 237, 239, 248, 272, 273, 283, 287, 304, 309, 326, 395, 397, 402, 482, 501, 505, 513, 628, 654, 691, 726, 752, 769, 771, 772, 780, 793, 795, 797, 800, 832, 838, 878, 886, 981, 995, 1023, 1051, 1080, 1094, 1217, 1259, 1271, 1279, 1280, 1289, 1290, 1294, 1295, 1303, 1315, 1317, 1346, 1360, 1389, 1396, 1403, 1420, 1438, 1441, 1442, 1450, 1478, 1491, 1496, 1507, 1520, 1522, 1529, 1558, 1560, 1564, 1565, 1682, 1714, 1720, 1753, 1761, 1762, 1763, 1777, 1796, 1831, 1838, 1910, 1911, 1916, 2067, 2133, 2134, 2135, 2137, 2139, 2142, 2144, 2145, 2153, 2154, 2166, 2168, 2169, 2180, 2182, 2191, 2192, 2195, 2202, 2204, 2235, 2240, 2248, 2262, 2263, 2264, 2265, 2267, 2272, 2274, 2277, 2289, 2290, 2295, 2310, 2314, 2328, 2332, 2347, 2348, 2349, 2365, 2380, 2409, 2414, 2417, 2430, 2433, 2439, 2464, 2517, 2519, 2520, 2533, 2544, 2550, 2551, 2553, 2554, 2558, 2560, 2562, 2567, 2579, 2590, 2606, 2627, 2628, 2635, 2637, 2639, 2659, 2675, 2690, 2704, 2706, 2708, 2709, 2713, 2715, 2718, 2735, 2746, 2758, 2761, 2816, 2826, 2850, 2858, 2871, 2894, 2906, 2908, 2942, 2963, 2966, 3136, 3137, 3148, 3149, 3150, 3151, 3152, 3153, 3154, 3155, 3156, 3157, 3158, 3159, 3160, 3161, 3162, 3163, 3164, 3165, 3166, 3167, 3168, 3169, 3170, 3171, 3172, 3173, 3174, 3175, 3176, 3177, 3178, 3179, 3180, 3181, 3182, 3183, 3184, 3185, 3186, 3187, 3188, 3189, 3190, 3191, 3192, 3193, 3194, 3195, 3196, 3197, 3198, 3199, 3200, 3201, 3202, 3203, 3204, 3205, 3206, 3207, 3208, 3209, 3210, 3211, 3212, 3213, 3214, 3215, 3216, 3217, 3218, 3219, 3220, 3221, 3222, 3223, 3224, 3225, 3226, 3227, 3228, 3229, 3230, 3231, 3232, 3233, 3234, 3235, 3236, 3237, 3238, 3239, 3240, 3241, 3242, 3243, 3244, 3245, 3246, 3247, 3248, 3249, 3250, 3251, 3252, 3253, 3254, 3255, 3256, 3257, 3258, 3259, 3260, 3261, 3262, 3263, 3264, 3265, 3266, 3267, 3268, 3269, 3270, 3271, 3272, 3273, 3274, 3275, 3276, 3277, 3278, 3279, 3280, 3281, 3282, 3283, 3284, 3285, 3286, 3287, 3288, 3289, 3290, 3291, 3292, 3293, 3294, 3295, 3296, 3297, 3298, 3299, 3300, 3301, 3302, 3303, 3304, 3305, 3306, 3307, 3308, 3309, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318, 3319, 3320, 3321, 3322, 3323, 3324, 3325, 3326, 3327, 3328, 3329, 3330, 3331, 3332, 3333, 3334, 3335, 3336, 3337, 3338, 3339, 3340, 3341, 3342, 3343, 3344, 3345, 3346, 3347, 3348, 3349, 3350, 3351, 3352, 3353, 3354, 3355, 3356, 3357, 3358, 3359, 3360, 3361, 3362, 3363, 3364, 3365, 3366, 3367, 3368, 3369, 3370, 3371, 3372, 3373, 3374, 3375, 3376, 3377, 3378, 3379, 3380, 3381, 3382, 3383, 3384, 3385, 3386, 3387, 3388, 3389, 3390, 3391, 3392, 3393, 3394, 3395, 3396, 3397, 3398, 3399, 3400, 3401, 3402, 3403, 3404, 3405, 3406, 3407, 3408, 3409, 3410, 3411, 3412, 3413, 3414, 3415, 3416, 3417, 3418, 3419, 3420, 3421, 3422, 3423, 3424, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3432, 3433, 3434, 3435, 3436, 3437, 3438, 3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 3456, 345The nucleotide sequence of any one of the sequences set forth in 3152 and 3153 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Tables 3 or 17, or the reverse complement thereof.

[0658] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 4, or the reverse complement thereof.

[0659] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 5, or the reverse complement thereof.

[0660] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 6 or Table 28, or the reverse complement thereof.

[0661] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 7 or Table 22, or the reverse complement thereof.

[0662] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a guide sequence set forth in any of Table 8 or Table 21, or the reverse complement thereof.

[0663] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 9, Table 29, Table 40, or Table 42, or the reverse complement thereof.

[0664] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 10, Table 20, Table 36, Table 37, Table 38, Table 49, or Table 50, or the reverse complement thereof.

[0665] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in SEQ ID NO: 26, or the reverse complement thereof.

[0666] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 11, or the reverse complement thereof.

[0667] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 12 or Table 23, or the reverse complement thereof.

[0668] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 13, or the reverse complement thereof.

[0669] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 14 or Table 18, or the reverse complement thereof.

[0670] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 24, or the reverse complement thereof.

[0671] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 25, or the reverse complement thereof.

[0672] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 26, or the reverse complement thereof.

[0673] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 27, or the reverse complement thereof.

[0674] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a guide sequence set forth in any of Table 30, or the reverse complement thereof.

[0675] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 31 or the reverse complement thereof.

[0676] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 32 or the reverse complement thereof.

[0677] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 33 or the reverse complement thereof.

[0678] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 34 or the reverse complement thereof.

[0679] In some embodiments of the disclosure, the targeting system comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of the guide sequences set forth in Table 35 or the reverse complement thereof.

[0680] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in any one of SEQ ID NOS: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416.

[0681] In some embodiments of the disclosure, the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918.

[0682] In some embodiments of the disclosure, the guide polynucleotide is comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to 62, 63, 69, 72, 119, 127, 133, 166, 167, 188, 211, 212, 213, 214, 236, 237, 239, 248, 272, 273, 283, 287, 304, 309, 326, 395, 397, 402, 482, 501, 505, 513, 628, 654, 691, 726, 752, 769, 771, 772, 780, 793, 795, 797, 800, 832, 838, 878, 886, 981, 995, 1023, 1051, 1080, 1094, 1217, 1259, 1271, 1279, 1280, 1289, 1290, 1294, 1295, 1303, 1315, 1317, 1346, 1360, 1389, 1396, 1403, 1420, 1438, 1441, 1442, 1450, 1478, 1491, 1496, 1507, 1520, 1522, 1529, 1558, 1560, 1564, 1565, 1682, 1714, 1720, 1753, 1761, 1762, 1763, 1777, 1796, 1831, 1838, 1910, 1911, 1916, 2067, 2133, 2134, 2135, 2137, 2139, 2142, 2144, 2145, 2153, 2154, 2166, 2168, 2169, 2180, 2182, 2191, 2192, 2195, 2202, 2204, 2235, 2240, 2248, 2262, 2263, 2264, 2265, 2267, 2272, 2274, 2277, 2289, 2290, 2295, 2310, 2314, 2328, 2332, 2347, 2348, 2349, 2365, 2380, 2409, 2414, 2417, 2430, 2433, 2439, 2464, 2517, 2519, 2520, 2533, 2544, 2550, 2551, 2553, 2554, 2558, 2560, 2562, 2567, 2579, 2590, 2606, 2627, 2628, 2635, 2637, 2639, 2659, 2675, 2690, 2704, 2706, 2708, 2709, 2713, 2715, 2718, 2735, 2746, 2758, 2761, 2816, 2826, 2850, 2858, 2871, 2894, 2906, 2908, 2942, 2963, 2966, 3136, 3137, 3148, 3149, 3150, 3151, 3152, 3153, 3154, 3155, 3156, 3157, 3158, 3159, 3160, 3161, 3162, 3163, 3164, 3165, 3166, 3167, 3168, 3169, 3170, 3171, 3172, 3173, 3174, 3175, 3176, 3177, 3178, 3179, 3180, 3181, 3182, 3183, 3184, 3185, 3186, 3187, 3188, 3189, 3190, 3191, 3192, 3193, 3194, 3195, 3196, 3197, 3198, 3199, 3200, 3201, 3202, 3203, 3204, 3205, 3206, 3207, 3208, 3209, 3210, 3211, 3212, 3213, 3214, 3215, 3216, 3217, 3218, 3219, 3220, 3221, 3222, 3223, 3224, 3225, 3226, 3227, 3228, 3229, 3230, 3231, 3232, 3233, 3234, 3235, 3236, 3237, 3238, 3239, 3240, 3241, 3242, 3243, 3244, 3245, 3246, 3247, 3248, 3249, 3250, 3251, 3252, 3253, 3254, 3255, 3256, 3257, 3258, 3259, 3260, 3261, 3262, 3263, 3264, 3265, 3266, 3267, 3268, 3269, 3270, 3271, 3272, 3273, 327...

Claims

1. A targeting system, wherein the system targets the expression of any one, two, or more genes selected from the group consisting of: ANGPTL3, LPA, ASGR1, PNPLA3, TTR, APOC3, PCSK9, INHBE, AGT, HSD17B13, APOB, DLG4, SLC30A8, RALA, GPR75, AGTR1, XDH, SLC2A9, ALAS1, EDNRA, and EDNRB.

2. The targeting system of claim 1, wherein the targeting system is a gene editing system, preferably a CRISPR gene editing system, or a CRISPR-like gene editing system; and / or, the gene is a mammalian gene, preferably a human gene; the targeting system knocks out the target gene, alters the nucleotide sequence of the target gene, suppresses the transcription of the target gene, or activates the transcription of the target gene; preferably, the alteration of the nucleotide sequence of the target gene includes, but is not limited to, nucleotide base substitution (including but not limited to single base editing), addition of at least one nucleotide, or deletion of at least one nucleotide on the nucleotide sequence; the suppression of the transcription of the target gene can be achieved by fusing the gene editing system with an epigenetic regulation domain, and then targeting the regulatory region of the target gene, thereby inducing chromatin state changes to achieve transcription suppression; the suppression of the transcription of the target gene can be achieved by fusing the gene editing system with a transcription regulation domain, and then targeting the promoter or enhancer region of the target gene, thereby directly inhibiting the transcription initiation or transcription elongation process; more preferably, the sequence of the gene is selected from the group consisting of: (a) including but not limited to exons, introns, and promoter regions located upstream of the transcription initiation site; (b) including but not limited to enhancers, silencers, and other cis-acting regulatory elements, as well as 5' untranslated regions (5' UTR), 3' untranslated regions (3' UTR), polyadenylation signals, and regulatory elements related to mRNA splicing, stability, and translation efficiency; (c) reverse complements of the above (a) to (b) sequences, as well as nucleic acid molecules modified by methylation, sulfuration, phosphorylation, or other artificial modifications; (d) derived from natural genomic cloning, cDNA synthesis, in vitro transcription, chemical synthesis, or vector construction.

3. The targeting system of claim 2, comprising: (i) a nuclease; such as Cas9, Cas12, TnpB, IscB, IsrB, Fanzor, or Tas; (ii) a guide polynucleotide for guiding the nuclease; such as gRNA, ωRNA, tigRNA; preferably, in (i), the nuclease is a Cas protein including Cas9, Cas12, such as spCas9, Cas12i; in (ii), the guide polynucleotide is a gRNA such as sgRNA; preferably sgRNA including a guide sequence, a direct repeat sequence; ​ more preferably, the nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in any one of SEQ ID NOs: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416; or more preferably, the nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in any one of SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918; or the nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 133, 119, 166, 211, 127, 167, 72, 212, 236, 273, 214, 239, 309, 304, 326, 213, 283, 237, 287, 248, 272, 505, 395, 397, 628, 402, 513, 654, 691, 482, 501, 752, 797, 838, 726, 1094, 995, 1080, 981, 1259, 1279, 1315, 1317, 1280, 1294, 1303, 1295, 1522, 1507, 1450, 1403, 1396, 1496, 1558, 1491, 1389, 1442, 1478, 1565, 1720, 1682, 1763, 1714, 1796, 1762, 1777, 1838, 1831, 1916, 3136, 3137, 3148, 3149, 3150, 3151, 3152, 3153, 1346, 1441, 1564, 1560, 1520, and 1529; or the nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2908, 2850, 2871, 2906, 2816, 2894, 2858, 2826, 2966, 2942, 2963, 1217, and 1360; or the nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 188, 63, 69, 62, 2067, 1911, 1910, 1438, 1420, 1023, 1051, 878, 886, 1753, 2154, 1761, 2153, 2139, 2145, 2142, 2144, 1763, 2166, 2191, 2182, 2192, 2180, 2202, 2169, 2204, 2168, 2195, 2248, 2264, 2277, 2265, 2235, 2240, 2274, 2262, 2263, 2267, 2272, 2295, 2310, 2347, 2348, 2289, 2314, 2349, 2328, 2332, 2290, 2380, 2365, 793, 771, 2409, 797, 780, 772, 832, 795, 800, 769, 2414, 1271, 1290, 1317, 2417, 1289, 2433, 2439, 2430, 2464, 2758, 2746, 2735, 2761, 2718, 2675, 2706, 2704, 2708, 2713, 2715, 2709, 2690, 2628, 2627, 2659, 2637, 2635, 2639, 2554, 2551, 2590, 2560, 2553, 2550, 2562, 2606, 2579, 2567, 2558, 2533, 2520, 2517, 2519, and 2544; or the nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 188, 63, 69, 62, 2067, 1911, 1910, 1438, 1420, 1023, 1051, 878, 886, 1753, 2154, 1761, 2153, 2139, 2145, 2142, 2144, 1763, 2166, 2191, 2182, 2192, 2180, 2202, 2169, 2204, 2168, 2195, 2248, 2264, 2277, 2265, 2235, 2240, 2274, 2262, 2263, 2267, 2272, 2295, 2310, 2347, the nuclease has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2134, 2137, 2135, and 2133.

4. The targeting system of any one of claims 1-3, wherein, (1) the sequence of the gene is set forth in any one of SEQ ID NOs: 25, 28-38, or any one of SEQ ID NOs: 25, 28-38 with T replaced by U; or the reverse complement thereof; (2) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 99.5% sequence identity to the sequence of (1), a synonymous mutant sequence, or a functionally equivalent derivative sequence.

5. The targeting system of any one of claims 1-4, wherein, (A) the targeting system targets the nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 of human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, and / or 43928819-43928876; or the complement thereof, or a nucleotide sequence not lower than 80%, 90%, 95%, 98%, or 99% homologous to the above-mentioned sequences, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (B) the targeting system targets the nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 of human chromosome 22 (RefSeq: NC_000022.11) positions 43923047-43923138, 43923047-43923115, 43923047-43923104, 43923047-43923073, 43923082-43923138, 43923082-43923115, 43923093-43923138, 43923229-43923304, 43923229-43923279, 43923229-43923256, 43923257-43923304, 43923257-43923296, 43923274-43923304, 43923345-43923431, 43923345-43923390, 43923345-43923367, 43923368-43923431, 43923396-43923431, 43923400-43923431, 43923484-43923553, 43923497-43923553, 43926921-43927125, 43926935-43927125, 43926921-43926987, 43926935-43926987, 43926993-43927044, 43927012-43927044, 43927060-43927125, 43927091-43927125, 43928819-43928903, 43928819-43928867, and / or 43928819-43928876; or the complement thereof, or a nucleotide sequence not lower than 80%, 90%, 95%, 98%, or 99% homologous to the above-mentioned sequences, including but not limited to splice variants, mutants, or conservatively substituted derivative sequences of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (B) the targeting system targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 of human chromosome 8 (RefSeq: NC_000008.11) at positions 117147040-117147098, 117152956-117153112, 117153001-117153058, 117153001-117153038, 117157672-117157715, 117161754-117161794, 117163469-117163526, 117163469-117163505, 117172544-117172661, 117172620-117172661, and / or 117172598-117172624; or the complement thereof, or a nucleotide sequence with not less than 80%, 90%, 95%, 98%, or 99% homology to the above sequences, including but not limited to splice variants, mutants, or conservatively substituted derivatives of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (C) the targeting system comprises a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 of human chromosome 7 (RefSeq: NC_000007.14) at positions 39686689-39686744, 39686700-39686732, 39690385-39690611, 39686760-39686797, 39686642-39686797, 39686642-39686684, 39686642-39686674, 39690469-39690506, 39690581-39690611, 39690469-39690496, 39696674-39696719, 39706209-39706264, 39696801-39696838, or 39696801-39696877; or the complement thereof, or a nucleotide sequence with not less than 80%, 90%, 95%, 98%, or 99% homology to the above sequences, including but not limited to splice variants, mutants, or conservatively substituted derivatives of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (D) the targeting system targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.P14 of human chromosome 3 (RefSeq: NC_000003.12) at positions 148741034-148741092, 148741120-148741225, 148741153-148741205, 148741153-148741225, 148741180-148741225, and / or 148741430-148741469; or the complement thereof, or a nucleotide sequence with not less than 80%, 90%, 95%, 98%, or 99% homology to the aforementioned sequences, including but not limited to splice variants, mutants, or conservatively substituted derivatives of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (E) the targeting system targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.P14 of human chromosome 2 (RefSeq: NC_000002.12) at positions 31414656-31414686, 31386496-31386559, 31375408-31375460, and / or 31370393-31370424; or the complement thereof, or a nucleotide sequence with not less than 80%, 90%, 95%, 98%, or 99% homology to the aforementioned sequences, including but not limited to splice variants, mutants, or conservatively substituted derivatives of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (F) the targeting system targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.P14 of human chromosome 4 (RefSeq: NC_000004.12) at positions 9908319-9908354, 9985758-9985796, 9996902-9996948, 10018991-10019086, and / or 10018991-10019028; or the complement thereof, or a nucleotide sequence with not less than 80%, 90%, 95%, 98%, or 99% homology to the aforementioned sequences, including but not limited to splice variants, mutants, or conservatively substituted derivatives of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (G) the targeting system comprises the nucleotide sequence of nucleotides 52199272-52199419, 52199272-52199333, 52199297-52199333, 52199309-52199333, or 52199384-52199419 of human chromosome 3 (RefSeq: NC_000003.12) of the NCBI Reference Genome assembly GRCh38.p14; or the complement thereof, or a nucleotide sequence with not less than 80%, 90%, 95%, 98%, or 99% homology to the above sequences, including but not limited to splice variants, mutants, or conservatively substituted derivatives of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (H) the targeting system comprises the nucleotide sequence of nucleotides 147485664-147486122, 147485664-14748569, 147485712-147485750, 147485712-147485736, 147485712-147485769, 147485728-147485769, 147485741-147485769, 147485801-147485875, 147485801-147485901, 147485801-147485845, 147485852-147485901, 147485979-147486021, 147485979-147486005, 147486055-147486122, 147486055-147486121, 147486055-147486115, 147486055-147486097, or 147486066-147486115 of human chromosome 4 (RefSeq: NC_000004.12) of the NCBI Reference Genome assembly GRCh38.p14; or the complement thereof, or a nucleotide sequence with not less than 80%, 90%, 95%, 98%, or 99% homology to the above sequences, including but not limited to splice variants, mutants, or conservatively substituted derivatives of the native sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (I) the targeting system targets a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 of human chromosome 13 (RefSeq: NC_000013.11) at positions 77899844-77899928; or the complement thereof, or a nucleotide sequence having not less than 80%, 90%, 95%, 98%, or 99% homology to the above sequence, including but not limited to a splice variant, a mutant, or a conservatively substituted derivative sequence of the natural sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof; (J) the targeting system comprises a nucleotide sequence of NCBI Reference Genome assembly GRCh38.p14 of human chromosome 17 (RefSeq: NC_000017.11) at positions 7179922-7179957, 7179398-7179442, 7178498-7178587, 7176834-7176920, 7176870-7176902, 7174370-7174458, 7174370-7174401, 7174196-7174287, 7174196-7174239, or 7174012-7174091; or the complement thereof, or a nucleotide sequence having not less than 80%, 90%, 95%, 98%, or 99% homology to the above sequence, including but not limited to a splice variant, a mutant, or a conservatively substituted derivative sequence of the natural sequence; preferably a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides thereof.

6. The targeting system as shown in any one of claims 3-5, wherein, The gRNA in the targeting system is selected from the group consisting of: (i) the nucleic acid constituting the gRNA sequence comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazanucleosides, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); (ii) the gRNA comprises chemically modified bases, biologically modified bases (e.g., methylated bases), inserted bases, modified sugars (e.g., 2’-fluoro-ribose, ribose, 2’-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5’-N-phosphoramidite linkages); Preferably, in (ii), (iii) the gRNA comprises a 5’ cap, a 3’ tail, or both. (a) the modification between nucleotides of the gRNA comprises phosphorothioate (PS) linkage; preferably, the first 3-4 nucleotides at the 5’ end and the last 3-4 nucleotides at the 3’ end of the gRNA are linked by phosphorothioate (PS) linkage; (b) the gRNA uses modified ribonucleotides, deoxyribonucleotides, other synthetic bases and synthetic backbones linkage, such as peptide nucleic acid (PNA), locked nucleic acid (LNA); More preferably, the gRNA further comprises a modified sequence at the 5’ of the guide RNA, preferably a sequence as set forth in SEQ ID NO:

5.

7. The targeting system of claim 6, wherein, the sequence of the guide RNA is as set forth in any of the guide sequences in Tables 3-14, 17, 18, 20-38, 40, 42, 46, 48 and 49; or a reverse complement thereof; or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto; and / or, the targeting system reduces the expression level of the gene in a cell 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 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and / or, the number of off-target genes that the targeting system binds to and cleaves when binding to and cleaving the gene is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1.

8. The targeting system as shown in any one of claims 3-7, wherein, In the targeting system, (1) the Cas protein comprises 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 15, or 16; (2) the sgRNA comprises a nucleotide sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of any of the guide sequences in Tables 3-14, 17, 18, 20-38, 40, 42, 46, 48 and 49; Preferably, the Cas protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416; or the Cas protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918; or the Cas protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in any one of SEQ ID NOs: 133, 119, 166, 211, 127, 167, 72, 212, 236, 273, 214, 239, 309, 304, 326, 213, 283, 237, 287, 248, 272, 505, 395, 397, 628, 402, 513, 654, 691, 482, 501, 752, 797, 838, 726, 1094, 995, 1080, 981, 1259, 1279, 1315, 1317, 1280, 1294, 1303, 1295, 1522, 1507, 1450, 1403, 1396, 1496, 1558, 1491, 1389, 1442, 1478, 1565, 1720, 1682, 1763, 1714, 1796, 1762, 1777, 1838, 1831, 1916, 3136, 3137, 3148, 3149, 3150, 3151, 3152, 3153, 1346, 1441, 1564, 1560, 1520, and 1529; or the Cas protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence set forth in any one of SEQ ID NOs: 2908, 2850, 2871, 2906, 2816, 2894, 2858, 2826, 2966, 2942, 2963, 1217, and 1360; or the Cas protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 188, 63, 69, 62, 2067, 1911, 1910, 1438, 1420, 1023, 1051, 878, 886, 1753, 2154, 1761, 2153, 2139, 2145, 2142, 2144, 1763, 2166, 2191, 2182, 2192, 2180, 2202, 2169, 2204, 2168, 2195, 2248, 2264, 2277, 2265, 2235, 2240, 2274, 2262, 2263, 2267, 2272, 2295, 2310, 2347, 2348, 2289, 2314, 2349, 2328, 2332, 2290, 2380, 2365, 793, 771, 2409, 797, 780, 772, 832, 795, 800, 769, 2414, 1271, 1290, 1317, 2417, 1289, 2433, 2439, 2430, 2464, 2758, 2746, 2735, 2761, 2718, 2675, 2706, 2704, 2708, 2713, 2715, 2709, 2690, 2628, 2627, 2659, 2637, 2635, 2639, 2554, 2551, 2590, 2560, 2553, 2550, 2562, 2606, 2579, 2567, 2558, 2533, 2520, 2517, 2519, and 2544; or the Cas protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 188, 63, 69, 62, 2067, 1911, 1910, 1438, 1420, 1023, 1051, 878, 886, 1753, 2154, 1761, 2153, 2139, 2145, 2142, 2144, 1763, 2166, 2191, 2182, 2192, 2180, 2202, 2169, 2204, 2168, 2195, 2248, 2264, 2277, 2265, 2235, 2240, 2274, 2262, 2263, 2267, 2272, 2295, 2310, 2347, the Cas protein has 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 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16; and the guide polynucleotide comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2134, 2137, 2135, and 2133.

9. The targeting system of any one of claims 3-7, wherein the amino acid sequence of the Cas protein is set forth in SEQ ID NO: 1; and the nucleotide sequence of the guide polynucleotide is set forth in any one of SEQ ID NOs: 1491, 1564, 1560, 1520, 1529, 1419, 1412, 1421, and 1416; or the amino acid sequence of the Cas protein is set forth in SEQ ID NO: 3; and the nucleotide sequence of the guide polynucleotide is set forth in any one of SEQ ID NOs: 2780, 2785, 2795, 2801, 2808, 2809, 2816, 2823, 2826, 2827, 2833, 2850, 2906, 2909, 2908, and 2918.

10. An isolated nucleic acid encoding the targeting system of any one of claims 1-9.

11. A vector comprising the nucleic acid of claim 10, and a regulatory sequence; Preferably, the vector is an mRNA or a viral vector; More preferably, the mRNA is delivered by LNP (lipid nanoparticle), the vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or an HIV viral vector.

12. A vector system, wherein, the vector system comprises the vector of claim 10; Preferably, the vector system comprises: (a) a polynucleotide sequence encoding a gRNA, and a first regulatory sequence regulating the expression of the gRNA; and (b) a polynucleotide sequence encoding the gRNA-guided nuclease, and a second regulatory sequence regulating the expression of the gRNA-guided nuclease; Preferably, the vector system comprises one or more vectors; More preferably, the vector system comprises a plurality of vectors, for example two vectors, the polynucleotide sequence encoding a gRNA, and a first regulatory sequence regulating the expression of the gRNA are located on a first vector, and the polynucleotide sequence encoding the gRNA-guided nuclease, and a second regulatory sequence regulating the expression of the gRNA-guided nuclease are located on a second vector.

13. A pharmaceutical composition, wherein, the pharmaceutical composition comprises the targeting system of any one of claims 1-9, the vector of claim 11, and / or the vector system of claim 12; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

14. Use of the targeting system according to any one of claims 1-9, the nucleic acid according to claim 10, the vector according to claim 11, the vector system according to claim 12 and / or the pharmaceutical composition according to claim 13 in the manufacture of a medicament for the diagnosis, treatment or prevention of a disease or disorder associated with a target nucleic acid; Preferably, the disease or disorder associated with a target nucleic acid comprises a disease or disorder caused by abnormal expression of the target nucleic acid. More preferably, the disease or disorder associated with a target nucleic acid comprises one or more of hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, coronary heart disease, stroke, myocardial infarction, non-alcoholic fatty liver disease, alcoholic fatty liver disease, obesity and amyloidosis.

15. A method of diagnosing, treating or preventing a disease or disorder associated with a target nucleic acid, comprising administering to a sample of a subject in need thereof or to a subject in need thereof an effective amount of the targeting system according to any one of claims 1-9, the vector according to claim 11, the vector system according to claim 12 and / or the pharmaceutical composition according to claim 13; Preferably, the disease or disorder associated with a target nucleic acid comprises a disease or disorder caused by abnormal expression of the target nucleic acid. More preferably, the disease or disorder associated with a target nucleic acid comprises one or more of hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, coronary heart disease, stroke, myocardial infarction, non-alcoholic fatty liver disease, alcoholic fatty liver disease, obesity and amyloidosis.

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