Genome editing compositions and methods

Abstract

The present disclosure provides CRISPR / Cas9-based fusion molecules and guide RNAs for use in the in vivo targeted reduction or ablation of the VEGFA gene product. The present disclosure also relates to formulations, methods of manufacture, and methods of use thereof.

Description

[Technical field]

[0001] The present invention relates to compositions and methods for genome editing. Description of electronically submitted text files

[01] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference herein. The name of the text file containing the sequence listing is "20230303_0300-PA-023JP_sequences.xml". The file is 702KB in size, was created on March 2, 2023, and has been submitted electronically via EFS-Web.

[0002] [Technical field]

[02] The present disclosure relates generally to the fields of molecular biology, immunology, and medicine. More specifically, the present invention relates to CRISPR / Cas9-based fusion molecules for use in targeted reduction or ablation of gene products in vivo, and methods of use thereof. [Background technology]

[0003]

[03] Recombinant DNA-binding proteins that can be customized to target any gene in mammalian cells have enabled rapid advances in biomedical research and are a promising platform for gene therapy. The RNA-guided CRISPR-Cas9 system has emerged as a promising platform for programmable targeted gene regulation. Fusing catalytically inactive, "death" Cas9 (dCas9) to a Kruppel-associated box (KRAB) domain generates a synthetic repressor capable of highly specific and potent regulation or silencing of target genes in cell culture experiments.

[0004]

[04] However, the sustained regulation and silencing of endogenous genes using synthetic dCas9-KRAB fusion proteins presents challenges for use in in vivo therapy. Synthetic repressors exceed the size package limit of viral vector delivery methods. Safety, toxicity, immunogenicity, and off-target effects are other challenges that limit the use of synthetic repressors in vivo. There is a need in the art for alternative approaches to generate engineered synthetic gene repressors and deliver them in vivo for use as therapeutic agents. The present disclosure addresses this unmet need in the art. Summary of the Invention

[0005]

[05] The present disclosure provides a construct of formula I: 5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -E-3'(I), wherein one of A and B is a polynucleotide encoding DNMT3A or a portion thereof, and the other of A and B is a polynucleotide encoding DNMT3L or a portion thereof, CasN is a polynucleotide encoding the N-terminal portion of dCas9, CasC is a polynucleotide encoding the C-terminal portion of dCas9, and E is a 5'-(A m5 -B m6 ) n3 -K r -D q -3' or 5'-K r -D q -(A m5 -B m6 ) n3-3', K is a polynucleotide encoding KRAB or a portion thereof, D is a polynucleotide encoding a modulator of gene expression, T comprises a polynucleotide encoding (i) an epitope capable of binding to an antibody or an antigen-binding fragment thereof, or (ii) a polypeptide sequence capable of binding to a nucleic acid structural element, m1, m2, m3, m4, m5, and m6 are each independently an integer selected from 0 to 3, n1, n2, and n3 are each independently an integer selected from 0 to 2, p is an integer selected from 0 to 20, q is an integer selected from 0 to 5, and r is an integer selected from 0 to 5, wherein when p is 0, at least one of m1, m2, m3, and m4 is not 0, at least one of n1 and n2 is not 0, and at least one of q and r is not 0.

[0006]

[06] In some embodiments, the construct has formula II: 5'-CasN-(A m3 -B m4 ) n2 -CasC-K r -D q In some embodiments, the construct comprises Formula IIa: 5'-CasN-(AB)-CasC-K-3' (II), where n2 is an integer selected from 1 and 2, r is an integer selected from 1 to 5, q is an integer selected from 0 to 5, and at least one of m3 and m4 is not 0. In some embodiments, the construct comprises Formula IIa: 5'-CasN-(AB)-CasC-K r -D q -3'(IIa).

[0007]

[07] In some embodiments, the construct has formula III: 5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -E-3'(III), where p is an integer selected from 1 to 20. In some embodiments, the construct comprises the formula IIIa: 5'-CasN-CasC-T p -3'(IIIa).

[0008]

[08] In some embodiments, the construct has formula IIIb:5'-(A m1 -B m2 )-CasN-CasC-T p -E-3'(IIIb), where at least one of m1 and m2 is not 0. In some embodiments, the construct comprises the formula IIIb-1:5'-(AB)-CasN-CasC-T p -K r -D q -3'(IIIb-1), in which r is an integer selected from 1 to 5, and q is an integer selected from 0 to 5.

[0009]

[09] In some embodiments, the construct has the formula IIIc: 5'-CasN-CasC-T p In some embodiments, the construct comprises the formula IIIc-1:5'-CasN-CasC-T p -(A m5 -B m6 ) n3 -K r -D q In some embodiments, the construct comprises the formula IIIc-2:5'-CasN-CasC-T p -K r -D q -(A m5 -B m6 ) n3 -3'(IIIc-2).

[0010] In some embodiments, T comprises (a) a polynucleotide encoding an epitope capable of binding to an antibody or antigen-binding fragment thereof, and further comprises (b) a polynucleotide at the 3' end of the polynucleotide of (a) encoding a self-cleaving peptide. In some embodiments, the 3' end of the nucleotide encoding the self-cleaving peptide further comprises a polynucleotide encoding (c) an antibody or antigen-binding fragment thereof capable of binding to the epitope.

[0011]

[0011] In some embodiments, T comprises (a) a polynucleotide encoding a polypeptide sequence capable of binding to a nucleotide sequence element, and further comprises a polynucleotide encoding a self-cleaving peptide at the 3' end of the polynucleotide (a).

[0012] In some embodiments, the auto-cleaving peptide is selected from the group consisting of T2A, P2A, E2A, and F2A auto-cleaving peptides. In some embodiments, the auto-cleaving peptide is T2A. In some embodiments, T2A comprises the amino acid sequence of SEQ ID NO:99.

[0013]

[0013] In some embodiments, DNMT3A comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the polynucleotide encoding DNMT3A comprises the nucleic acid sequence of SEQ ID NO: 83.

[0014]

[0014] In some embodiments, DNMT3L comprises Homo sapiens DNMT3L, Mus musculus DNMT3L, Mus caroli DNMT3L, Mus Pahari DNMT3L, Rattus norvegicus DNMT3L, Rattus Rattus DNMT3L, Arvicanthis niloticus DNMT3L, Grammomys surdaster DNMT3L, or Mastomys coucha DNMT3L. In some embodiments, DNMT3L comprises any one of the amino acid sequences of SEQ ID NOs: 74-82. In some embodiments, the polynucleotide encoding DNMT3L comprises any one of the nucleic acid sequences of SEQ ID NOs: 84-92.

[0015] In some embodiments, KRAB comprises the amino acid sequence of SEQ ID NO: 51, 53, or 230-241. In some embodiments, a polynucleotide encoding KRAB comprises the nucleic acid sequence of SEQ ID NO: 52, 54, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, or 228.

[0016] In some embodiments, the modulator of gene expression comprises Kruppel-associated repression box (KRAB), enhancer of zete homolog 2 (EZH2), G9A, lysine-specific histone demethylase 1A (LSD1), heterochromatin protein 1 (HP1), friend of GATA protein 1 (FOG1), histone deacetylase (HDAC3), and / or DOT1L. In some embodiments, the modulator of gene expression comprises the amino acid sequence of SEQ ID NO: 51, 53, 55, 57, 59, 61, 63, 65, or 67. In some embodiments, the polynucleotide encoding the modulator of gene expression comprises the nucleic acid sequence of SEQ ID NO: 52, 54, 56, 58, 60, 62, 64, 66, or 68.

[0017]

[0017] In some embodiments, the dCas9 is Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g. strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvibaculum lavamentivorans dCas9, Nitratifractor salsuginis (e.g. strain DSM 16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, and Streptococcus thermophilus (e.g., strain LMD-9) dCas9. In some embodiments, the dCas9 comprises the amino acid sequence of any one of SEQ ID NOs: 106-122. In some embodiments, the polynucleotide encoding dCas9 comprises the nucleic acid sequence of SEQ ID NO: 26.

[0018] In some embodiments, dCas9 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the N-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24. In some embodiments, the C-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25.

[0019] In some embodiments, a polynucleotide encoding dCas9 comprises the nucleic acid sequence of SEQ ID NO: 26. In some embodiments, a polynucleotide encoding an N-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, a polynucleotide encoding a C-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0020] In some embodiments, the polypeptide capable of binding to an antibody or antigen-binding fragment thereof is selected from the group consisting of GCN4, T2A, 10x GCN4, and 10XGFP-11. In some embodiments, GCN4 comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the polynucleotide encoding GCN4 comprises the nucleic acid sequence of SEQ ID NO: 101.

[0021] In some embodiments, the antibody or antigen-binding fragment is a single domain antibody, scFv, Fab, VH, VHH, or an antibody mimetic. In some embodiments, the antigen-binding fragment is an scFv.

[0022] In some embodiments, the polypeptide sequence capable of binding to a nucleic acid structural element is selected from the group consisting of MS2 bacteriophage coat protein (MCP), PP7, and PCP. In some embodiments, the polypeptide sequence capable of binding to a nucleic acid structural element is MCP. In some embodiments, the MCP comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the polynucleotide encoding MCP comprises the nucleic acid sequence of SEQ ID NO: 105.

[0023] In some embodiments, the nucleic acid structural element is an RNA hairpin motif. In some embodiments, the RNA hairpin motif is selected from the group consisting of MS2 and PP7. In some embodiments, the RNA hairpin motif is an MS2 RNA hairpin motif. In some embodiments, the MS2 RNA hairpin motif comprises the nucleic acid sequence of SEQ ID NO: 104.

[0024]

[0024] In some embodiments, the construct comprises a nucleic acid sequence of SEQ ID NO:134-162.

[0025]

[0025] The present disclosure provides a polypeptide expressed by any one of the constructs described herein. The present disclosure provides a vector comprising any one of the constructs described herein.

[0026] In some embodiments, the vector further comprises a polynucleotide encoding a single guide RNA (sgRNA). In some embodiments, the polynucleotide encoding the sgRNA further comprises 2 to 20 copies of a nucleic acid structural element.

[0027]

[0027] The present disclosure provides a cell comprising any one of the constructs described herein. The present disclosure provides a cell comprising any one of the polypeptides described herein. The present disclosure provides a cell comprising any one of the vectors described herein.

[0028] In some embodiments, the cell further comprises at least one sgRNA. In some embodiments, the at least one sgRNA further comprises 2-20 copies of the nucleic acid structural element.

[0029]

[0029] The present disclosure provides a composition comprising any one of the constructs described herein. The present disclosure provides a composition comprising any one of the polypeptides described herein. The present disclosure provides a composition comprising any one of the vectors described herein.

[0030] In some embodiments, the composition further comprises at least one sgRNA. In some embodiments, the sgRNA further comprises 2-20 copies of the nucleic acid structural element. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier.

[0031]

[0031] The present disclosure provides a method for modifying expression of a gene product and minimizing off-target modifications in a population of cells, comprising the step of introducing into the population of cells i) any one of the constructs described herein, or a polypeptide expressed by the construct, and ii) at least one sgRNA, wherein a KRAB and / or a modulator of gene expression provides a modification of at least one nucleotide near the gene and / or within a control element of the gene, thereby modifying expression of the gene product.

[0032] In some embodiments, (a) the 5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -3' portion, 5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -3' portion, 5'-(A m1 -B m2 )-CasN-CasC-T p -3' portion, 5'-(AB)-CasN-CasC-T of formula IIIb-1 p - 3' part, 5'-CasN-CasC-T of formula IIIc p -3' portion, 5'-CasN-CasC-T of formula IIIc-1 p -3' portion, or 5'-CasN-CasC-T of formula IIIc-2 p(b) a polypeptide of i) comprising a 5'-E-3' portion of Formula I, a 5'-E-3' portion of Formula III, a 5'-E-3' portion of Formula IIIb, a 5'-K of Formula IIIb-1, and a sgRNA of ii) are recruited to a genomic locus; r -D q -3' moiety of formula IIIc, 5'-E-3' moiety of formula IIIc-1, 5'-(A m5 -B m6 ) n3 -K r -D q -3' portion or the 5'-K of formula IIIc-2 r -D q -(A m5 -B m6 ) n3 Multiple copies of the polypeptide of i) including the -3' portion are recruited to a genomic locus by binding of the antibody or antigen-binding fragment thereof to the epitope, thereby recruiting the polypeptide expressed by the construct to the genomic locus and altering expression of the gene product in a population of cells.

[0033] In some embodiments, the method further comprises administering to the cell: iii) a 5′-(A m5 -B m6 ) n3 -K r -D q -3' or 5'-K r -D q -(A m5 -B m6 ) n3 5'-CasN-CasC-T of formula IIIa or a polypeptide expressed by the second construct, p The polypeptide of i) comprising the -3' portion and the sgRNA are recruited to a genomic locus, where multiple copies of the polypeptide of iii) are recruited to the genomic locus by binding of the antibody or antigen-binding fragment thereof to the epitope, thereby recruiting the polypeptide expressed by the construct of i) and the construct of iii) to the genomic locus and altering expression of a gene product in a population of cells.

[0034] In some embodiments, (a) the 5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -3' portion, 5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -3' portion, 5'-(A m1 -B m2 )-CasN-CasC-T p -3' portion, 5'-(AB)-CasN-CasC-T of formula IIIb-1 p - 3' part, 5'-CasN-CasC-T of formula IIIc p -3' portion, 5'-CasN-CasC-T of formula IIIc-1 p -3' portion, or 5'-CasN-CasC-T of formula IIIc-2 p (b) a polypeptide of i) comprising a 5'-E-3' portion of Formula I, a 5'-E-3' portion of Formula III, a 5'-E-3' portion of Formula IIIb, a 5'-K of Formula IIIb-1, and a sgRNA of ii) are recruited to a genomic locus; r -D q -3' moiety of formula IIIc, 5'-E-3' moiety of formula IIIc-1, 5'-(A m5 -B m6 ) n3 -K r -D q -3' portion or the 5'-K of formula IIIc-2 r -D q -(A m5 -B m6 ) n3 Multiple copies of the polypeptide of i) including the -3' portion are recruited to a genomic locus by binding of the polypeptide sequence capable of binding to a nucleic acid structural element to the nucleic acid structural element, thereby recruiting the polypeptide expressed by the construct to the genomic locus and modifying expression of a gene product in a population of cells.

[0035] In some embodiments, the method further comprises administering to the cell: iii) a 5′-(A m5 -B m6 ) n3 -K r -D q -3' or 5'-K r -D q -(A m5 -B m6 ) n3 5'-CasN-CasC-T of formula IIIa or a polypeptide expressed by the second construct, p The polypeptide of i) comprising the -3' portion, and the sgRNA are recruited to a genomic locus, where multiple copies of iii) are recruited to the genomic locus by binding of the polypeptide sequence capable of binding to a nucleic acid structural element to the nucleic acid structural element, thereby recruiting the polypeptide expressed by the construct to the genomic locus and altering expression of a gene product in a population of cells.

[0036]

[0036] The present disclosure provides an in vivo method of reducing or eliminating expression of a gene product in a subject, comprising the step of introducing into cells of the subject i) any one of the constructs described herein, or a polypeptide expressed by the construct, and ii) at least one sgRNA, wherein the KRAB and / or a modulator of gene expression provides a modification of at least one nucleotide near the gene and / or within a control element of the gene, thereby modifying expression of the gene product in the subject.

[0037]

[0037] The present disclosure provides a method for treating or alleviating a symptom of a gene product associated disorder in a subject, comprising the step of introducing into a cell of the subject i) any one of the constructs described herein, or a polypeptide expressed by the construct, and ii) at least one sgRNA, wherein a KRAB and / or a modulator of gene expression provides an alteration of at least one nucleotide near the gene and / or within a control element of the gene, thereby altering expression of the gene product and treating or alleviating a symptom of the gene product associated disorder in the subject.

[0038] In some embodiments, expression of the gene product is reduced by 50-100% in the plurality of modified cells compared to a wild-type cell population. In some embodiments, the ratio of on-site modifications of the gene product to off-site modifications of the gene product is about 10:1.

[0039] In some embodiments, the modification of the at least one nucleotide is DNA methylation or a histone modification. In some embodiments, the modification of the at least one nucleotide is DNA methylation.

[0040]

[0040] In some embodiments, the gene control element is a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, or a locus control region.

[0041]

[0041] In some embodiments, at least one nucleotide modification near a gene and / or within a control element of the gene is located within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp upstream of the transcription start site of the gene.

[0042]

[0042] In some embodiments, at least one nucleotide modification near a gene and / or within a control element of the gene is located within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp downstream of the transcription start site of the gene.

[0043] In some embodiments, the construct is deoxyribonucleic acid (DNA). In some embodiments, the construct is messenger ribonucleic acid (mRNA). In some embodiments, the construct is formulated in a liposome or lipid nanoparticle. In some embodiments, the construct and sgRNA are formulated in a liposome or lipid nanoparticle. In some embodiments, the construct and sgRNA are formulated in the same liposome or lipid nanoparticle. In some embodiments, the construct and sgRNA are formulated in different liposomes or lipid nanoparticles.

[0044] In some embodiments, the liposome or lipid nanoparticle comprises an ionizable lipid (20%-70%, molar ratio), a PEGylated lipid (0%-30%, molar ratio), a supporting lipid (30%-50%, molar ratio), and cholesterol (10%-50%, molar ratio). In some embodiments, the ionizable lipid is selected from the group consisting of a pH-responsive ionizable lipid, a thermoresponsive ionizable lipid, and a light-responsive ionizable lipid.

[0045] In some embodiments, the construct is formulated in an AAV vector. In some embodiments, the construct and the sgRNA are formulated in an AAV vector. In some embodiments, the construct and the sgRNA are formulated in the same AAV vector. In some embodiments, the construct and the sgRNA are formulated in different AAV vectors.

[0046]

[0046] In some embodiments, the construct or the polypeptide expressed by the construct is delivered to cells by local injection, systemic injection, or a combination thereof.

[0047]

[0047] In some embodiments, the gene product is selected from the group consisting of a VEGFA gene product, a PCSK9 gene product, an ANGPTL3 gene product, a PTBP1 gene product, a TTR gene product, a Ube3a-ATS gene product, a Ptp1b gene product, an APOC3 gene product, an hsd17b13 gene product, a bcl11a gene product, and a TGF-β gene product.

[0048] In some embodiments, the subject is a human. In some embodiments, the disease or disorder is selected from the group consisting of familial hypercholesterolemia (FH), nonalcoholic steatohepatitis (NASH), Parkinson's disease, liver fibrosis (HF), age-related macular disease (AMD), Angelman syndrome (AS), type 2 diabetes, β-thalassemia, and hepatocellular carcinoma. [Brief description of the drawings]

[0049]

[0049] [Figure 1A] Schematic showing the interactions of constructs containing DNMT3A, DNMT3L, KRAB, and either intact or split dCas9 with DNA. [Figure 1B] A schematic diagram of the construct is shown. [Figure 1C]The editing efficiency in suppressing the expression of two targeted genes by gRNAs targeting CD81 or CD151, respectively, for the constructs shown in Figures 1A-1B is shown. In the figure, Dnmt-dCas9-Krab represents Dnmt3A-Dnmt3L-dCas9-Krab, 768 INS represents dCas9N-2-Dnmt3A-Dnmt3L-dCas9C-2-Krab, 776 INS represents dCas9N-3-Dnmt3A-Dnmt3L-dCas9C-3-Krab, 1009 INS represents dCas9N-4-Dnmt3A-Dnmt3L-dCas9C-4-Krab, and 1048-1063 INS represents dCas9N-5-Dnmt3A-Dnmt3L-dCas9C-6-Krab. INS represents dCas9N-5-Dnmt3A-Dnmt3L-dCas9C-5-Krab, 1072 INS represents dCas9N-6-Dnmt3A-Dnmt3L-dCas9C-6-Krab, 1246 INS represents dCas9N-7-Dnmt3A-Dnmt3L-dCas9C-7-Krab, 1248 INS represents dCas9N-8-Dnmt3A-Dnmt3L-dCas9C-8-Krab, 1260 INS represents dCas9N-10-Dnmt3A-Dnmt3L-dCas9C-10-Krab, and the control represents Dnmt3A-Dnmt3L-dCas9-Krab containing Nt gRNA.

[0050] [Figure 2A] Schematic diagram showing recruitment of DNMT3A, DNMT3L, and KRAB to dCas9 via scFv bound to 10xGCN4. [Figure 2B] A schematic diagram of the construct is shown. [Fig. 2C-2E]The editing efficiency in suppressing the expression of two targeted genes by gRNAs targeting CD81 or CD151, respectively, for the constructs shown in Figures 2A-2B is shown. In the figure, Suntag represents Dnmt3A-Dnmt3L-dCas9-10×GCN4-T2A-scFv-KRAB, and Dnmt-dCas9+scFv-Krab represents Dnmt3A-Dnmt3L-dCas9- Also represents 10xGCN4-T2A-scFv-KRAB, dCas9+scFv-Dnmt-Krab represents dCas9-10xGCN4-T2A-scFv-Dnmt3A-Dnmt3L-KRAB, dCas9+scFv-Dnmt+scFv-Krab represents dCas9-10xGCN4-T2A-scFv-Dnmt3A-Dnmt3L-scFv-KRAB, and the control represents Dnmt3A-Dnmt3L-dCas9-Krab in Figure 1, containing Nt gRNA.

[0051] [Figure 3A] FIG. 1 is a schematic diagram showing a construct containing dCas9 and a guide RNA containing MS2 that binds to DNMT3A, DNMT3L, and KRAB via MCP. [Figure 3B] A schematic diagram of the construct containing DNMT3A, DNMT3L, MCP, dCas9, and KRAB is shown.

[0052] [Figure 4A] Schematic diagram showing the interaction of complexes comprising dCas9, DNMT3A, and DNMT3L, as well as modulators of gene expression selected from Kruppel-associated repression box (KRAB), enhancer of zete homolog 2 (EZH2), G9A, lysine-specific histone demethylase 1A (LSD1), heterochromatin protein 1 (HP1), friend of GATA protein 1 (FOG1), histone deacetylase (HDAC3), and / or DOT1L, with their DNA target sites. [Figure 4B]Schematic diagram of a construct comprising dCas9, DNMT3A, and DNMT3L, as well as a modulator of gene expression selected from Kruppel-associated repression box (KRAB), enhancer of zete homolog 2 (EZH2), G9A, lysine-specific histone demethylase 1A (LSD1), heterochromatin protein 1 (HP1), friend of GATA protein 1 (FOG1), histone deacetylase (HDAC3), and / or DOT1L. [Fig. 4C-4D] The editing efficiency in suppressing target gene expression for the constructs shown in Figures 4A-4B is shown, where the modulator of gene expression is selected from the types listed to the right of the figure. [Fig. 4E-4F] The editing efficiency in suppressing target gene expression for the constructs shown in Figures 4A-4B is shown, where the KRAB modulator is selected from the sources listed to the right of the figure. The control represents Dnmt3A-Dnmt3L-dCas9-Krab in Figure 1, which contains Nt gRNA.

[0053] [Figure 5A] FIG. 1 is a schematic showing interactions between complexes containing dCas9, DNMT3A, and DNMT3L from various species. [Figure 5B] A schematic diagram of the construct is shown. [Fig. 5C-5D] The editing efficiency in suppressing target gene expression for the constructs shown in Figures 5A-5B is shown, where DNMT3A and DNMT 3L are selected from the sources listed on the right of the figure. The control represents Dnmt3A-Dnmt3L-dCas9-Krab in Figure 1, which contains Nt gRNA.

[0054] [Figure 6A] Three examples of constructs of the present application are shown. [Figure 6B] 1 shows the editing efficiency in suppressing targeted gene expression for the three constructs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050]

[0055] The present disclosure overcomes problems associated with current technology by providing constructs comprising DNMT3A, DNMT3L, dCas9, and KRAB for targeted modification of expression of gene products, e.g., targeted modification of gene products in cells for use in in vivo gene therapy.

[0051]

[0056] I. Definition

[0052]

[0057] As used herein, the term "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that encodes a protein. The coding sequence may further comprise initiation and termination signals operably linked to control elements, including a promoter and polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized.

[0053]

[0058] The terms "complement" or "complementary" as used herein with reference to nucleic acids can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule. "Complementarity" refers to the property shared between two nucleic acid sequences such that when they are aligned antiparallel to one another, the nucleotide bases at every position are complementary.

[0054]

[0059] The terms "correction", "genome editing" and "restoration" refer to altering a mutated gene to code for a mutant protein, a truncated protein, or no protein at all, resulting in a full-length functional or partially full-length functional protein. Correcting or restoring a mutated gene can include replacing a region of the gene with a mutation, or replacing the entire mutated gene with a copy of the gene without the mutation by a repair mechanism such as homology-directed repair (HDR). Correcting or restoring a mutated gene can also include repairing a frameshift mutation that causes a premature stop codon, an aberrant splice acceptor site, or an aberrant splice donor site by generating a double-strand break in the gene and then repairing it using non-homologous end joining (NHEJ). NHEJ can add or delete at least one base pair during repair, which can restore the proper reading frame and remove the premature stop codon. Correcting or restoring a mutated gene can also include disrupting the aberrant splice acceptor site or splice donor sequence. Correcting or restoring a mutated gene can also involve deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA strand to remove the DNA between the two nuclease target sites and restore the proper reading frame by repairing the DNA break by NHEJ.

[0055]

[0060] As used herein, the terms "donor DNA," "donor template," and "repair template" refer to a double-stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA can encode a fully functional protein, or a partially functional protein.

[0056]

[0061] As used herein, the terms "frameshift" or "frameshift mutation" are used interchangeably and refer to a type of genetic mutation that causes a shift in the reading frame of an mRNA codon by the addition or deletion of one or more nucleotides. A shift in reading frame can result in a change in the amino acid sequence during protein translation, such as a missense mutation or a premature stop codon.

[0057]

[0062] As used herein, the terms "functional" and "fully functional" describe a protein that has biological activity. A "functional gene" means a gene that is transcribed into mRNA that is translated into a functional protein.

[0058]

[0063] As used herein, the term "fusion protein" refers to a chimeric protein created directly or indirectly by the covalent or non-covalent joining of two or more genes that originally coded for separate proteins. In some embodiments, translation of the fusion gene results in a single polypeptide that possesses functional properties derived from each of the original proteins.

[0059]

[0064] As used herein, the term "genetic construct" refers to a DNA or RNA molecule that comprises a nucleotide sequence that encodes a protein. The coding sequence includes initiation and termination signals operably linked to control elements, including a promoter and polyadenylation signal, capable of directing expression in a cell.

[0060]

[0065] The term "homologous recombination repair" or "HDR", used interchangeably herein, refers to a mechanism that repairs double-stranded DNA damage in cells when a homologous piece of DNA is present in the nucleus, mainly in the G2 and S phases of the cell cycle. HDR uses a donor DNA template to guide repair, which can be used to create specific sequence changes to the genome, including targeted addition of the entire genome. When the donor template is provided with a site-specific nuclease, such as by a CRISPR / Cas9-based system, the cellular machinery repairs the break by homologous recombination, which increases by several orders of magnitude in the presence of DNA breaks. In the absence of a homologous DNA piece, non-homologous end joining may occur instead.

[0061]

[0066] As used herein, the term "genome editing" refers to altering a gene. Genome editing can include correcting or restoring a mutated gene. Genome editing can include knocking out a gene, such as a mutated gene or a normal gene. Genome editing can be used to treat a disease by altering a gene of interest.

[0062]

[0067] The term "identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are the same over a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences at a particular region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment creates one or more staggered ends and the particular region being compared contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using computer sequence algorithms such as BLAST or BLAST 2.0. The identity of related peptides can be easily calculated by known methods.Such methods include those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al, SIAM J. Applied Math. 48, 1073 (1988), the entireties of which are incorporated herein by reference.

[0063]

[0068] As used herein, the terms "mutated gene" or "mutated gene", used interchangeably herein, refer to a gene that has undergone a detectable mutation. A mutant gene has undergone an alteration, such as a loss, gain, or exchange of genetic material, that affects the normal transmission and expression of the gene. As used herein, a "disrupted gene" refers to a mutant gene that has a mutation that causes a premature stop codon. A disrupted gene product is truncated compared to the full-length, undisrupted gene product.

[0064]

[0069] As used herein, the term "modulator of epigenetic modification" refers to an agent that targets gene expression by epigenetic modification (e.g., by histone acetylation or methylation, or DNA methylation at the control elements of the target gene, such as promoters, enhancers, or transcription start sites). Chromatin remodeling and DNA methylation are two main mechanisms for controlling gene transcription. Specific epigenetic marks (e.g., DNA methylation) structurally or biochemically direct gene transcription or gene silencing / suppression. For example, DNA methylation of regions that control transcriptional activity alters gene expression without changing the underlying DNA sequence. Transcriptional control using epigenetic modification (e.g., DNA methylation) allows targeted regulation of gene expression without affecting the expression of other gene products.

[0065]

[0070] As used herein, the term "non-homologous end joining (NHEJ) pathway" refers to a pathway that repairs double-strand breaks in DNA by directly ligating the broken ends without the need for a homologous template. Template-independent religation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random microinsertions and microdeletions (indels) at the breaks in DNA. This method can be used to intentionally disrupt, delete, or change the reading frame of a targeted gene sequence. NHEJ typically uses short homologous DNA sequences, called microhomologies, to guide repair. These microhomologies are often present in the single-stranded overhangs at the ends of double-strand breaks. Although NHEJ usually repairs the breaks accurately when the overhangs are perfectly matched, imprecise repairs that result in loss of nucleotides can still occur, but this is much more common when the overhangs are mismatched.

[0066]

[0071] As used herein, the term "normal gene" refers to a gene that has not undergone alteration, such as loss, gain, or exchange of genetic material. A normal gene undergoes normal gene transmission and gene expression.

[0067]

[0072] As used herein, the term "nuclease-mediated NHEJ" refers to NHEJ that is initiated after a nuclease, such as cas9, breaks double-stranded DNA.

[0068]

[0073] As used herein, the term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA, or hybrids, and can contain combinations of deoxyribonucleotides and ribonucleotides and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0069]

[0074] As used herein, the term "operably linked" means that the expression of a gene is under the control of a promoter with which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. As is well known in the art, variations in this distance may be accommodated without loss of promoter function.

[0070]

[0075] As used herein, the term "partially functional" describes a protein encoded by a mutant gene that has less biological activity than a functional protein, but more biological activity than a non-functional protein. In one embodiment, a partially functional protein exhibits less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the biological activity of the corresponding functional protein.

[0071]

[0076] The terms "premature stop codon" or "out-of-frame stop codon," as used interchangeably herein, refer to a nonsense mutation in a sequence of DNA that results in a stop codon at a position not normally found in the wild-type gene. A premature stop codon may cause a protein to be truncated or to be shorter than the full-length version of the protein.

[0072]

[0077] As used herein, the term "promoter" refers to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcription control sequences that further enhance expression and / or alter the spatial and / or temporal expression of the nucleic acid. A promoter may contain distal enhancer or repressor elements, which can be located thousands of base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially control the expression of genetic components with respect to the cell, tissue, or organ in which expression occurs, with respect to the developmental stage in which expression occurs, or in response to an external stimulus such as a physiological stress, a pathogen, a metal ion, or an inducer. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, and the CMV IE promoter.

[0073]

[0078] As used herein, the term "target gene" refers to any nucleotide sequence that encodes a known or putative gene product. A target gene may be a mutated gene involved in a genetic disease or disorder.

[0074]

[0079] As used herein, the term "target region" means the region of a target gene to which a site-specific nuclease is designed to bind.

[0075]

[0080] As used herein, the term "transgene" refers to a gene or genetic material containing a genetic sequence that is isolated from one organism and introduced into a different organism. Alternatively, the term "transgene" also refers to a gene or genetic material that is chemically synthesized and introduced into an organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or may alter the normal function of the genetic code of the transgenic organism. Introduction of a transgene has the potential to alter the phenotype of the organism.

[0076]

[0081] As used herein, the term "variant" when used in reference to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence, (ii) a complement of the referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto. "Variant" refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity.

[0077]

[0082] Variant may also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing one amino acid with another amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982), the entirety of which is incorporated herein by reference. The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is well known in the art that amino acids with similar hydropathic indices can be substituted and still retain the function of the protein. In one aspect, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. Consideration of the hydrophilicity of an amino acid in the context of a peptide allows one to calculate the maximum local average hydrophilicity of that peptide. Substitutions can be made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0078]

[0083] As used herein, the term "vector" refers to a nucleic acid sequence that contains a replication origin. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, such as a DNA plasmid.

[0079]

[0084] As used herein, the terms "gene transfer," "gene delivery," and "gene transduction" refer to a method or system for reliably inserting specific nucleotide sequences (e.g., DNA or RNA), fusion proteins, polypeptides, etc., into targeted cells.

[0080]

[0085] As used herein, the terms "adenovirus-associated virus (AAV) vector," "AAV gene therapy vector," and "gene therapy vector" refer to a vector having functional or partially functional ITR sequences and a transgene. As used herein, the term "ITR" refers to an inverted terminal repeat (ITR). The ITR sequences can be derived from adeno-associated virus serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, and AAV-6. However, the ITRs need not be wild-type nucleotide sequences and can be modified (e.g., by insertion, deletion, or substitution of nucleotides) so long as the sequences retain the ability to provide functional rescue, replication, and packaging. An AAV vector can have one or more of the AAV wild-type genes, preferably the rep and / or cap genes, deleted in whole or in part, but retain functional flanking ITR sequences. Functional ITR sequences function, for example, to rescue, replicate, and package AAV virions or particles. Thus, an "AAV vector" is defined herein to include at least those sequences necessary to insert a transgene into the cells of a subject, optionally including those sequences necessary in cis for viral replication and packaging (e.g., functional ITRs).

[0081]

[0086] As used herein, the term "gene therapy" refers to a method of treating a patient in which a polypeptide or nucleic acid sequence is transferred to the patient's cells such that the activity and / or expression of a particular gene is modulated. In certain embodiments, expression of the gene is inhibited. In certain embodiments, expression of the gene is enhanced. In certain embodiments, the temporal or spatial pattern of expression of the gene is modulated.

[0082]

[0087] A "transgene" can contain a transgenic sequence or a natural or wild-type DNA sequence. A transgene can become part of the genome of a primate subject. A transgenic sequence can be partially or wholly species-heterologous, i.e., the transgenic sequence, or a portion thereof, can originate from a different species than the cell into which it is introduced.

[0083]

[0088] As used herein, the term "stably maintained" refers to the characteristic of a transgenic subject (e.g., a human or non-human primate) that maintains at least one of their transgenic elements (i.e., the desired element) through multiple generations of cells. For example, the term is intended to encompass many cell division cycles of the originally transfected cell. The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of a cell. The term "stable transfectant" refers to a cell that has foreign DNA stably integrated into its genomic DNA.

[0084]

[0089] As used herein, the terms "transgene encoding," "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides can determine, for example, the order of amino acids along a polypeptide (protein) chain. Thus, a DNA sequence can encode an amino acid sequence.

[0085]

[0090] As used herein, the term "wild-type" (wt) refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is therefore arbitrarily designated the "normal" or "wild-type" form of the gene. In contrast, the term "altered" or "mutant" refers to a gene or gene product that exhibits altered sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It should be noted that it is possible to isolate naturally occurring mutants, which are identified by the acquisition of altered characteristics when compared to the wild-type gene or gene product.

[0086]

[0091] As used herein, the term "transfection" refers to the uptake of foreign nucleic acid (e.g., DNA or RNA) by a cell. A cell is "transfected" when an exogenous nucleic acid (DNA or RNA) is introduced into the cell membrane. Numerous transfection techniques are generally known in the art (see, e.g., Graham et al., Virol., 52:456 (1973); Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier, (1986); and Chu et al., Gene 13:197 (1981), which are incorporated herein by reference in their entireties). Using such techniques, one or more exogenous DNA moieties, such as gene transfer vectors and other nucleic acid molecules, can be introduced into suitable recipient cells.

[0087]

[0092] As used herein, the terms "stable transfection" and "stably transfected" refer to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has foreign DNA stably integrated into its genomic DNA.

[0088]

[0093] As used herein, the term "transient transfection" or "transiently transfected" refers to the introduction of foreign DNA into a cell, where the foreign DNA is not integrated into the genome of the transfected cell, but is maintained as an episome. During this time, the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in chromosomes. The term "transient transfectant" refers to a cell that has taken up foreign DNA, but has failed to integrate this DNA. As used herein, the term "transduction" refers to the delivery of DNA molecules to a recipient cell, either in vivo or in vitro, by a replication-defective viral vector, e.g., a recombinant AAV virion.

[0089]

[0094] As used herein, the term "recipient cell" refers to a cell that has been or can be transfected or transduced with a nucleic acid construct or vector having a selected nucleotide sequence of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent, provided that the selected nucleotide sequence is present. Recipient cells can be cells of a subject to which gene therapy particles and / or gene therapy vectors have been administered.

[0090]

[0095] As used herein, the term "recombinant DNA molecule" means a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.

[0091]

[0096] As used herein, the term "control element" refers to a genetic element capable of controlling the expression of a nucleic acid sequence. For example, a promoter is a control element that facilitates the initiation of transcription of an operably linked coding region. Other control elements are splicing signals, polyadenylation signals, termination signals, etc.

[0092]

[0097] The term DNA "regulatory sequences" collectively refers to control elements, such as promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these regulatory sequences need be present.

[0093]

[0098] Transcriptional control signals in eukaryotes generally include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987), incorporated herein by reference in its entirety). Promoter and enhancer elements have been isolated from a wide variety of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (analogous regulatory sequences, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on the type of recipient cell. Some eukaryotic promoters and enhancers have a broad host range, while others are functional in a limited subset of cell types (see, e.g., Voss et al., Trends Biochem. Sci., 11:287 (1986); and Maniatis et al., supra, for review, incorporated herein by reference in its entirety). For example, the SV40 early gene enhancer is highly active in a variety of cell types from many mammalian species and is used to express proteins in a wide range of mammalian cells (Dijkema et al, EMBO J. 4:761 (1985), incorporated herein by reference in its entirety). Promoter and enhancer elements derived from the human elongation factor 1 alpha gene (Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene 91:217 (1990); and Mizushima and Nagata, Nucl. Acids. Res., 18:5322 (1990)), Rous sarcoma virus long terminal repeat (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)), and human cytomegalovirus (Boshart et al., Cell 41:521 (1985)) are also useful for expression of proteins in a variety of mammalian cells and are incorporated by reference in their entireties.Promoters and enhancers can be found in nature, either alone or together. For example, retroviral-type long terminal repeats contain both promoter and enhancer elements. Generally, promoters and enhancers function independently of the gene to be transcribed or translated. Thus, the enhancers and promoters used can be "endogenous," "exogenous," or "heterologous" with respect to the gene to which they are operably linked. An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is juxtaposed to a gene by genetic engineering (i.e., molecular biology techniques) such that transcription of the gene is directed by the linked enhancer / promoter.

[0094]

[0099] As used herein, the term "tissue-specific" refers to control elements or regulatory sequences, e.g., promoters, enhancers, and the like, which direct expression of a nucleic acid sequence to be substantially greater than in a particular cell type or tissue.

[0095]

[0100] The presence of "splicing signals" in an expression vector often results in high levels of expression of recombinant transcripts. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of splice donor and acceptor sites (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7-16.8, which is incorporated herein by reference in its entirety). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

[0096]

[0101] Transcription termination signals are usually found downstream of polyadenylation signals and are several hundred nucleotides long. As used herein, the term "poly A site" or "poly A sequence" refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of recombinant transcripts is desirable because transcripts lacking a poly A tail are unstable and rapidly degraded. The poly A signal utilized in an expression vector can be "heterologous" or "endogenous." An endogenous poly A signal is one that is naturally found at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one that has been isolated from one gene and operably linked to the 3' end of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 polyA signal is contained on a 237 bp BamHI / BclI restriction fragment and directs both termination and polyadenylation (Sambrook et al., supra, at 16.6-16.7, incorporated herein by reference in its entirety).

[0097]

[0102] As used herein, the terms "subject" and "patient" are used interchangeably herein to refer to both human and non-human animals. The term "non-human animal" in this disclosure refers to all vertebrates, including mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0098]

[0103] As defined herein, a "therapeutically effective amount" or "therapeutically effective dose" is an amount or dose of a fusion protein, polypeptide, nucleic acid, lipid nanoparticle, liposome, AAV particle, or virion that can produce a sufficient amount of a desired protein to modulate the activity of the protein in a desired manner, thereby providing a palliative tool for clinical intervention. In some embodiments, a therapeutically effective amount or dose of a transfected fusion protein, polypeptide, nucleic acid, AAV particle, or virion described herein is sufficient to confer suppression of the gene targeted by the fusion protein / gene therapy construct.

[0099]

[0104] As used herein, for example, the term "treating" a disorder means that a subject (e.g., a human) having, at risk of having, and / or experiencing symptoms of the disorder when a fusion molecule, or a nucleic acid encoding a fusion molecule, and / or a gRNA, or a nucleic acid encoding a gRNA, e.g., as described herein, is administered, suffers from less severe symptoms and / or recovers more quickly than if the nucleic acid fusion molecule, or a nucleic acid encoding a fusion molecule, and / or a gRNA, or a nucleic acid encoding a gRNA, e.g., as described herein, is never administered.

[0100]

[0105] II. Constructs

[0101]

[0106] In one aspect, provided herein is a construct comprising a polynucleotide encoding DNMT3A or a portion thereof, a polynucleotide encoding DNMT3L or a portion thereof, a polynucleotide encoding dCas9, a polynucleotide encoding KRAB or a portion thereof, a polynucleotide encoding a modulator of gene expression, and a polynucleotide encoding (i) an epitope capable of binding to an antibody or antigen-binding fragment thereof, or (ii) a polypeptide sequence capable of binding to a nucleic acid structural element. In some embodiments, provided herein is a construct comprising a polynucleotide having Formula I:5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -E-3'(I)

[0102]

[0107] In formula I, one of A and B is a polynucleotide encoding DNMT3A or a part thereof, and the other of A and B is a polynucleotide encoding DNMT3L or a part thereof. CasN and CasC are polynucleotides encoding the N-terminal portion of dCas9 and the C-terminal portion of dCas9, respectively. E is 5'-(A m5-B m6 ) n3 -K r -D q -3' or 5'-K r -D q -(A m5 -B m6 ) n3 -3'. K is a polynucleotide encoding KRAB or a portion thereof. D is a polynucleotide encoding a modulator of gene expression. The modulator of gene expression can be, for example, Kruppel-associated repression box (KRAB), enhancer of zete homolog 2 (EZH2), G9A, lysine-specific histone demethylase 1A (LSD1), heterochromatin protein 1 (HP1), friend of GATA protein 1 (FOG1), histone deacetylase (HDAC3), and / or DOT1L. T comprises a polynucleotide encoding (i) an epitope capable of binding to an antibody or antigen-binding fragment thereof (e.g., a single domain antibody, scFv, Fab, VH, VHH, or antibody mimetic), or (ii) a polypeptide sequence capable of binding to a nucleic acid structural element. The polypeptide capable of binding to an antibody or antigen-binding fragment thereof can be, for example, GCN4. A polypeptide sequence capable of binding to a nucleic acid structural element can be, for example, the MS2 bacteriophage coat protein (MCP). Polypeptides encoding DNMT3A, DNMT3L, CasN, CasC, KRAB, and modulators of gene expression are described in more detail below.

[0103]

[0108] In formula I, m1, m2, m3, m4, m5, and m6 are each independently an integer selected from 0 to 3, n1, n2, and n3 are each independently an integer selected from 0 to 2, p is an integer selected from 0 to 20, q is an integer selected from 0 to 5, and r is an integer selected from 0 to 5; when p is 0, at least one of m1, m2, m3, and m4 is not 0, at least one of n1 and n2 is not 0, and at least one of q and r is not 0.

[0104]

[0109] In some embodiments, the construct provided herein has the formula II: 5'-CasN-(A m3 -B m4 ) n2 -CasC-K r -D q In some embodiments, the construct has the structure of Formula IIa: 5'-CasN-(AB)-CasC-K-3', where n2 is an integer selected from 1 and 2, r is an integer selected from 1 to 5, q is an integer selected from 0 to 5, and at least one of m3 and m4 is not 0. In some embodiments, the construct has the structure of Formula IIa: 5'-CasN-(AB)-CasC-K r -D q -3' structure. Exemplary constructs having the structure of Formula IIa are shown in the second, third, and fourth schematic diagrams of FIG.

[0105]

[0110] In some embodiments, the constructs provided herein have the formula III: 5'-(A m1 -B m2 ) n1 -CasN-(A m3 -B m4 ) n2 -CasC-T p -E-3', where p is an integer selected from 1 to 20. Exemplary constructs having the structure of formula III are shown in Figures 2B and 3B. In some embodiments, the construct has the structure of formula IIIa: 5'-CasN-CasC-T p -3' In the case of the construct of formula IIIa, DNMT3A, DNMT3L, and KRAB are expressed in separate constructs.

[0106]

[0111] In some embodiments, the construct provided herein has the formula IIIb:5'-(A m1 -B m2 )-CasN-CasC-T p 5'-(AB)-CasN-CasC-T-E-3', wherein at least one of m1 and m2 is not 0. Exemplary constructs having the structure of formula IIIb are shown in the second schematic diagram of FIG. 2B and in the second schematic diagram of FIG. 3B. In some embodiments, the construct has the structure of formula IIIb-1:5'-(AB)-CasN-CasC-Tp -K r -D q -3', in which r is an integer selected from 1 to 5, and q is an integer selected from 0 to 5.

[0107]

[0112] In some embodiments, the construct provided herein has the formula IIIc: 5'-CasN-CasC-T p 3B , wherein n3 is an integer selected from 1 and 2, r is an integer selected from 1 to 5, q is an integer selected from 0 to 5, and at least one of m5 and m6 is not 0. Exemplary constructs having the structure of formula IIIc are shown in the third and fourth schematic diagrams of FIG. 2B and the third and fourth schematic diagrams of FIG. 3B . In some embodiments, the construct has the structure of formula IIIc-1:5'-CasN-CasC-T p -(A m5 -B m6 ) n3 -K r -D q In some embodiments, the construct has the structure of formula IIIc-2:5'-CasN-CasC-T p -K r -D q -(A m5 -B m6 ) n3 -3' structure.

[0108]

[0113] The sequences of the constructs described herein are:

[0109]

[0114] As described in Table 1, dCas9 is shown in bold underline and DNMT3A is shown in italics. DNMT3L is underlined. , KRAB is shown in bold italics, and EZH2, G9A, LSD1, HP1, FOG1, DHAC3, DOT1L, 10x GCN4, MCP, and ScFV are shown in bold.

[0110]

[0115] Table 1: Exemplary sequences of constructs TIFF2025507990000001.tif212170TIFF2025507990000002.tif220170TIFF2025507990000003.tif220170TIFF2025507990000004.tif220170TIFF2025507990000005.tif220170TIFF2025507990000006.tif221170TIFF2025507990000007.tif220170TIFF2025507990000008.tif220170TIFF2025507990000009.tif220170TIFF2025507990000010.tif220170TIFF2025507990000011.tif221170TIFF2025507990000012.tif220170TIFF2025507990000013.tif220170TIFF2025507990000014.tif220170TIFF2025507990000015.tif220170TIFF2025507990000016.tif220170TIFF2025507990000017.tif221170TIFF2025507990000018.tif220170TIFF2025507990000019.tif220170TIFF2025507990000020.tif220170TIFF2025507990000021.tif220170TIFF2025507990000022.tif221170TIFF2025507990000023.tif220170TIFF2025507990000024.tif220170TIFF2025507990000025.tif220170TIFF2025507990000026.tif220170TIFF2025507990000027.tif220170TIFF2025507990000028.tif221170TIFF2025507990000029.tif220170TIFF2025507990000030.tif220170TIFF2025507990000031.tif220170TIFF2025507990000032.tif220170TIFF2025507990000033.tif220170TIFF2025507990000034.tif220170TIFF2025507990000035.tif221170TIFF2025507990000036.tif220170TIFF2025507990000037.tif220170TIFF2025507990000038.tif220170TIFF2025507990000039.tif220170TIFF2025507990000040.tif220170TIFF2025507990000041.tif220170TIFF2025507990000042.tif220170TIFF2025507990000043.tif221170TIFF2025507990000044.tif220170TIFF2025507990000045.tif220170TIFF2025507990000046.tif220170TIFF2025507990000047.tif220170TIFF2025507990000048.tif220170TIFF2025507990000049.tif220170TIFF2025507990000050.tif221170TIFF2025507990000051.tif220170TIFF2025507990000052.tif220170TIFF2025507990000053.tif220170TIFF2025507990000054.tif220170TIFF2025507990000055.tif220170TIFF2025507990000056.tif221170TIFF2025507990000057.tif220170TIFF2025507990000058.tif220170TIFF2025507990000059.tif220170TIFF2025507990000060.tif220170TIFF2025507990000061.tif220170TIFF2025507990000062.tif220170TIFF2025507990000063.tif220170TIFF2025507990000064.tif221170TIFF2025507990000065.tif220170TIFF2025507990000066.tif220170TIFF2025507990000067.tif220170TIFF2025507990000068.tif220170TIFF2025507990000069.tif220170TIFF2025507990000070.tif221170TIFF2025507990000071.tif220170TIFF2025507990000072.tif220170TIFF2025507990000073.tif220170TIFF2025507990000074.tif220170TIFF2025507990000075.tif220170TIFF2025507990000076.tif220170TIFF2025507990000077.tif220170TIFF2025507990000078.tif220170TIFF2025507990000079.tif221170TIFF2025507990000080.tif220170TIFF2025507990000081.tif220170TIFF2025507990000082.tif220170TIFF2025507990000083.tif220170TIFF2025507990000084.tif220170TIFF2025507990000085.tif220170TIFF2025507990000086.tif220170TIFF2025507990000087.tif221170TIFF2025507990000088.tif220170TIFF2025507990000089.tif220170TIFF2025507990000090.tif220170TIFF2025507990000091.tif220170TIFF2025507990000092.tif220170TIFF2025507990000093.tif220170TIFF2025507990000094.tif220170TIFF2025507990000095.tif221170TIFF2025507990000096.tif220170TIFF2025507990000097.tif220170TIFF2025507990000098.tif220170TIFF2025507990000099.tif220170TIFF2025507990000100.tif220170TIFF2025507990000101.tif220170TIFF2025507990000102.tif220170TIFF2025507990000103.tif221170TIFF2025507990000104.tif220170TIFF2025507990000105.tif220170TIFF2025507990000106.tif220170TIFF2025507990000107.tif220170TIFF2025507990000108.tif220170TIFF2025507990000109.tif220170TIFF2025507990000110.tif220170TIFF2025507990000111.tif220170TIFF2025507990000112.tif221170TIFF2025507990000113.tif220170TIFF2025507990000114.tif220170TIFF2025507990000115.tif220170TIFF2025507990000116.tif220170TIFF2025507990000117.tif220170TIFF2025507990000118.tif220170TIFF2025507990000119.tif221170TIFF2025507990000120.tif220170TIFF2025507990000121.tif220170TIFF2025507990000122.tif220170TIFF2025507990000123.tif220170TIFF2025507990000124.tif220170TIFF2025507990000125.tif221170TIFF2025507990000126.tif221170TIFF2025507990000127.tif220170TIFF2025507990000128.tif220170TIFF2025507990000129.tif220170TIFF2025507990000130.tif220170TIFF2025507990000131.tif221170TIFF2025507990000132.tif220170TIFF2025507990000133.tif221170TIFF2025507990000134.tif220170TIFF2025507990000135.tif220170TIF F2025507990000136.tif221170TIFF2025507990000137.tif220170TIFF2025507990000138.tif221170TIFF202550799000 0139.tif221170TIFF2025507990000140.tif220170TIFF2025507990000141.tif220170TIFF2025507990000142.tif2201 70TIFF2025507990000143.tif220170TIFF2025507990000144.tif217170TIFF2025507990000145.tif220170TIFF2025507 990000146.tif221170TIFF2025507990000147.tif220170TIFF2025507990000148.tif221170TIFF2025507990000149.ti f220170TIFF2025507990000150.tif220170TIFF2025507990000151.tif220170TIFF2025507990000152.tif220170TIFF20 25507990000153.tif220170TIFF2025507990000154.tif221170TIFF2025507990000155.tif220170TIFF20255079900001 56.tif220170TIFF2025507990000157.tif220170TIFF2025507990000158.tif220170TIFF2025507990000159.tif173170.

[0111]

[0116] The present disclosure provides a method for the preparation of a compound of formula: 5'-(AB)-CasN-CasC-K r -D q -3', wherein r is an integer selected from 0 to 5, and q is an integer selected from 0 to 5, and at least one of q and r is not 0.

[0112]

[0117] In some embodiments, the construct comprises the following amino acid sequence: KRAB is underlined ):

[0113]

[0118] (1)DNMT3A-DNMT3L-dCas9-KRAB(KOX1)

[0114] TIFF2025507990000160.tif215170TIFF2025507990000161.tif220170TIFF2025507990000162.tif29170In the formula, the nucleic acid sequence of KRAB (KOX1) is

[0115] The file is TIFF2025507990000163.tif51170.

[0116]

[0119] (2)DNMT3A-DNMT3L-dCas9-KRAB(ZIM3)

[0117] TIFF2025507990000164.tif215170TIFF2025507990000165.tif220170In the formula, the nucleic acid sequence of KRAB (ZIM3) is

[0118] The file is TIFF2025507990000166.tif50170.

[0119]

[0120] (3)DNMT3A-DNMT3L-dCas9-KRAB(ZNF680)

[0120] TIFF2025507990000167.tif215170TIFF2025507990000168.tif220170In the formula, the nucleic acid sequence of KRAB (ZNF680) is

[0121] The file is TIFF2025507990000169.tif50170.

[0122]

[0121] (4)DNMT3A-DNMT3L-dCas9-KRAB(ZNF554)

[0123] TIFF2025507990000170.tif215170TIFF2025507990000171.tif220170TIFF2025507990000172.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF554) is

[0124] The file is TIFF2025507990000173.tif50170.

[0125]

[0122] (5)DNMT3A-DNMT3L-dCas9-KRAB(ZNF264)

[0126] TIFF2025507990000174.tif215170TIFF2025507990000175.tif220170TIFF2025507990000176.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF264) is

[0127] The file is TIFF2025507990000177.tif50170.

[0128]

[0123] (6)DNMT3A-DNMT3L-dCas9-KRAB(ZNF582)

[0129] TIFF2025507990000178.tif215170TIFF2025507990000179.tif220170TIFF2025507990000180.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF582) is

[0130] The file is TIFF2025507990000181.tif48170.

[0131]

[0124] (7)DNMT3A-DNMT3L-dCas9-KRAB(ZNF324)

[0132] TIFF2025507990000182.tif215170TIFF2025507990000183.tif220170TIFF2025507990000184.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF324) is

[0133] The file is TIFF2025507990000185.tif49170.

[0134]

[0125] (8)DNMT3A-DNMT3L-dCas9-KRAB(ZNF669)

[0135] TIFF2025507990000186.tif215170TIFF2025507990000187.tif220170TIFF2025507990000188.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF669) is

[0136] The file is TIFF2025507990000189.tif48170.

[0137]

[0126] (9)DNMT3A-DNMT3L-dCas9-KRAB(ZNF354A)

[0138] TIFF2025507990000190.tif215170TIFF2025507990000191.tif220170TIFF2025507990000192.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF354A) is

[0139] The file is TIFF2025507990000193.tif50170.

[0140]

[0127] (10)DNMT3A-DNMT3L-dCas9-KRAB(ZNF82)

[0141] TIFF2025507990000194.tif215170TIFF2025507990000195.tif220170TIFF2025507990000196.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF82) is

[0142] The file is TIFF2025507990000197.tif50170.

[0143]

[0128] (11)DNMT3A-DNMT3L-dCas9-KRAB(ZNF595)

[0144] TIFF2025507990000198.tif215170TIFF2025507990000199.tif220170TIFF2025507990000200.tif29170The nucleic acid sequence of KRAB (ZNF595) is

[0145] The file is TIFF2025507990000201.tif49170.

[0146]

[0129] (12)DNMT3A-DNMT3L-dCas9-KRAB(ZNF419)

[0147] TIFF2025507990000202.tif215170TIFF2025507990000203.tif220170TIFF2025507990000204.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF419) is

[0148] The file is TIFF2025507990000205.tif50170.

[0149]

[0130] (13)DNMT3A-DNMT3L-dCas9-KRAB(ZNF566)

[0150] TIFF2025507990000206.tif215170TIFF2025507990000207.tif220170TIFF2025507990000208.tif29170In the formula, the nucleic acid sequence of KRAB (ZNF566) is

[0151] The file is TIFF2025507990000209.tif50170.

[0152]

[0131] (14)DNMT3A-DNMT3L-dCas9-KRAB(ZIM2)

[0153] TIFF2025507990000210.tif215170TIFF2025507990000211.tif220170TIFF2025507990000212.tif29170In the formula, the nucleic acid sequence of KRAB (ZIM2) is

[0154] The file is TIFF2025507990000213.tif49170.

[0155] The construct can be DNA or RNA. In some embodiments, the construct is mRNA. In some embodiments, the construct is double stranded DNA. In some embodiments, the construct is double stranded RNA. In some embodiments, the construct is single stranded DNA. In some embodiments, the construct is single stranded RNA.

[0156]

[0133] CRISPR-Cas system

[0134] The present disclosure provides a CRISPR / Cas9-based recombination system for use in genome editing and the treatment of genetic diseases. The CRISPR / Cas9-based recombination system can be designed to target any gene, including genes involved in angiogenesis, such as VEGFA. The present disclosure provides a CRISPR-Cas system that includes an engineered Cas protein and / or guide RNA with a desired specificity and activity (e.g., reducing or eliminating expression of the VEGFA gene product). The CRISPR / Cas9-based system can include a Cas9 protein, a mutant Cas9 protein, or a Cas9 fusion protein (e.g., a DNMT3A-DNMT3L (3A3L)-dCas9-KRAB fusion molecule), and at least one sgRNA (e.g., a VEGFA sgRNA). The Cas9 fusion protein can include, for example, a domain (e.g., DNMT3A, DNMT3L, or KRAB) that has an activity different from that endogenous to Cas9.

[0157]

[0135] The Cas9 protein can be divided into an N-terminal portion (CasN) and a C-terminal portion (CasC).

[0158]

[0136] Generally, a Cas protein (used interchangeably herein as CRISPR protein, CRISPR enzyme, CRISPR-Cas protein, CRISPR-Cas enzyme, Cas, CRISPR effector, or Cas effector protein) and / or a guide sequence are components of the CRISPR-Cas system. CRISPR-Cas system, or CRISPR system, collectively refers to the transcripts and other elements involved in expression or directing activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA, or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or "RNAs" as that term is used herein (e.g., RNAs that guide Cas, e.g., CRISPR RNA and trans-activating (tracr) RNA, or single guide RNAs (sgRNAs, also referred to as chimeric RNAs), or other sequences and transcripts derived from a CRISPR locus.

[0159]

[0137] In general, CRISPR systems are characterized by elements (also called protospacers in the context of endogenous CRISPR systems) that promote the formation of a CRISPR complex at the site of the target sequence. In the recombinant system of the present disclosure, the direct repeats can include naturally occurring or non-naturally occurring sequences. The direct repeats of the present disclosure are not limited to naturally occurring lengths and sequences. In addition, the direct repeats of the present disclosure can include insertions of nucleotides such as aptamers or sequences that bind to adaptor proteins (for associating with functional domains). In certain embodiments, one end of the direct repeat containing such an insertion is approximately the first half of the short DR, and the other end is approximately the second half of the short DR.

[0160] In the context of the formation of a CRISPR complex, a "target sequence" or a "target polynucleotide" refers to a sequence to which a guide sequence is designed to have complementarity, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. A target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0161] In general, the guide sequence (or spacer sequence) can be any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0162] In certain embodiments, the adjustment of cleavage efficiency can be utilized by introducing one or more mismatches, such as one or two mismatches, between the spacer sequence and the target sequence, including the position of the mismatch along the spacer / target. The more centrally located (i.e., not 3' or 5') the double mismatch is, for example, the greater the impact on cleavage efficiency. Thus, the position of the mismatch along the spacer can be selected to adjust the cleavage efficiency. For example, if less than 100% cleavage of the target is desired (e.g., in a cell population), one or more, such as preferably two, mismatches between the spacer and the target sequence can be introduced into the spacer sequence. The more centrally located the mismatch position is along the spacer, the lower the cleavage percentage.

[0163]

[0141] The CRISPR-Cas system or its components can be used to introduce one or more mutations into a target locus or nucleic acid sequence. The mutations can include the introduction, deletion, or substitution of one or more nucleotides in each target sequence of a cell by a guide, RNA, or sgRNA. The mutations can include the introduction, deletion, or substitution of 1-75 nucleotides in each target sequence of the cell by a guide RNA.

[0164]

[0142] In the context of endogenous CRISPR-Cas systems, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) typically results in cleavage at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence), which may depend, for example, on secondary structure, particularly in the case of RNA targets. In some cases, in the context of endogenous CRISPR systems, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) typically results in cleavage at or near the target nucleic acid sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence) of one or both strands (if applicable).

[0165]

[0143] In some embodiments, a guide RNA (capable of guiding Cas to a target locus) can include (1) a guide sequence capable of hybridizing to a target locus (a polynucleotide target locus, such as an RNA target locus) in a eukaryotic cell, and (2) direct repeat (DR) sequences present in a single RNA, i.e., sgRNA (arranged in a 5' to 3' direction) or crRNA.

[0166]

[0144] For general information regarding the production and use of CRISPR-Cas systems, including methods, materials, delivery vehicles, vectors, particles, AAV, components thereof, and delivery of such components, as well as amounts and combinations thereof, all useful in the practice of the present disclosure, see U.S. Patent Nos. 8,999,641; 8,993,233; 8,945,839; 8,932,814; 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359; U.S. Patent Publication No. US 2005 / 01231144; and US 2005 / 01231144. 2014-0310830, US 2014-0287938 A1, US 2014-0273234 A1, US 2014-0273232 A1, US 2014-0273231 A1, US 2014-0256046 A1, US 2014-0248702 A1, US 2014-0242700 A1, US 2014-0242699 A1, US 2014-0242664 A1, US 2014-0234972 A1, US 2014-0227787 A1 No. US 2014-0189896 A1, US 2014-0186958, US 2014-0186919 A1, US 2014-0186843 A1, US 2014-0179770 A1, and US 2014-0179006 A1, US 2014-0170753, European Patent Applications EP 2784162 B1 and EP 2771468 B1, European Patent Applications EP 2771468, EP 2764103 and EP 2784162, and PCT Patent Publication No. WO No. WO 2014 / 093694 (PCT / US2013 / 074790), No. WO 2014 / 093595 (PCT / US2013 / 074611), No. WONo. WO 2014 / 093718 (PCT / US2013 / 074825), No. WO 2014 / 093709 (PCT / US2013 / 074812), No. WO 2014 / 093622 (PCT / US2013 / 074667), No. WO 2014 / 093635 (PCT / US2013 / 074691), No. WO 2014 / 093655 (PCT / US2013 / 074736), No. WO 2014 / 093712 (PCT / US2013 / 074819), No. WO 2014 / 093701 (PCT / US2013 / 074800), No. WO No. WO 2014 / 018423 (PCT / US2013 / 051418), No. WO 2014 / 204723 (PCT / US2014 / 041790), No. WO 2014 / 204724 (PCT / US2014 / 041800), No. WO 2014 / 204725 (PCT / US2014 / 041803), No. WO 2014 / 204726 (PCT / US2014 / 041804), No. WO 2014 / 204727 (PCT / US2014 / 041806), No. WO 2014 / 204728 (PCT / US2014 / 041808), No. WO See PCT / US2014 / 204729 (PCT / US2014 / 041809), each of which is incorporated by reference in its entirety.

[0167]

[0145] Cas protein

[0168] A Cas protein (e.g., a recombinant Cas protein) can have a nuclease activity that is substantially the same (e.g., 80%-100%, 90%-100%, 95%-100%, 98%-100%, 99%-100%, 99.9%-100%, or about 100%) as a wild-type corresponding Cas protein. In certain cases, a recombinant Cas protein has a nuclease activity that is higher (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher) than a wild-type corresponding Cas protein.

[0169] Alternatively, or in addition, a Cas protein (e.g., a recombinant Cas protein) can have a specificity that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than a wild-type counterpart Cas protein. In certain embodiments, a Cas protein (e.g., a recombinant Cas protein) can have a specificity that is at least 30% higher than a wild-type counterpart Cas protein. As used herein, the term "specificity" of a Cas can correspond to the number or percentage of on-target polynucleotide cleavage events relative to the number or percentage of all polynucleotide cleavage events, including on-target and off-target events. The activity and specificity of the Cas proteins are consistent with that described in Hsu PD et al., DNA targeting specificity of RNA-guided Cas9 nucleases, Nat Biotechnol. 2013 Sep;31(9):827- 832; and Slaymaker IM, et al., Rationally engineered Cas9 nucleases with improved specificity, Science. 2016 Jan l;351(6268):84-88, which also describe exemplary methods for detecting the activity and specificity of the Cas proteins, which are incorporated by reference in their entirety and are described in detail elsewhere herein.

[0170] In some embodiments, the Cas protein (e.g., its RuvC domain) can slide one base upstream (relative to the PAM) to create a staggered cleavage that can be filled, resulting in a single base duplication (i.e., +1 insertion). Examples of +1 insertion positions are described in Zuo, Z., and Liu, J. (2016). Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations. Scientific Reports 6, 37584. In some embodiments, the recombinant Cas protein has a different +1 insertion frequency than the wild-type counterpart Cas protein. For example, the frequency of +1 insertion when a guanine is at the -2 position relative to the PAM is higher than +1 insertion when a thymidine, cytidine, or adenine is at the -2 position relative to the PAM. In some cases, the +1 insertion is dependent on the host machinery in human cells. In some examples, the Cas protein can create a staggered cleavage. The staggered cut can be a 1-bp or 1-nucleotide 5' overhang. The staggered cut can be a 1-bp or 1-nucleotide 3' overhang.

[0171]

[0149] The nucleic acid molecule encoding Cas can be codon-optimized. An example of a codon-optimized sequence is one that is optimized for expression in a eukaryote, such as a human (i.e., optimized for expression in a human), or another eukaryote, animal, or mammalian organism discussed herein, see, for example, the SaCas9 human codon-optimized sequence in WO 2014 / 093622 (PCT / US2013 / 074667). While this is preferred, other examples are possible, and it will be understood that codon optimization for host species other than humans, or for specific organs, is well known. In some embodiments, the enzyme coding sequence encoding Cas is codon-optimized for expression in a specific cell, such as a eukaryotic cell. The eukaryotic cells can be of or derived from a particular life form, such as, but not limited to, a human or a non-human eukaryote or animal or mammal discussed herein, such as a mammal, including a mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, processes that modify the genetic identity of the germline of a human and / or an animal that is likely to afflict them without any substantial medical effect on the human or animal, and also the genetic identity of the animal resulting from such a process, can be excluded. In general, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the natural sequence with a codon that is more frequently or most frequently used in the genes of that host cell, while maintaining the natural amino acid sequence. Different species show a particular bias for certain codons of certain amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the nature of the codons being translated and on the availability of specific transfer RNA (tRNA) molecules.The predominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be applied in a variety of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, for example Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in a sequence encoding a Cas correspond to the most frequently used codon for a particular amino acid.

[0172] In some embodiments, Cas protein may have nucleic acid cleavage activity. Cas protein may have RNA binding and DNA cleavage functions. In some embodiments, Cas may direct the cleavage of one or two nucleic acid strands at or near the target sequence, such as within a target sequence and / or within the complement of the target sequence or within a sequence associated with the target sequence, for example, within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the Cas protein may direct more than one cut (such as 1, 2, 3, 4, 5 or more cuts) of one or two strands within the target sequence and / or within the complement of the target sequence or within a sequence related to the target sequence and / or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the cuts can be blunt, i.e., generate blunt ends. In some embodiments, the cuts can be staggered, i.e., generate sticky ends.

[0173] In some embodiments, the vector encodes a nucleic acid-targeted Cas protein that is mutable relative to the corresponding wild-type enzyme, such that the mutated, nucleic acid-targeted Cas protein lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence, e.g., due to an alteration or mutation in the HNH domain, to produce a mutant Cas that lacks substantially all DNA cleavage activity, e.g., the DNA cleavage activity of the mutant enzyme is about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme, an example being where the nucleic acid cleavage activity of the mutant form is nonexistent or negligible compared to the non-mutated form. As used herein, the term "derived from" in reference to an enzyme means that the derived enzyme is based largely on the wild-type enzyme, in the sense of having a high degree of sequence homology to the wild-type enzyme, but has been mutated (modified) in some manner known in the art or described herein.

[0174] Typically, in the context of an endogenous nucleic acid targeting system, formation of a nucleic acid targeting complex (comprising a guide RNA or crRNA hybridized to a target sequence and complexed with one or more nucleic acid targeting effector proteins) results in DNA strand cleavage within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of the target sequence). As used herein, the term "sequence associated with a target locus of interest" refers to a sequence near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of the target sequence, where the target sequence is included in the target locus of interest).

[0175] It will be understood that the term "effector protein" includes, in some embodiments, "enzymes" since effector proteins are based on or derived from enzymes. However, it will also be understood that effector proteins may have activities including DNA or RNA binding (but not necessarily cleaving or nicking), death Cas protein function, as appropriate in some embodiments.

[0176] In some embodiments, the Cas protein can form a component of an inducible system. The inducible nature of the system allows for spatiotemporal control of gene editing or gene expression using forms of energy, including but not limited to electromagnetic radiation, sound energy, chemical energy, and thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), or light-inducible systems (Phytochrome, LOV domain, or Cryptochrome). In one embodiment, the CRISPR effector protein can be part of a Light Inducible Transcriptional Effector (LITE), which directs changes in transcriptional activity in a sequence-specific manner. The light component can include a CRISPR effector protein, a light-responsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcription activation / repression domain. Further examples of inducible DNA binding proteins and methods of their use are provided in US 61 / 736465 and US 61 / 721,283, as well as WO 2014018423 A2, which are incorporated by reference in their entireties.

[0177] In some embodiments, the mutant Cas can have one or more mutations resulting in reduced off-target effects, for example, an improved CRISPR enzyme used to affect modifications to the target locus, but with reduced or eliminated activity against the off-target, such as when complexed with a guide RNA, as well as an improved CRISPR enzyme to increase the activity of the CRISPR enzyme, such as when complexed with a guide RNA. It should be understood that the mutant enzymes described herein below can be used in any of the methods according to the disclosures described elsewhere herein. Any of the methods, products, compositions, and uses described elsewhere herein are equally compatible with the mutant CRISPR enzymes further detailed below.

[0178]

[0156] Methods and mutations that can be used in various combinations to increase or decrease on-target and off-target activity and / or specificity of activity, or to increase or decrease on-target and off-target binding and / or specificity of binding, can be used to compensate for or enhance mutations or modifications made to promote other effects. Such mutations or modifications made to promote other effects include mutations or modifications to Cas or mutations or modifications made to guide RNA. The methods and mutations disclosed herein are used to modulate Cas nuclease activity and / or binding to chemically modified guide RNA.

[0179] In certain embodiments, the catalytic activity of the Cas protein of the present disclosure is altered or modified. A mutated Cas should be understood to have an altered or modified catalytic activity if the catalytic activity is different from that of the corresponding wild-type Cas protein (e.g., unmutated Cas protein). Catalytic activity can be measured by methods well known in the art. By way of example and not limitation, catalytic activity can be measured in vitro or in vivo by measuring the indel percentage (e.g., after a given time or at a given dose). In certain embodiments, catalytic activity is increased. In certain embodiments, catalytic activity is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In certain embodiments, catalytic activity is decreased. In certain embodiments, catalytic activity is reduced by at least 5%, preferably at least 10%, more preferably at least 20%, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or (substantially) 100%. One or more mutations herein may inactivate catalytic activity, which may reduce substantially all catalytic activity, reduce activity to below detectable levels, or reduce to the point where there is no measurable catalytic activity at all.

[0180] One or more properties of the recombinant Cas protein may differ from the corresponding wild-type Cas protein. Examples of such properties include catalytic activity, gRNA binding, specificity of the Cas protein (e.g., editing specificity of a defined target), stability of the Cas protein, off-target binding, target binding, protease activity, nickase activity, PFS recognition. In some examples, the recombinant Cas protein may include one or more mutations of the corresponding wild-type Cas protein. In some embodiments, the catalytic activity of the recombinant Cas protein is increased compared to the corresponding wild-type Cas protein. In some embodiments, the catalytic activity of the recombinant Cas protein is decreased compared to the corresponding wild-type Cas protein. In some embodiments, the gRNA binding of the recombinant Cas protein is increased compared to the corresponding wild-type Cas protein. In some embodiments, the gRNA binding of the recombinant Cas protein is decreased compared to the corresponding wild-type Cas protein. In some embodiments, the specificity of the Cas protein is increased compared to the corresponding wild-type Cas protein. In some embodiments, the specificity of the Cas protein is decreased compared to the corresponding wild-type Cas protein. In some embodiments, the stability of the Cas protein is increased compared to the corresponding wild-type Cas protein. In some embodiments, the stability of the Cas protein is decreased compared to the corresponding wild-type Cas protein. In some embodiments, the recombinant Cas protein further comprises one or more mutations that inactivate catalytic activity. In some embodiments, the off-target binding of the Cas protein is increased compared to the corresponding wild-type Cas protein. In some embodiments, the off-target binding of the Cas protein is decreased compared to the corresponding wild-type Cas protein. In some embodiments, the target binding of the Cas protein is increased compared to the corresponding wild-type Cas protein. In some embodiments, the target binding of the Cas protein is decreased compared to the corresponding wild-type Cas protein.In some embodiments, the recombinant Cas protein has increased protease activity or polynucleotide binding ability compared to the corresponding wild-type Cas protein, hi some embodiments, PFS recognition is altered compared to the corresponding wild-type Cas protein.

[0181] Examples of Cas Proteins

[0182]

[0160] Examples of Cas proteins include class I (e.g., types I, III, and IV) and class 2 (e.g., types II, V, and VI) Cas proteins, such as Cas9, Cas12 (e.g., Cas12a, Cas12, Cas12c, Cas12d), Cas13 (e.g., Cas13a, Cas13b, Cas13c, Cas13d), CasX, CasY, Cas14, variants (e.g., mutant forms, truncated forms), homologs, and orthologs. The terms "ortholog" and "homolog" are well known in the art. With further guidance, as used herein, a "homolog" of a protein is a protein of the same species that performs the same or similar function as the protein that is the homolog. Homologous proteins may be, but are not necessarily, structurally related or are only partially structurally related. As used herein, an "ortholog" of a protein is a protein in a different species that performs the same or similar function as the protein it is an ortholog of. Orthologous proteins may, but are not necessarily, structurally related, or may be only partially structurally related.

[0183]

[0161] Class 2 Cas proteins

[0184] In some embodiments, the Cas protein is a class 2 Cas protein, i.e., a Cas protein of a class 2 CRISPR-Cas system. The class 2 CRISPR-Cas system can be of subtype, for example, type II-A, type II-B, type II-C, type VA, type VB, type VC, or type VU. In some embodiments, the Cas protein is Cas9, Cas12a, Cas12, Cas12c, or Cas12d. In some embodiments, the Cas9 can be SpCas9, SaCas9, StCas9, and other Cas9 orthologues. The Cas12 can include Cas12a, Cas12, and Cas12c, including FnCas12a, or homologs or orthologues thereof. Definitions and exemplary elements of CRISPR-Cas systems include those described in Kira S. Makarova and Eugene V. Koonin, Annotation and Classification of CRISPR-Cas systems, Methods Mol Biol. 2015;1311:47-75; and Sergey Shmakov et al., Diversity and evolution of class 2 CRISPR-Cas systems, Nat Rev Microbial. 2017 Mar;15(3):169-182.

[0185]

[0163] Cas protein linker

[0186]

[0164] In some embodiments, the Cas protein comprises at least one RuvC domain and at least one HNH domain. The Cas protein may further comprise a first and a second linker domain connecting the RuvC domain and the HNH domain. The first linker (L1) and the second linker (L2) connecting the HNH and RuvC domains in Cas9 are described in the study by Nishimasu, H. et al. "Crystal structure of Cas9 in complex with guide RNA and target RNA" Cell 156 (Feb. 27, 2014): 935-949 and Ribeiro, L. et al. (2018) "Protein engineering strategies to expand CRISPR-Cas9 applications" International Journal of Genomics Volume 2018, Article ID 1652567 (doi.org / 10.1155 / 2018 / 1652567). Figure 1 of Ribeiro shows the overall organization, structure and function of Cas9 and is specifically incorporated herein by reference. Specifically, Figure 1A shows a schematic diagram of the domain organization of SpCas9, showing the genetic structure of the HNH and RuvC domains, including linkers L1 (spanning amino acids 765-780) and L2 (spanning amino acids 906-918) as described herein.

[0187] Similarly, the domain organization of Staphylococcus aureus Cas9 (SaCas9) can be utilized when referring to the first and second linker domains. In one aspect, the linker 1 domain region spans residues 481-519 and connects the RuvC-II domain to the HNH domain of SaCas9. In some embodiments, the linker 2 region spans residues 629-649 and connects the RuvC-III and HNH domains of SaCas9. Thus, the first and / or second linker domains can be mutated in a Cas9 ortholog and can refer to amino acid residues that correspond to those of wild-type SaCas9. See Nishimasu, Cell. 2015 Aug 27; 162(5): 1113-1126; doi: 10.1016 / j.cell.2015.08.007, the entirety of which is incorporated herein by reference. In particular, Figure 1, S1-S3 of Nishimasu detail the domain organization of the Cas9 protein, the teachings of which are specifically incorporated by reference herein.

[0188] The first and second linkers can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more amino acids. The first and second linkers can correspond to wild-type linkers. In some embodiments, the first and second linkers comprise one or more mutations in the first and / or second linker. In one embodiment, the first and / or second linker comprise one or more mutations that improve the specificity of the Cas9 protein.

[0189] In some embodiments, the linkers L1 and L2 connecting the HNH and RuvC domains of Cas9 contain wild-type amino acid sequences. In some embodiments, the linkers connecting the HNH and RuvC domains contain one or more amino acid mutations. In an exemplary embodiment, the first linker (L1) contains a mutation corresponding to amino acid T769I of SpCas9, and / or the second linker (L2) contains a mutation corresponding to amino acid G915M of SpCas9. In an exemplary embodiment, one or more linker mutations, e.g., T769I and G915M, confer improved specificity to the Cas9 protein.

[0190] In one embodiment, one or more mutations in the first and second linkers can be combined with one or more mutations in other portions of the Cas9 protein as described herein to retain further improved specificity and / or activity that is substantially the same as the wild-type Cas9 protein. In one embodiment, mutations in the linkers and / or further mutations in the Cas protein can be identified using the methods detailed herein that enhance / improve specificity for wild-type Cas9 and retain substantially wild-type activity for wild-type Cas9.

[0191]

[0169] Class 2, type II Cas proteins (e.g., Cas9)

[0192] In some embodiments, the Cas protein can be a class 2, type II Cas protein of a CRISPR-Cas system (type II Cas protein). In some embodiments, the Cas protein can be a class 2, type II Cas protein, such as Cas9. In some embodiments, the CRISPR / Cas9-based system can include a Cas9 protein or fragment thereof, a Cas9 fusion protein, a nucleic acid encoding a Cas9 protein or fragment thereof, or a nucleic acid encoding a Cas9 fusion protein. "Cas9 (CRISPR associated protein 9)" refers to a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_269215 and having RNA-binding activity, DNA-binding activity, and / or DNA-cleavage activity (e.g., endonuclease or nickase activity). "Cas9 function" can be defined by any of a number of assays, including, but not limited to, fluorescence polarization-based nucleic acid binding assays, fluorescence polarization-based strand invasion assays, transcription assays, EGFP disruption assays, DNA cleavage assays, and / or Surveyor assays, for example, as described herein. "Cas9 nucleic acid molecule" refers to a polynucleotide encoding a Cas9 polypeptide or a fragment thereof. An exemplary Cas9 nucleic acid molecule sequence is provided in genomic SEQ ID NO: NC_002737. In some embodiments, inhibitors of Cas9, such as naturally occurring Cas9 in S. pyogenes (SpCas9) or S. aureus (SaCas9), or variants thereof, are disclosed herein. Cas9 recognizes foreign DNA using a protospacer adjacent motif (PAM) sequence and base pairing of the target DNA with a guide RNA (gRNA). Cas9 allows for efficient genome editing in multiple cell types and organisms by allowing the relative ease of inducing targeted strand breaks at any genomic locus. Cas9 derivatives can also be used as transcriptional activators / repressors.

[0193] In some cases, the CRISPR-Cas protein is Cas9 or a variant thereof. In some embodiments, the Cas9 can be a wild-type Cas9, including any naturally occurring bacterial Cas9. Cas9 orthologs typically share the common organization of 3-4 RuvC domains and an HNH domain. The 5'-most RuvC domain cleaves the non-complementary strand and the HNH domain cleaves the complementary strand. All notations refer to the guide sequence. The catalytic residues of the 5' RuvC domain were identified by homology comparison of the subject Cas9 with other Cas9 orthologues (from S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPR locus) and the conserved Asp residue (D10) was mutated to alanine to convert the Cas9 into a complementary strand nicking enzyme. Thus, the Cas enzyme can be a wild-type Cas9, including any naturally occurring bacterial Cas9. The CRISPR, Cas, or Cas9 enzyme can be codon-optimized or can be an engineered version, including any chimera, mutant, homolog, or orthologue. In further aspects of the disclosure, the Cas9 enzyme can include one or more mutations and can be used as a genetic DNA binding protein with or without fusion to a functional domain.

[0194] The mutations can be artificially introduced mutations or gain-of-function or loss-of-function mutations. In some embodiments, the transcriptional activation domain can be VP64. In some embodiments, the transcriptional repressor domain can be KRAB or SID4X. Other aspects of the present disclosure relate to mutant Cas9 enzymes fused to domains, including but not limited to nucleases, transcriptional activators, repressors, recombinases, transposases, histone remodelers, demethylases, DNA methyltransferases, cryptochromes, light-inducible / regulatory domains, or chemical-inducible / regulatory domains. The present disclosure can be accompanied by sgRNAs or tracrRNAs, or guide or chimeric guide sequences that can enhance the performance of these RNAs in cells. The type II CRISPR enzyme can be any Cas enzyme. In some cases, the Cas9 enzyme is from or derived from SpCas9 or SaCas9. As used herein, the term "derived from" in reference to an enzyme means that the derived enzyme is based largely on the wild-type enzyme in the sense of having a high degree of sequence homology with the wild-type enzyme, but has been mutated (modified) in some manner known in the art or described herein. In one example, the mutations can include one or more mutations in the first linker domain, the second linker domain, and / or other portions of the protein. A high degree of sequence homology can include at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more compared to the wild-type enzyme.

[0195]

[0173] The Cas enzyme may be the identified Cas9, since it can refer to a general class of enzymes that share homology to the largest nucleases with multiple nuclease domains of the Type II CRISPR system. In some cases, the Cas9 enzyme is from or derived from SpCas9 (S. pyogenes Cas9) or saCas9 (S. aureus Cas9). "StCas9" refers to wild-type Cas9 from S. thermophilus (UniProt ID: G3ECR1). Similarly, "SpCas9" refers to wild-type Cas9 from S. pyogenes (UniProt ID: Q99ZW2). As used herein, the term "derived from" in reference to an enzyme means that the derived enzyme is based very much on the wild-type enzyme, in the sense of having a high degree of sequence homology with the wild-type enzyme, but has been mutated (modified) in some manner known in the art or described herein. It will be understood that the terms Cas and CRISPR enzyme are used interchangeably herein unless otherwise clear. As noted above, much of the residue numbering used herein refers to the Cas9 enzyme from the Type II CRISPR locus in Streptococcus pyogenes.

[0196]

[0174] In specific embodiments, Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacte, Carnobacterium, Rhodobacter, Listeria, Paludiba cter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, B acillus, Brevibacilus, Methylobacterium or Acidaminococcus, Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma,or a Cas9 effector protein from or derived from an organism from a genus including Campylobacter.

[0197] In some embodiments, the Cas9 protein is S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia, C. jejuni, C. coli; N salsuginis, N tergarcus; S. auricularis, S. carnosus; N meningitides, N gonorrhoeae, L.monocytogenes, L.ivanovii; C.botulinum, C.difficile, C.tetani, or C.sordellii, Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2_33_10, Parcubacteria bacterium GW2011 GWC2_44_17, Smithella sp.SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae. In some embodiments, the Cas9 effector protein is from an organism selected from Streptococcus pyogenes, Staphylococcus aureus, or Streptococcus thermophilus Cas9 or derived from such an organism.

[0198]

[0176] In a more preferred embodiment, the Cas9 protein is derived from a bacterial species selected from Streptococcus pyogenes, Staphylococcus aureus, or Streptococcus thermophilus Cas9. In certain embodiments, the Cas9 is activated by Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC20171, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 JO, Parcubacteria bacterium GW2011 GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp.BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae. In certain embodiments, the Cas9 protein is derived from a bacterial species selected from Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020. In certain embodiments, the effector protein is derived from a subspecies of Francisella tularensis 1, including but not limited to Francisella tularensis subsp. Novicida.

[0199]

[0177] Cas9 enzymes include S.pyogenes serotype M1 (UniProt ID: Q99ZW2), S. aureus Cas9 (UniProt ID: J7RUA5), Eubacterium ventriosum Cas9 (UniProt ID: A5Z395), Azospirillum (strain B510) Cas9 (UniProt ID: D3NT09), Gluconacetobacter diazotrophicus (strain ATCC 49037) Cas9 (UnitProt ID: A9HKP2), Nisseria cinerea Cas9 (UniProt ID: D0W2Z9), Roseburia intestinalis Cas9 (UniProt ID: C7G697), Parvibaculum lavamentivorans (strain DS-1) Cas9 (UniProt ID: A7HP89), Nitratifractor salsuginis(DSM Co., Ltd.) 16511) Cas9 (UniProt ID: E6WZS9), Campylobacter lari Cas9 (UniProt ID: G1UFN3), but are not limited to these.

[0200]

[0178] Enzymatic action by Cas9 from Streptococcus pyogenes or any closely related Cas9 generates a double-stranded break at the target site sequence that hybridizes to 20 nucleotides of the guide sequence and has a protospacer adjacent motif (PAM) sequence (examples include NGG / NRG or PAM, which can be determined as described herein) after 20 nucleotides of the target sequence. CRISPR activity by Cas9 for site-specific DNA recognition and cleavage is defined in part by the guide sequence, which is a tracr sequence that hybridizes to the guide sequence and the PAM sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defense in bacteria and archaea, Mole Cell 2010, January 15;37(1):7. The type II CRISPR locus from Streptococcus pyogenes SF370 contains a cluster of four genes, Cas9, Cas1, Cas2, and Csnl, plus two non-coding RNA elements, tracrRNA, and a characteristic array of repeated sequences (direct repeats) spaced with short stretches of non-repetitive sequences (spacers, each about 30 bp). In this system, targeted DNA double-strand breaks (DSBs) are generated in four successive steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of the pre-crRNA, which are then processed into mature crRNAs containing distinct spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the DNA target, which consists of the protospacer and the corresponding PAM, by the formation of a heteroduplex between the crRNA and the spacer-region of the protospacer DNA. Finally, Cas9 mediates cleavage of the target DNA upstream of the PAM, creating a DSB within the protospacer. Pre-crRNA arrays, consisting of a single spacer flanked by two direct repeats (DRs), are also encompassed by the term "tracr-mate sequence."In certain embodiments, Cas9 can be constitutively present, inducibly present, conditionally present, or administered or delivered. Optimization of Cas9 can be used to enhance or develop new functions. Chimeric Cas9 proteins can be generated, and Cas9 can be used as a general DNA-binding protein. The structural information provided for Cas9 can be used to further modify and optimize the CRISPR-Cas system, which can also be extrapolated to confirm the structure-function relationship in other CRISPR enzyme systems, particularly in other type II CRISPR enzymes or Cas9 orthologs. The crystal structure information (U.S. Provisional Application Nos. 61 / 915,251, filed December 12, 2013, 61 / 930,214, filed January 22, 2014, and 61 / 980,012, filed April 15, 2014; and Nishimasu et al, “Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA,” Cell 156(5):935- 949, DOI: http: / / dx.doi.org / 10.1016 / j.cell.2014.02.001 (2014), each and every one of which is incorporated herein by reference) provides structural information for cleaving and creating modular, or multipart, CRISPR enzymes that can be incorporated into inducible CRISPR-Cas systems. In particular, structural information has been provided for S. pyogenes Cas9 (SpCas9), which can be extrapolated to other Cas9 orthologues or other type II CRISPR enzymes. The Cas9 gene is found in several diverse bacterial genomes, typically at the same locus as the casl, cas2, and cas4 genes, and CRISPR cassettes. Furthermore, the Cas9 protein contains a readily identifiable C-terminal region that is homologous to transposon ORF-B and contains an active RuvC-like nuclease, an arginine-rich region.

[0201]

[0179] dCas9

[0202]

[0180] The Cas9 protein can be mutated to inactivate nuclease activity. An inactivated Cas9 protein from S. pyogenes (iCas9, also called "dCas9"), which lacks endonuclease activity, has recently been targeted to genes in bacteria, yeast, and human cells by gRNA to silence gene expression by steric hindrance. As used herein, "dCas molecule" may refer to a dCas protein, or a fragment thereof. As used herein, "dCas9 molecule" may refer to a dCas9 protein, or a fragment thereof. As used herein, the terms "iCas" and "dCas" are used interchangeably to refer to a catalytically inactive, CRISPR-associated protein. In one embodiment, the dCas molecule comprises one or more mutations in the DNA cleavage domain. In one embodiment, the dCas molecule comprises one or more mutations in the RuvC or HNH domain. In one embodiment, the dCas molecule comprises one or more mutations in both the RuvC and HNH domains. In one embodiment, the dCas molecule is a fragment of a wild-type Cas molecule. In one embodiment, the dCas molecule comprises a functional domain from a wild-type Cas molecule, the functional domain being selected from a Reel domain, a bridge helix domain, or a PAM interaction domain. In one embodiment, the nuclease activity of the dCas molecule is reduced by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the nuclease activity of the corresponding wild-type Cas molecule. An exemplary amino acid sequence of a wild-type dCas9 protein is set forth in SEQ ID NO:1. An exemplary nucleic acid sequence encoding wild-type dCas9 is set forth in SEQ ID NO:26.

[0203]

[0181] A suitable dCas molecule can be derived from a wild-type Cas molecule. The Cas molecule can be from a type I, type II, or type III CRISPR-Cas system. In one embodiment, a suitable dCas molecule can be derived from a Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, or Cas10 molecule. In one embodiment, the dCas molecule is derived from a Cas9 molecule. The dCas9 molecule can be obtained, for example, by introducing a point mutation (e.g., a substitution, deletion, or addition) into the Cas9 molecule in a DNA cleavage domain, for example, in a nuclease domain, for example, in the RuvC and / or HNH domain. See, for example, Jinek et al., Science (2012) 337:816-21, the entire contents of which are incorporated herein by reference. For example, two point mutations in the RuvC and HNH domains are introduced to reduce Cas9 nuclease activity while maintaining Cas9 sgRNA and DNA binding activity. In one embodiment, the two point mutations in the RuvC and HNH active sites are D10A and H840A mutations in the S.pyogenes Cas9 molecule. Alternatively, D10 and H840 in the S.pyogenes Cas9 molecule can be deleted to eliminate Cas9 nuclease activity while maintaining its sgRNA and DNA binding activity. In one embodiment, the two point mutations in the RuvC and HNH active sites are D10A and N580A mutations in the S.pyogenes Cas9 molecule.

[0204] In one embodiment, the dCas molecule is a S. aureus dCas9 molecule comprising a mutation at D10 and / or N580, numbered according to SEQ ID NO: 1. In one embodiment, the dCas molecule is a S. aureus dCas9 molecule comprising a D10A and / or N580A mutation, numbered according to SEQ ID NO: 1.

[0205]

[0183] In one embodiment, the dCas9 molecule is a S. aureus dCas9 molecule comprising the amino acid sequence of SEQ ID NO:1, a sequence substantially identical to SEQ ID NO:1 (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity), or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:1, or a fragment of any of these.

[0206]

[0184] Similar mutations can also be applied to any other naturally occurring Cas9 (e.g., Cas9 from other species) or recombinant Cas9 molecules. In certain embodiments, the dCas9 molecule is a Streptococcus pyogenes dCas9 molecule, a Staphylococcus aureus dCas9 molecule, a Campylobacterjejuni dCas9 molecule, a Corynebacterium diphtheria dCas9 molecule, a Eubacterium ventriosum dCas9 molecule, a Streptococcus pasteurianus dCas9 molecule, a Lactobacillusfarciminis dCas9 molecule, a Sphaerochaeta globus dCas9 molecule, an Azospirillum (strain B 510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria cinerea dCas9 molecule, a Roseburia intestinalis dCas9 molecule, a Parvibaculum lavamentivorans dCas9 molecule, a Nitratifractor salsuginis (strain DSM 1651 1) A dCas9 molecule, a Campylobacter lari (strain CF89-12) dCas9 molecule, a Streptococcus thermophilus (strain LMD-9) dCas9 molecule, or a fragment thereof.

[0207] In certain embodiments, the present disclosure provides a dCas9 molecule for a Streptococcus pyogenes dCas9 molecule, a Staphylococcus aureus dCas9 molecule, a Campylobacterjejuni dCas9 molecule, a Corynebacterium diphtheria dCas9 molecule, a Eubacterium ventriosum dCas9 molecule, a Streptococcus pasteurianus dCas9 molecule, a Lactobacillus farciminis dCas9 molecule, a Sphaerochaeta globus dCas9 molecule, an Azospirillum (strain B510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria cinerea dCas9 molecule, a Roseburia intestinalis dCas9 molecule, a Parvibaculum lavamentivorans dCas9 molecule, a Nitratifractor salsuginis (strain DSM 1651 1) Provided is a vector comprising a nucleotide encoding a dCas9 molecule, a Campylobacter lari (strain CF89-12) dCas9 molecule, a Streptococcus thermophilus (strain LMD-9) dCas9 molecule, or a fragment thereof.

[0208]

[0186] In some embodiments, the dCas9 protein comprises an array set forth in any one of SEQ ID NOs: 106-122.

[0209]

[0187] The dCas9 molecules of the constructs provided herein can be continuous or divided into an N-terminal portion (dCas9N) and a C-terminal portion (dCas9C). In some embodiments, the N-terminal and C-terminal portions are separated by DNMT3A and / or DNMT3L. Thus, provided herein are constructs that include, in order from N-terminus to C-terminus, dCas9N, DNMT3A, DNMT3L, and dCas9C.

[0210]

[0188] Those skilled in the art will understand that any dCas9 protein can be split at various points in the protein sequence, so long as the fusion protein can be refolded into a functional dCas9 molecule. Table 2 shows exemplary sequences of dCas9-N and dCas9-C sequences. It is preferred to pair the N- and C-terminal sequences according to their numbers, e.g., dCas9N-1 is used with dCas9C-1, dCas9N-2 is used with dCas9C-2, and so on. In some embodiments, the constructs provided herein include a dCas9N comprising the sequence set forth in SEQ ID NO:2, and a dCas9C comprising the sequence set forth in SEQ ID NO:3. In some embodiments, the constructs provided herein include a dCas9N comprising the sequence set forth in SEQ ID NO:4, and a dCas9C comprising the sequence set forth in SEQ ID NO:5. In some embodiments, the constructs provided herein include a dCas9N comprising the sequence set forth in SEQ ID NO:6, and a dCas9C comprising the sequence set forth in SEQ ID NO:7. In some embodiments, constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO:8, and a dCas9C comprising the sequence set forth in SEQ ID NO:9. In some embodiments, constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO:10, and a dCas9C comprising the sequence set forth in SEQ ID NO:11. In some embodiments, constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO:12, and a dCas9C comprising the sequence set forth in SEQ ID NO:13. In some embodiments, constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO:14, and a dCas9C comprising the sequence set forth in SEQ ID NO:15. In some embodiments, constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO:16, and a dCas9C comprising the sequence set forth in SEQ ID NO:17. In some embodiments, constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO:18, and a dCas9C comprising the sequence set forth in SEQ ID NO:19. In some embodiments, the constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO:20, and a dCas9C comprising the sequence set forth in SEQ ID NO:21.In some embodiments, the constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO: 22, and a dCas9C comprising the sequence set forth in SEQ ID NO: 23. In some embodiments, the constructs provided herein comprise a dCas9N comprising the sequence set forth in SEQ ID NO: 24, and a dCas9C comprising the sequence set forth in SEQ ID NO: 25.

[0211] In some embodiments, dCas9N is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. In some embodiments, dCas9C is encoded by a polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

[0212] Table 2: Exemplary dCas9 and split dCas9 sequences. TIFF2025507990000214.tif209170TIFF2025507990000215.tif218170TIFF20255079900 00216.tif217170TIFF2025507990000217.tif218170TIFF2025507990000218.tif217170 TIFF2025507990000219.tif217170TIFF2025507990000220.tif220170TIFF20255079900 00221.tif217170TIFF2025507990000222.tif217170TIFF2025507990000223.tif216170

[0213] Cas9 fusion protein

[0214]

[0191] The CRISPR / Cas9-based system can include a fusion molecule (e.g., DNMT3A-DNMT3L (3A3L)-dCas9-KRAB). In some embodiments, the fusion molecule includes dCas9, KRAB, DNMT3A, and DNMT3L.

[0215] In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to DNMT3A or a fragment thereof. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to DNMT3L or a fragment thereof. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to DNMT3L and DNMT3L, or a fragment thereof. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to a DNMT3A-DNMT3L fusion peptide.

[0216]

[0193] DNMT3A

[0217] TIFF2025507990000224.tif66170

[0218]

[0194] DNMT3L

[0219] TIFF2025507990000225.tif50170

[0220]

[0195] DNMT3A-DNMT3L fusion peptide

[0221] TIFF2025507990000226.tif120170

[0222] In some embodiments, DNMT3A is human DNMTA. In some embodiments, DNMT3A is non-human DNMT3A, for example, rodent DNMT3A. In some embodiments, DNMT3A is selected from Mus musculus DNMT3A, Mus caroli DNMT3A, Mus Pahari DNMT3A, Rattus norvegicus DNMT3A, Rattus Rattus DNMT3A, Arvicanthis niloticus DNMT3A, Grammomys surdaster DNMT3A, Mastomys coucha DNTM3A.

[0223] In some embodiments, the DNMT3L is human DNMT3L (SEQ ID NO: 74). In some embodiments, the DNMT3L is non-human DNMT3L, for example, rodent DNMT3L. In some embodiments, the DNMT3L is selected from Mus musculus DNMT3L (SEQ ID NO: 75), Mus caroli DNMT3L (SEQ ID NO: 76), Mus Pahari DNMT3L (SEQ ID NO: 77), Rattus norvegicus DNMT3L (SEQ ID NO: 78), Rattus Rattus DNMT3L (SEQ ID NO: 79), Arvicanthis niloticus DNMT3L (SEQ ID NO: 80), Grammomys surdaster DNMT3L (SEQ ID NO: 81), and Mastomys coucha DNMT3L (SEQ ID NO: 82).

[0224]

[0198] In some embodiments, DNMT3L is encoded by a polynucleotide comprising a nucleic acid sequence set forth in one of SEQ ID NOs: 83-92.

[0225]

[0200] In one embodiment, the Cas9 fusion protein also includes a nuclear localization sequence (NLS), e.g., n NLSs fused to the N-terminus and / or C-terminus of Cas9.

[0226] Nuclear localization sequences are well known in the art. In one embodiment, the NLS comprises the amino acid sequence of SEQ ID NO: 72, 73, 125, or 126, a sequence substantially identical to SEQ ID NO: 72, 73, 125, or 126 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% or more sequence identity), or a sequence having one, two, three, four, five or more changes, such as amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 72 or 73, or any fragment thereof. SEQ ID NO:72 (Exemplary nuclear localization sequence): APKKKRKVGIHGVPAA SEQ ID NO:73 (Exemplary nuclear localization sequence): KRPAATKKAGQAKKKK SEQ ID NO: 125 (BPNLS): KRTADGSEFESPKKKRKV SEQ ID NO:126 (SV40 NLS): PKKKRKV

[0227] The construct may further comprise a modulator of gene expression. Various modulators of gene expression are well known in the art, see, for example, Thakore et al., Nat Methods. 2016; 13: 127-37, the entirety of which is incorporated herein by reference. Non-limiting examples of modulators of gene expression include Kruppel-associated repression box (KRAB), enhancer of zeste homolog 2 (EZH2), G9A, lysine-specific histone demethylase 1A (LSD1), heterochromatin protein 1 (HP1), friend of GATA protein 1 (FOG1), histone deacetylase (HDAC3), and DOT1L, each of which is described in more detail below.

[0228] In some embodiments, the modulator of an epigenetic modification can have DNA methylase activity. For example, the modulator of an epigenetic modification can have methylase activity that involves transferring a methyl group to DNA, RNA, a protein, a small molecule, cytosine, or adenine.

[0229] In some embodiments, the CRISPR / Cas9-based system can include a dCas9 molecule and a modulator of gene expression, or a nucleic acid encoding the dCas9 molecule and the modulator of gene expression. In one embodiment, the dCas9 molecule and the modulator of gene expression are covalently linked. In one embodiment, the modulator of gene expression is covalently fused directly to the dCas9 molecule. In one embodiment, the modulator of gene expression is covalently fused indirectly to the dCas9 molecule, for example, via a non-modulator or linker, or via a second modulator. In one embodiment, the modulator of gene expression is present at the N-terminus and / or C-terminus of the dCas9 molecule. In one embodiment, the dCas9 molecule and the modulator of gene expression are non-covalently linked. Exemplary sequences include, but are not limited to, those listed in Table 3. In some embodiments, the linker between the dCas9 and at least one modulator of gene expression comprises an amino acid sequence corresponding to a linker listed in Table 3.

[0230] Table 3: Exemplary linker sequences TIFF2025507990000227.tif149170

[0231] In one embodiment, the dCas9 molecule is fused to a first tag, e.g., a first peptide tag. In one embodiment, the modulator of gene expression is fused to a second tag, e.g., a second peptide tag. In one embodiment, the first and second tags, e.g., the first peptide tag and the second peptide tag, interact with each other non-covalently, thereby bringing the dCas9 molecule and the modulator of gene expression into close proximity.

[0232] In one embodiment, the CRISPR / Cas9 based system comprises a fusion molecule or a nucleic acid encoding a fusion molecule, in one embodiment, the fusion molecule comprises a sequence comprising dCas9 fused to a modulator of gene expression. In one embodiment, the dCas9 molecule is selected from the group consisting of a Streptococcus pyogenes dCas9 molecule, a Staphylococcus aureus dCas9 molecule, a Campylobacterjejuni dCas9 molecule, a Corynebacterium diphtheria dCas9 molecule, a Eubacterium ventriosum dCas9 molecule, a Streptococcus pasteurianus dCas9 molecule, a Lactobacillus farciminis dCas9 molecule, a Sphaerochaeta globus dCas9 molecule, an Azospirillum (strain B510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria cinerea dCas9 molecule, a Roseburia intestinalis dCas9 molecule, a Parvibaculum lavamentivorans dCas9 molecule, a Nitratifractor salsuginis (strain DSM 16511) dCas9 molecule, Campylobacter lari (strain CF89-12) dCas9 molecule, Streptococcus thermophilus (strain LMD-9) dCas9 molecule, or a fragment thereof.

[0233] Modulators of Gene Expression

[0234] In some embodiments, the constructs provided herein include one or more modulators of gene expression. The modulator of gene expression can be a repressor of gene expression or an activator of gene expression. The repressor can be any known repressor of gene expression, such as a repressor selected from Kruppel-associated box (KRAB) domain, mSin3 interaction domain (SID), MAX-interacting protein 1 (MXI1), chromoshadow domain, EAR repression domain (SRDX), eukaryotic translation termination factor 1 (ERFl), eukaryotic translation termination factor 3 (ERF3), tetracycline repressor, lad repressor, Catharanthus roseus G-box binding factor 1 and 2, Drosophila Groucho, Tripartite motif-containing 28 (TRTM28), nuclear receptor corepressor 1, nuclear receptor corepressor 2, or fragments or fusions thereof. The activator can be any known activator of gene expression, such as a VP16 activation domain, a VP64 activation domain, a p65 activation domain, an Epstein-Barr virus R transactivator Rta molecule, or a fragment thereof. Activators that can be used with dCas9 molecules are well known in the art. See, for example, Chavez et al., Nat Methods. (2016) 13:563-67, the entire contents of which are incorporated herein by reference.

[0235] In certain embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to a modulator of gene expression. In some embodiments, the modulator of gene expression comprises a modulator of epigenetic modification. In one embodiment, the fusion molecule regulates target gene expression by epigenetic modification, for example, by histone acetylation or methylation, or DNA methylation, at a control element of the target gene, for example, a promoter, enhancer, or transcription start site. The modulator can be any known modulator of epigenetic modification, for example, histone acetyltransferase (e.g., p300 catalytic domain), histone deacetylase, histone methyltransferase (e.g., SUV39H1 or G9a (EHMT2)), histone demethylase (e.g., LSD1), DNA methyltransferase (e.g., DNMT3a or DNMT3a-DNMT3L), DNA demethylase (e.g., TET1 catalytic domain or TDG), or a fragment thereof.

[0236]

[0209] Kruppel related box (KRAB)

[0237]

[0210] The KRAB domain is a type of transcriptional repression domain present in the N-terminal portion of many zinc finger protein-based transcription factors. When tethered to target DNA by the DNA binding domain, the KRAB domain functions as a transcriptional repressor. The KRAB domain is rich in charged amino acids and can be divided into subdomains A and B. The KRAB A and B subdomains can be separated by a variable spacer segment, and many KRAB proteins contain only the A subdomain. The 45 amino acid sequence of the KRAB A subdomain has been shown to be important for transcriptional repression. The B subdomain does not repress transcription by itself, but enhances the repression exerted by the KRAB A subdomain. The KRAB domain recruits the corepressors KAP1 (KRAB-associated protein 1), also known as transcription mediator 1β, KRAB-A interacting protein, and triad motif protein 28), and heterochromatin protein 1 (HP1), as well as other chromatin regulatory proteins, resulting in transcriptional repression through heterochromatin formation. In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to a KRAB domain or fragment thereof. In one embodiment, the KRAB domain or fragment thereof is fused to the N-terminus of the dCas9 molecule. In one embodiment, the KRAB domain or fragment thereof is fused to the C-terminus of the dCas9 molecule. In one embodiment, the KRAB domain or fragment thereof is fused to both the N-terminus and the C-terminus of the dCas9 molecule. In one embodiment, the fusion molecule comprises a KRAB domain comprising a sequence of SEQ ID NO: 51, 53, or 230-241, a sequence substantially identical to SEQ ID NO: 51, 53, or 230-241 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 51, 53, or 230-241, or a fragment of any of these.

[0238] Exemplary KRAB Domain Sequences RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP (sequence number 51).

[0239]

[0212] In one embodiment, the fusion molecule is a DNMT3A-DNMT3L (3A3L)-dCas9-KRAB fusion molecule comprising a DNMT3A-DNMT3L fusion peptide (3A3L), a dCas9 peptide, and a KRAB peptide domain fused directly or indirectly (e.g., via a linker) from the N-terminus to the C-terminus.

[0240]

[0213] In one embodiment, the fusion molecule is a DNMT3A-DNMT3L (3A3L)-dCas9-KRAB fusion molecule comprising a DNMT3A-DNMT3L fusion peptide (3A3L), a dCas9 peptide, and a KRAB peptide domain fused directly or indirectly (e.g., via a linker) from the N-terminus to the C-terminus.

[0241]

[0214] In one embodiment, the fusion molecule comprises the amino acid sequence of SEQ ID NO: 96, a sequence substantially identical to SEQ ID NO: 97 (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity), or a sequence having one, two, three, four, five or more changes, e.g., substitutions, insertions, or deletions, relative to SEQ ID NO: 96, or any fragment thereof.

[0242]

[0215] DNMT3A-DNMT3L(3A3L)-dCas9-KRAB

[0243] TIFF2025507990000228.tif215170TIFF2025507990000229.tif220170TIFF2025507990000230.tif5170

[0244] In some embodiments, the KRAB domain is a zinc finger imprint 3 (ZIM3) KRAB domain. The ZIM3 KRAB domain is a potent repressor of gene expression. In some embodiments, the ZIM3 KRAB domain comprises a sequence of SEQ ID NO:53, a sequence substantially identical to SEQ ID NO:53 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% or more sequence identity), or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:53, or any fragment thereof. In some embodiments, the ZIM3 KRAB domain is encoded by a sequence set forth in SEQ ID NO:54.

[0245]

[0217] Enhancer of Zeste Homolog 2 (EZH2)

[0246] In one embodiment, the construct provided herein comprises an enhancer of Zeste homolog 2 (EZH2) domain. EZH2 is a histone methyltransferase that controls several aspects of cell cycle progression. It removes methyl groups from mono- and dimethylated lysine 4 and / or lysine 9 in histone H3 (H3K4me1 / 2 and H3K9me1 / 2).

[0247] In some embodiments, the EZH2 domain comprises the sequence of SEQ ID NO:55, a sequence substantially identical to SEQ ID NO:55 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence that has one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:55, or any fragment thereof. In some embodiments, the EZH2 domain is encoded by the sequence set forth in SEQ ID NO:56.

[0248]

[0220] G9a (Euchromatin histone lysine methyltransferase 2, EHMT2)

[0249] In some embodiments, the constructs provided herein comprise a G9A or EhMT2 domain. EHMT2 is a methyltransferase that methylates lysine residues on histone H3. In some embodiments, the EHMT2 domain is fused to the C-terminus of a dCas9 protein. In some embodiments, the EHMT2 domain is fused to the C-terminus of a KRAB domain.

[0250] In some embodiments, the EHMT2 domain comprises the sequence of SEQ ID NO:57, a sequence substantially identical to SEQ ID NO:57 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence that has one, two, three, four, five or more alterations, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:57, or any fragment thereof. In some embodiments, the EZH2 domain is encoded by the sequence set forth in SEQ ID NO:58.

[0251] Lysine-specific histone demethylase 1A (LSD1)

[0252] In some embodiments, the constructs provided herein comprise an LSD1 domain. LSD1 is a histone demethylase that removes methyl groups from mono- and di-methylated lysine 4 and / or lysine 9 in histone H3 (H3K4me1 / 2 and H3K9me1 / 2). In some embodiments, the LSD1 domain is fused to the C-terminus of a dCas9 protein. In some embodiments, the LSD1 domain is fused to the C-terminus of a KRAB domain.

[0253] In some embodiments, the LSD1 domain comprises the sequence of SEQ ID NO:59, a sequence substantially identical to SEQ ID NO:59 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence having one, two, three, four, five or more alterations, such as amino acid substitutions, insertions or deletions, relative to SEQ ID NO:59, or any fragment thereof. In some embodiments, the LSD1 domain is encoded by the sequence set forth in SEQ ID NO:60.

[0254] Heterochromatin Protein 1 (HP1)

[0255] In some embodiments, the constructs provided herein comprise an HP1 domain. HP1 contributes to the formation of heterochromatin structures. In some embodiments, the HP1 domain is fused to the C-terminus of a dCas9 protein. In some embodiments, the HP1 domain is fused to the C-terminus of a KRAB domain.

[0256] In some embodiments, the HP1 domain comprises the sequence of SEQ ID NO:61, a sequence substantially identical to SEQ ID NO:61 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence that has one, two, three, four, five or more alterations, such as amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:61, or any fragment thereof. In some embodiments, the LSD1 domain is encoded by the sequence set forth in SEQ ID NO:62.

[0257] Friend of GATA Protein 1 (FOG1)

[0258] In some embodiments, the constructs provided herein comprise a Friend of GATA protein 1 (FOG1). FOG1 is a cofactor of the GATA1 transcription factor and controls cell differentiation. In some embodiments, the FOG1 domain is fused to the C-terminus of the dCas9 protein. In some embodiments, the FOG1 domain is fused to the C-terminus of the KRAB domain.

[0259] In some embodiments, the FOG1 domain comprises the sequence of SEQ ID NO:63, a sequence substantially identical to SEQ ID NO:63 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:63, or any fragment thereof. In some embodiments, the FOG1 domain is encoded by the sequence set forth in SEQ ID NO:64.

[0260] Histone deacetylase (HDAC3)

[0261] In some embodiments, the constructs provided herein comprise HDAC3. HDAC3 is responsible for deacetylation of lysine residues at the N-terminal portion of core histones (H2A, H2B, H3, and H4). In some embodiments, the HDAC3 domain is fused to the C-terminus of the dCas9 protein. In some embodiments, the HDAC3 domain is fused to the C-terminus of the KRAB domain.

[0262] In some embodiments, the HDAC3 domain comprises the sequence of SEQ ID NO:65, a sequence substantially identical to SEQ ID NO:65 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence having one, two, three, four, five or more alterations, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:65, or any fragment thereof. In some embodiments, the HDAC3 domain is encoded by the sequence set forth in SEQ ID NO:66.

[0263]

[0235] DOT1-like histone lysine methyltransferase (DOT1L)

[0264] In some embodiments, the constructs provided herein comprise DOT1L. DOT1L is a histone methyltransferase that methylates lysine-79 of histone H3. In some embodiments, the DOT1L domain is fused to the C-terminus of a dCas9 protein. In some embodiments, the DOT1L domain is fused to the C-terminus of a KRAB domain.

[0265] In some embodiments, the DOT1L domain comprises the sequence of SEQ ID NO:67, a sequence substantially identical to SEQ ID NO:67 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% or more sequence identity), or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO:67, or any fragment thereof. In some embodiments, the DOT1L domain is encoded by the sequence set forth in SEQ ID NO:68.

[0266] Histone-modifying activity

[0267] In some embodiments, a modulator of an epigenetic modification can have histone modifying activity, which can include, but is not limited to, histone deacetylase, histone acetyltransferase, histone demethylase, or histone methyltransferase activity.

[0268] In some embodiments, the modulator of epigenetic modification can have histone acetyltransferase activity. The histone acetyltransferase can be a p300 or CREB binding protein (CBP) protein, or a fragment thereof. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to an acetyltransferase p300 or a fragment thereof, such as the catalytic core of p300. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to a CREB binding protein (CBP) or a fragment thereof.

[0269] In some embodiments, the modulator of epigenetic modification can have histone demethylase activity. For example, the modulator of epigenetic modification can include an enzyme that removes methyl (CH3-) groups from nucleic acids or proteins (e.g., histones). In some embodiments, the methods and compositions disclosed herein include a fusion molecule that includes a dCas9 molecule fused to Lys-specific histone demethylase 1 (LSD1) or a fragment thereof.

[0270] In some embodiments, the modulator of epigenetic modification can have histone methyltransferase activity. In some embodiments, the methods and compositions disclosed herein comprise a fusion molecule comprising a dCas9 molecule fused to SUV39H1 or a fragment thereof. In some embodiments, the methods and compositions disclosed herein comprise a fusion molecule comprising a dCas9 molecule fused to G9a (EHMT2) or a fragment thereof.

[0271] DNA demethylase activity

[0272] In some embodiments, the modulator of epigenetic modification can have DNA demethylase activity. For example, the modulator of epigenetic modification can convert a methyl group to hydroxymethylcytosine as a mechanism for demethylating DNA. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to 10-11 translocation methylcytosine dioxygenase 1 (TET1) or a fragment thereof. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to thymine DNA glycosylase (TDG) or a fragment thereof.

[0273]

[0245] gRNA

[0274]

[0246] As used herein, the term "guide sequence" in the context of the CRISPR-Cas system includes any polynucleotide sequence that hybridizes with a target nucleic acid sequence and has sufficient complementarity with the target nucleic acid sequence to direct sequence-specific binding of a nucleic acid target complex to the target nucleic acid sequence. The guide sequence can form a duplex with the target sequence. The duplex can be a DNA duplex, an RNA duplex, or an RNA / DNA duplex. The terms "guide molecule" and "guide RNA" and "single guide RNA" are used interchangeably herein and refer to an RNA-based molecule that can form a complex with a CRISPR-Cas protein, including a guide sequence that hybridizes with a target nucleic acid sequence and has sufficient complementarity with the target nucleic acid sequence to direct sequence-specific binding of the complex to the target nucleic acid sequence. Guide molecules or guide RNAs specifically encompass RNA-based molecules that have one or more chemical modifications (e.g., by chemically linking two ribonucleotides or by replacing one or more ribonucleotides with one or more deoxyribonucleotides), as described herein.

[0275] A guide molecule or guide RNA for a CRISPR-Cas protein can include a tracr-mate sequence (including a "direct repeat" in the context of an endogenous CRISPR system) and a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system). In some embodiments, the CRISPR-Cas systems or complexes described herein do not include and / or do not depend on the presence of a tracer sequence. In certain embodiments, a guide molecule can include, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or a spacer sequence.

[0276] In general, CRISPR-Cas system is characterized by the element that promotes the formation of CRISPR complex at the site of target sequence.In the context of the formation of CRISPR complex, " target sequence " refers to the sequence that guide sequence is designed to have complementarity with, and the hybridization of target DNA sequence and guide sequence promotes the formation of CRISPR complex.

[0277] In certain embodiments, the length of the spacer of the guide sequence or guide molecule is 15-50 nucleotides. In certain embodiments, the length of the spacer of the guide RNA is at least 15 nucleotides. In certain embodiments, the length of the spacer is 15-17 nucleotides, 17-20 nucleotides, 20-24 nucleotides, 23-25 ​​nucleotides, 24-27 nucleotides, 27-30 nucleotides, 30-35 nucleotides, or longer than 35 nucleotides.

[0278] In some embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length.

[0279] In some embodiments, the sequence of the guide molecule (direct repeats and / or spacers) is selected to reduce the degree of secondary structure within the guide molecule. In some embodiments, less than about or about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less of the nucleotides of the nucleic acid target guide RNA participate in self-complementary base pairing when optimally folded. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold, described in Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, e.g., A. R. Gruber et al., 2008, Cell 106(1):23-24; and P. A. Carr and G. M. Church, 2009, Nature Biotechnology 27(12):1151-62).

[0280] As mentioned above, the CRISPR / Cas9 system utilizes a gRNA that provides targeting for the CRISPR / Cas9-based system. The gRNA is a fusion of two non-coding RNAs: crRNA and tracrRNA. The sgRNA can target any desired DNA sequence by replacing the sequence encoding the 20 bp protospacer, which confers target specificity through complementary base pairing, with the desired DNA target. The gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in type II effector systems. This duplex, which may include, for example, a 42 nucleotide crRNA and a 75 nucleotide tracrRNA, serves as a guide for Cas9 to cleave the target nucleic acid.

[0281]

[0253] As used interchangeably herein, the terms "target region," "target sequence," or "protospacer" refer to the region of a target gene targeted by a CRISPR / Cas9-based system. A CRISPR / Cas9-based system can include at least one gRNA, where the gRNAs target different DNA sequences. The target DNA sequences may overlap. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different type II systems have different PAM requirements. For example, the S. pyogenes type II system uses a "NGG" sequence, where "N" can be any nucleotide.

[0282] In some embodiments, the number of gRNAs administered to a cell can be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs, at least 7 different gRNAs, at least 8 different gRNAs, at least 9 different gRNAs, at least 10 different gRNAs, at least 11 different gRNAs, at least 12 different gRNAs, at least 13 different gRNAs, at least 14 different gRNAs, at least 15 different gRNAs, at least 16 different gRNAs, at least 17 different gRNAs, at least 18 different gRNAs, at least 19 different gRNAs, at least 20 different gRNAs, at least 25 different gRNAs, at least 30 different gRNAs, at least 35 different gRNAs, at least 40 different gRNAs, at least 45 different gRNAs, or at least 50 different gRNAs.

[0283] In some embodiments, the number of gRNAs administered to a cell is at least 1 gRNA to at least 50 different gRNAs, at least 1 gRNA to at least 45 different gRNAs, at least 1 gRNA to at least 40 different gRNAs, at least 1 gRNA to at least 35 different gRNAs, at least 1 gRNA to at least 30 different gRNAs, at least 1 gRNA to at least 25 different gRNAs, at least 1 gRNA to at least 20 different gRNAs, at least 1 gRNA to at least 16 different gRNAs, at least 1 gRNA to at least 12 different gRNAs, at least 1 gRNA to at least 8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs, at least 4 gRNA to at least 50 different gRNAs, at least 4 different gRNA to at least 45 different gRNAs, at least 4 different gRNA to at least 40 different gRNAs, at least 4 different gRNA to at least 35 different gRNAs, or at least At most, the number of different gRNAs can be 4 to at least 30 different gRNAs, at least 4 to at least 25 different gRNAs, at least 4 to at least 20 different gRNAs, at least 4 to at least 16 different gRNAs, at least 4 to at least 12 different gRNAs, at least 4 to at least 8 different gRNAs, at least 8 to at least 50 different gRNAs, at least 8 to at least 45 different gRNAs, at least 8 to at least 40 different gRNAs, at least 8 to at least 35 different gRNAs, 8 to at least 30 different gRNAs, at least 8 to at least 25 different gRNAs, 8 to at least 20 different gRNAs, at least 8 to at least 16 different gRNAs, or 8 to at least 12 different gRNAs.

[0284] In some embodiments, the gRNA is selected to increase or decrease the transcription of the target gene. In some embodiments, the gRNA targets a region upstream of the transcription start site (TSS) of the target gene (e.g., VEGFA), for example, 0-1000 bp upstream of the transcription start site of the target gene. In some embodiments, the gRNA targets a region 0-50 bp, 0-100 bp, 0-150 bp, 0-200 bp, 0-250 bp, 0-300 bp, 0-350 bp, 0-400 bp, 0-450 bp, 0-500 bp, 0-550 bp, 0-600 bp, 0-650 bp, 0-700 bp, 0-750 bp, 0-800 bp, 0-850 bp, 0-900 bp, 0-950 bp, or 0-1000 bp upstream of the transcription start site of the target gene. In some embodiments, the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp upstream of the transcription start site of the target gene. In one embodiment, the gRNA targets a region 0-300 bp upstream of the TSS of the target gene.

[0285]

[0257] In some embodiments, the gRNA targets a region downstream of the transcription start site of the target gene, for example, 0 to 1000bp downstream of the transcription start site of the target gene. In some embodiments, the gRNA targets a region downstream of the transcription start site of the target gene, 0 to 50bp, 0 to 100bp, 0 to 150bp, 0 to 200bp, 0 to 250bp, 0 to 300bp, 0 to 350bp, 0 to 400bp, 0 to 450bp, 0 to 500bp, 0 to 550bp, 0 to 600bp, 0 to 650bp, 0 to 700bp, 0 to 750bp, 0 to 800bp, 0 to 850bp, 0 to 900bp, 0 to 950bp, or 0 to 1000bp. In some embodiments, the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp downstream of the transcription start site of the target gene. In one embodiment, the gRNA targets a region 0-300 bp downstream of the TSS of the target gene.

[0286]

[0258] The present disclosure provides sgRNA sequences that target the human VEGFA target gene, as well as sgRNA sequences that target the mouse VEGFA target gene. The sequence of human VEGFA is shown in NCBI Reference Sequence: NG_008732.1. The sequence of mouse VEGFA is shown in NCBI Reference Sequence: NC_000083.7. The sequence of rhesus VEGFA is shown in NCBI Reference Sequence: NC_041757.1. The present disclosure also provides sgRNA sequences that target CD151, CD81, and PCSK9 target genes. Exemplary sgRNAs include, but are not limited to, those listed in Table 4.

[0287] Table 4. Exemplary sgRNAs TIFF2025507990000231.tif123170

[0288] In one embodiment, the gRNA targets the promoter region of the target gene. In one embodiment, the gRNA targets the enhancer region of the target gene. The gRNA can be divided into a target binding region, a Cas9 binding region, and a transcription termination region. The target binding region hybridizes with a target region in the target gene. Methods for designing such target binding regions are well known in the art, see, for example, Doench et al., Nat Biotechnol. (2014) 32: 1262-7; and Doench et al., Nat Biotechnol. (2016) 34: 184-91, which are incorporated herein by reference in their entirety. Design tools are available, for example, from the Feng Zhang Laboratory's target Finder, the Michael Boutros Laboratory's Target Finder (E-CRISP), RGEN Tools (Cas-OF Finder), CasFinder, and CRISPR Optimal Target Finder. In certain embodiments, the target binding region can be about 15 to about 50 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length). In certain embodiments, the target binding region can be about 19 to about 21 nucleotides in length. In one embodiment, the target binding region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0289] In one embodiment, the target binding region is complementary, for example, completely complementary, to the target region of the target gene. In one embodiment, the target binding region is substantially complementary to the target region of the target gene. In one embodiment, the target binding region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less nucleotides that are not complementary to the target region of the target gene.

[0290] In one embodiment, the target binding region is engineered to improve stability or extend half-life, for example, by incorporating unnatural or modified nucleotides in the target binding region, by removing or modifying RNA destabilizing sequence elements, by adding RNA stabilizing sequence elements, or by increasing the stability of the Cas9 / gRNA complex. In one embodiment, the target binding region is engineered to enhance its transcription. In one embodiment, the target binding region is engineered to reduce the formation of secondary structures. In one embodiment, the Cas9 binding region of the gRNA is engineered to enhance transcription of the gRNA. In one embodiment, the Cas9 binding region of the gRNA is engineered to improve stability or assembly of the Cas9 / gRNA complex.

[0291]

[0263] Delivery system

[0292]

[0264] The present disclosure also provides delivery systems for introducing components of the systems and compositions herein into a cell, tissue, organ, or organism. The delivery systems can include one or more delivery vehicles and / or cargoes.

[0293]

[0265] Cargo

[0294]

[0266] The delivery system can include one or more cargos. The cargo can include one or more components of the systems and compositions herein. The cargo can include one or more of the following: i) a plasmid encoding one or more Cas proteins; ii) a plasmid encoding one or more guide RNAs; iii) mRNA of one or more Cas proteins; iv) one or more guide RNAs; v) one or more Cas proteins; vi) any combination thereof. In some examples, the cargo can include a plasmid encoding one or more Cas proteins and one or more (e.g., multiple) guide RNAs. In some embodiments, the cargo can include mRNA encoding one or more Cas proteins and one or more guide RNAs.

[0295] In some embodiments, the cargo can include one or more Cas proteins and one or more guide RNAs, for example in the form of a ribonucleoprotein complex (RNP). The ribonucleoprotein complex can be delivered by the methods and systems herein. In some cases, the ribonucleoprotein can be delivered by a polypeptide-based shuttle agent. In one embodiment, the ribonucleoprotein can be delivered using a synthetic peptide that includes a cell-penetrating domain (CPD), a histidine-rich domain, and an endosomal leakage domain (ELD) operably linked to the CPD, for example as described in WO2016161516.

[0296] Physical Delivery

[0297]

[0269] In some embodiments, the cargo can be introduced into the cell by a physical delivery method. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery.

[0298] Microinjection

[0299] High efficiency, e.g., greater than 90% or about 100%, can be achieved by microinjecting cargo directly into cells. In some embodiments, microinjection can be performed using a microscope and a needle (e.g., 0.5-5.0 μm in diameter) to puncture the cell membrane and deliver cargo directly to a target site within the cell. Microinjection can be used for in vitro and ex vivo delivery.

[0300] Plasmids containing coding sequences for Cas proteins and / or guide RNAs, mRNA, and / or guide RNAs can be microinjected. In some cases, microinjection can be used to i) deliver DNA directly to the cell nucleus and / or ii) deliver (e.g., in vitro transcribed) mRNA to the cell nucleus or cytoplasm. In certain instances, microinjection can be used to deliver sgRNA directly to the nucleus and Cas-encoding mRNA to the cytoplasm, for example to facilitate translation and shuttling of Cas to the nucleus.

[0301] Microinjection can be used to generate genetically modified animals. For example, gene editing cargo can be injected into fertilized eggs to allow efficient germline modification. Such an approach can produce normal embryos and full-term pregnant mouse pups with the desired modifications. Microinjection can also be used to cause the transient up- or down-regulation of specific genes in the genome of cells, for example, using CRISPRa and CRISPRi.

[0302] Electroporation

[0303] In some embodiments, the cargo and / or delivery vehicle can be delivered by electroporation. Electroporation uses high-voltage pulsed current to temporarily open nanometer-sized pores in the cell membrane of cells suspended in a buffer solution, allowing components with hydrodynamic diameters of tens of nanometers to flow into the cells. In some cases, electroporation can be used in various cell types to efficiently transfer cargo into cells. Electroporation can be used for in vitro and ex vivo delivery.

[0304] Electroporation can be used to deliver cargo to the nucleus of mammalian cells by applying certain voltages and reagents, for example, by nucleofection. Such approaches include those described in Wu Y, et al. (2015). Cell Res 25:67-79; Ye L, et al. (2014). Proc Natl Acad Sci USA 111:9591-6; Choi PS, Meyerson M. (2014). Nat Commun 5:3728; Wang J, Quake SR. (2014). Proc Natl Acad Sci 111:13157-62. Electroporation can also be used to deliver cargo in vivo, for example, with the method described in Zuckermann M, et al. (2015). Nat Commun 6:7391.

[0305]

[0277] Hydrodynamic delivery

[0306] Hydrodynamic delivery can also be used for delivery of cargo, e.g., for in vivo delivery. In some examples, hydrodynamic delivery can be performed by rapidly pushing a large volume (8-10% body weight) of solution containing the gene editing cargo into the bloodstream of a subject (e.g., animal or human), e.g., a mouse, through the tail vein. Because blood is incompressible, a large bolus of liquid can increase hydrodynamic pressure and temporarily increase permeability to endothelial and parenchymal cells, allowing cargo that normally cannot pass through cell membranes to pass through to the cells. This approach can be used to deliver naked DNA plasmids and proteins. The delivered cargo can be abundant in the liver, kidney, lung, muscle, and / or heart.

[0307] Transfection

[0308] Cargo, for example, nucleic acid, can be introduced into cells by the transfection method of introducing nucleic acid into cells.Examples of transfection methods include calcium phosphate mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, impalefection, optical transfection, and uptake of nucleic acid enhanced by special agents.

[0309] Delivery Vehicle

[0310]

[0282] The delivery system can include one or more delivery vehicles. The delivery vehicle can deliver the cargo to a cell, tissue, organ, or organism (e.g., an animal or plant). The cargo can be packaged, carried, or otherwise associated with the delivery vehicle. The delivery vehicle can be selected based on the type of cargo to be delivered and / or the delivery is in vitro and / or in vivo. Examples of delivery vehicles include vectors, viruses, non-viral vehicles, and other delivery reagents described herein.

[0311] A delivery vehicle according to the present disclosure may have a maximum dimension (e.g., diameter) of less than 100 micrometers (μm). In some embodiments, a delivery vehicle has a maximum dimension of less than 10 μm. In some embodiments, a delivery vehicle may have a maximum dimension of less than 2000 nanometers (nm). In some embodiments, a delivery vehicle may have a maximum dimension of less than 1000 nanometers (nm). In some embodiments, a delivery vehicle may have a maximum dimension (e.g., diameter) of less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150 nm, or less than 100 nm, less than 50 nm. In some embodiments, a delivery vehicle may have a maximum dimension in the range of 25 nm to 200 nm.

[0312] In some embodiments, the delivery vehicle can be or can include a particle. For example, the delivery vehicle can be or can include a nanoparticle (e.g., a particle having a maximum dimension (e.g., diameter) of 1000 nm or less). The particles can be provided in different forms, for example, as solid particles (e.g., metals such as silver, gold, iron, titanium, non-metals, lipid-based solids, polymers), suspensions of particles, or combinations thereof. Metallic, dielectric, and semiconductor particles as well as hybrid structures (e.g., core-shell particles) can be prepared.

[0313]

[0285] Vector

[0314]

[0286] The system, composition, and / or delivery system may include one or more vectors. The present disclosure may also include a vector system. A vector system may include one or more vectors. In some embodiments, a vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded, nucleic acid molecules with one or more free ends, or without free ends (e.g., circular), nucleic acid molecules that include DNA, RNA, or both, and various other polynucleotides known in the art. Vectors may be plasmids, e.g., circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors may be capable of autonomously replicating in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Some vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and are thereby replicated along with the host genome. In certain examples, vectors may be expression vectors, e.g., capable of directing the expression of genes to which they are operably linked. In some cases, the expression vector is for expression in a eukaryotic cell. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0315]

[0287] Examples of vectors include pGEX, pMAL, pRIT5, E. coli expression vectors (e.g., pTrc, pET l ld), yeast expression vectors (e.g., pYepSecl, pMFa, pJRY88, pYES2, and picZ), baculovirus vectors (e.g., for expression in insect cells such as SF9 cells) (e.g., the pAc series and pVL series), and mammalian expression vectors (e.g., pCDM8 and pMT2PC).

[0316] A vector may comprise i) a Cas coding sequence, and / or ii) a single or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 32, at least 48, at least 50 guide RNA coding sequences. There may be a promoter for each RNA coding sequence in a single vector. Alternatively, or in addition, there may be promoters controlling (e.g., driving transcription and / or expression of) multiple RNA coding sequences in a single vector.

[0317] Control elements

[0318] The vector can include one or more control elements. The control elements can be operably linked to a coding sequence of a Cas protein, an accessory protein, a guide RNA (e.g., a single guide RNA, a crRNA, and / or a tracrRNA), or a combination thereof. The term "operably linked" is intended to mean that a nucleotide sequence of interest is linked to a control element to allow expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). In certain examples, the vector can include a first control element operably linked to a nucleotide sequence encoding a Cas protein and a second control element operably linked to a nucleotide sequence encoding a guide RNA.

[0319]

[0291] Examples of control elements 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). Such control elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990). Control elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of a nucleotide sequence (e.g., tissue-specific control sequences) only in certain host cells. Tissue-specific promoters can direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a specific cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, e.g., cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.

[0320]

[0292] Exemplary promoters include one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof. Exemplary pol III promoters include, but are not limited to, U6 and HI promoters. Exemplary pol II promoters include, but are not limited to, retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), SV40 promoter, dihydrofolate reductase promoter, actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1a promoter.

[0321] Viral Vectors

[0322] Cargo can be delivered by virus. In some embodiments, viral vectors are used. Viral vectors can include virus-derived DNA or RNA sequences for packaging into viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by the virus for transfection into host cells. Viruses and viral vectors can be used for in vitro, ex vivo, and / or in vivo delivery.

[0323]

[0295] Adeno-associated virus (AAV)

[0324] The systems and compositions herein can be delivered by adeno-associated virus (AAV). AAV vectors can be used for such delivery. AAV, which belongs to the Dependovirus genus and Parvoviridae family, is a single-stranded DNA virus. In some embodiments, AAV can provide a continuous source of donated DNA because the genomic material delivered by AAV can be integrated directly into the host DNA, for example, either as exogenous DNA or with minor modifications, and thus can remain in the cell indefinitely. In some embodiments, AAV does not cause or is not associated with any disease in humans. The virus itself can efficiently infect cells with little or no innate or adaptive immune response or associated toxicity.

[0325] Examples of AAV that can be used herein include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, and AAV-9. The type of AAV can be selected for the cells to be targeted. For example, AAV serotypes 1, 2, 5, or hybrid capsids AAV1, AAV2, AAV5, or any combination thereof can be selected to target brain or neuronal cells, and AAV4 can be selected to target cardiac tissue. AAV8 is useful for delivery to the liver. AAV-2-based vectors were originally proposed for CFTR delivery to CF airways, and other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9 show improved gene transfer efficiency in various models of lung epithelium. Examples of cell types targeted by AAV are described in Grimm, D. et al, J. Virol. 82:5887-5911 (2008) and WO 2021 / 183807A1, the entireties of which are incorporated by reference herein.

[0326] CRISPR-Cas AAV particles can be produced in HEK 293 T cells. Once particles with a particular tropism are produced, they can be used to infect target cell lines in much the same way that natural virus particles do. This can allow for persistent presence of CRISPR-Cas components in the infected cell type, making this version of delivery particularly suitable for cases where long-term expression is desired. Examples of AAV doses and formulations that can be used include those described in U.S. Patent Nos. 8,454,972 and 8,404,658.

[0327]

[0299] Various methods can be used for delivery of the systems and compositions herein by AAV. In some examples, the coding sequences for Cas and gRNA can be directly packaged into one DNA plasmid vector and delivered via one AAV particle. In some examples, AAV can be used to deliver gRNA to cells that have previously been engineered to express Cas. In some examples, the coding sequences for Cas and gRNA can be made into two separate AAV particles, which are used to co-transfect target cells. In some examples, markers, tags, and other sequences can be packaged into the same AAV particle as the coding sequences for Cas and / or gRNA.

[0328]

[0300] Lentivirus

[0329] The system and composition of the present specification can be delivered by lentivirus.Lentivirus vectors can be used for such delivery.Lentivirus is a complex retrovirus that has the ability to infect and express genes in both mitotic and postmitotic cells.

[0330] Examples of lentiviruses include the human immunodeficiency virus (HIV), which can use the envelope glycoproteins of other viruses to target a wide range of cell types, and the equine infectious anemia virus (EIAV)-based minimal non-primate lentiviral vectors that can be used to treat the eye. In certain embodiments, self-inactivating lentiviral vectors (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) carrying siRNAs targeting common exons shared by HIV tat / rev, nucleolus-localizing TAR decoys, and anti-CCR5 specific hammerhead ribozymes can be used and / or adapted to the nucleic acid targeting system herein.

[0331] Lentivirus can be pseudotyped with other viral proteins, such as the G protein of vesicular stomatitis virus. In this way, the cell chemotaxis of lentivirus can be broadly or narrowly changed as desired. In some cases, to improve safety, second and third generation lentivirus systems divide essential genes into three plasmids, which can reduce the possibility of accidental reconstitution of live viral particles in cells.

[0332]

[0304] In some embodiments, the integration activity can be exploited to use lentiviruses to generate libraries of cells containing different genetic modifications, for example, to screen and / or study genes and signaling pathways.

[0333] Adenovirus

[0334] The system and composition herein can be delivered by adenovirus. Adenovirus vectors can be used for such delivery. Adenoviruses include non-enveloped viruses that contain icosahedral nucleocapsids containing double-stranded DNA genomes. Adenoviruses can infect dividing and non-dividing cells. In some embodiments, adenoviruses do not integrate into the genome of host cells, which can be used to limit the off-target effects of CRISPR-Cas systems in gene editing applications.

[0335] Non-viral vehicles

[0336]

[0308] The delivery vehicle can include a non-viral vehicle. In general, any method and vehicle capable of delivering nucleic acid and / or protein can be used to deliver the system composition herein. Examples of non-viral vehicles include lipid nanoparticles, cell membrane penetrating peptides (CPPs), DNA nanoclues, gold nanoparticles, streptolysin 0, multifunctional enveloped nanodevices (MENDs), lipid-coated mesoporous silica particles, and other inorganic nanoparticles.

[0337]

[0309] Lipid particles

[0338]

[0310] The delivery vehicle can include lipid particles, such as lipid nanoparticles (LNPs) and liposomes.

[0339] Lipid Nanoparticles (LNPs)

[0340]

[0312] LNPs can encapsulate nucleic acids in cationic lipid particles (e.g., liposomes) and can be delivered to cells relatively easily. In some embodiments, the lipid nanoparticles do not contain any viral components, which helps to minimize safety and immunogenicity concerns. The lipid particles can be used for in vitro, ex vivo, and in vivo delivery. The lipid particles can be used for cell populations at various scales.

[0341] In some examples, LNPs can be used to deliver DNA molecules (e.g., those containing coding sequences for Cas and / or gRNA) and / or RNA molecules (e.g., Cas mRNA, gRNA). In certain cases, LNPs can be used to deliver RNP complexes of Cas / gRNA.

[0314] In some embodiments, LNPs are used to deliver mRNA and gRNA (e.g., an mRNA fusion molecule comprising DNMT3A-DNMT3L (3A-3L)-dCas9-KRAB and at least one sgRNA targeting VEGFA).

[0342]

[0315] Components of LNPs can include cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2-DMA), 3-o-[2(methoxypolyethylene glycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), R-3-[(ro-methoxy-poly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DOMG), and any combination thereof. Preparation and encapsulation of LNPs can be adapted from Conway et al., Molecular Therapy, vol. 27, no. 4, pages 866-877, Apr. 2019, and Rosin et al., Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011.

[0343] In some embodiments, the LNP can include ionizable lipids. In some embodiments, the ionizable lipids include, but are not limited to, pH-responsive ionizable lipids, thermoresponsive ionizable lipids, and light-responsive ionizable lipids. In some embodiments, the ionizable lipids include, but are not limited to, cationic lipids and anionic lipids that are ionized under certain conditions, such as, but not limited to, pH, temperature, or light. In some embodiments, the molar ratio of ionized lipid in the LNP is from 20% to about 70% (e.g., from about 20% to about 70%, from about 20% to about 65%, from about 20% to about 60%, from about 20% to about 55%, from about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, from about 20% to about 25%, from about 30% to about 70%, from about 30% to about 65%, from about 30% to about 60%, from about 30% to about 7 ...70%, from about 30% to about 65%, from about 30% to about 60%, from about 30% to about 70%, from about 30% to about % to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 60% to about 70%, or about 60% to about 65%).

[0344]

[0317] In some embodiments, the LNP can include a PEGylated lipid. In some embodiments, the molar ratio of the PEGylated lipid in the LNP is 0% to about 30% (e.g., about 0% to about 30%, about 0% to about 25%, about 0% to about 20%, about 0% to about 15%, about 0% to about 10%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 20% to about 30%, or about 20% to about 25%).

[0345]

[0318] In some embodiments, the LNP can include a supporting lipid. In some embodiments, the molar ratio of the supporting lipid in the LNP is 30% to about 50% (e.g., about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 40% to about 50%, or about 40% to about 45%).

[0346]

[0319] In some embodiments, the LNP can contain cholesterol. In some embodiments, the molar ratio of cholesterol in the LNP is 10% to about 50% (e.g., about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 40% to about 50%, or about 40% to about 45%).

[0347]

[0320] In some embodiments, the LNP can include a mixture of ionizable lipids (20%-70%, molar ratio), PEGylated lipids (0%-30%, molar ratio), supporting lipids (30%-50%, molar ratio), and cholesterol (10%-50%, molar ratio).

[0348]

[0321] Liposome

[0349] In some embodiments, the lipid particle can be a liposome. Liposomes are spherical vesicular structures composed of a unilamellar or multilamellar lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible and non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect these cargoes from degradation from plasma enzymes, and transport these loads across biological membranes and the blood-brain barrier (BBB).

[0350] Liposomes can be made from a number of different types of lipids, such as phospholipids. Liposomes can include natural phospholipids and lipids, such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialoganglioside, or any combination thereof.

[0351]

[0324] Several other additives can be added to liposomes to modify their structure and properties. For example, liposomes can further contain cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for example, to increase stability and / or prevent leakage of the liposome inner cargo.

[0352]

[0325] Stable nucleic acid lipid particles (SNALP)

[0353] In some embodiments, the lipid particle can be a stable nucleic acid lipid particle (SNALP). The SNALP can include ionizable lipids (DLinDMA) (e.g., cationic at low pH), neutral helper lipids, cholesterol, diffusible polyethylene glycol (PEG)-lipids, or any combination thereof. In some examples, the SNALP can include synthetic cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxypolyethylene glycol)2000)carbamoyl]-l,2-dimylestyroyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane. In some examples, the SNALP can include synthetic cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

[0354]

[0327] Other lipids

[0355]

[0328] The lipid particles can also contain one or more other types of lipids, such as cationic lipids, such as the amino lipids 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), DLin-KC2-DMA4, Cl2-200, and the co-lipids disteroylphosphatidylcholine, cholesterol, and PEG-DMG.

[0356] Lipoplexes and / or polyplexes

[0357] In some embodiments, the delivery vehicle comprises a lipoplex and / or a polyplex. Lipoplexes can bind to negatively charged cell membranes and induce endocytosis into cells. Examples of lipoplexes can be complexes that include lipid and non-lipid components. Examples of lipoplexes and polyplexes include FuGENE-6 reagent, non-liposomal solutions that include lipids and other components, zwitterionic amino lipids (ZAL), (e.g., DNA / Ca 2 + forming microcomplexes), Ca2p, polyetheneimine (PEI) (e.g., branched PEI), and poly(L-lysine) (PLL).

[0358]

[0331] Cell membrane penetrating peptide

[0359] In some embodiments, the delivery vehicle comprises a cell membrane penetrating peptide (CPP). CPPs are short peptides that facilitate cellular uptake of a variety of molecular cargoes (e.g., ranging from nano-sized particles to small chemical molecules and large fragments of DNA).

[0360] CPPs can be of various sizes, amino acid sequences, and charges. In some embodiments, CPPs can translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or organelles. CPPs can be introduced into cells by different mechanisms, such as direct penetration through the membrane, endocytosis-mediated entry, and translocation by the formation of temporary structures.

[0361] CPPs can have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine, or has a sequence that contains a varying pattern of polar / charged and non-polar hydrophobic amino acids. These two types of structures are called polycationic or amphipathic, respectively. A third class of CPPs are hydrophobic peptides that contain only non-polar residues with low net charge, or have hydrophobic amino acid groups that are important for cellular uptake. Another type of CPP is the transactivating transcription activator (Tat) from the human immunodeficiency virus I (HIV-I). Examples of CPPs include penetratin, Tat(48-60), Transportan, and (R-AhX-R4) (Ahx means aminohexanoyl). Examples of CPPs and related uses are also described in U.S. Pat. No. 8,372,951.

[0362] CPPs are very easy to use for in vitro and ex vivo work, and expensive optimization for each cargo and cell type is usually required. In some embodiments, CPPs can be directly covalently linked to Cas proteins, which are then complexed with gRNA and delivered to cells. In some embodiments, separate delivery of CPP-Cas and CPP-gRNA to multiple cells can be performed. CPPs can also be used to deliver RNPs.

[0363]

[0336] DNA Nano Crew

[0364] In some embodiments, the delivery vehicle comprises a DNA nanocleu. A DNA nanocleu refers to a sphere-like structure of DNA (e.g., having a shape like a spherical yam). The nanocleu can be synthesized by rolling circle amplification using a palindromic sequence that aids in the self-assembly of the structure. The sphere can then be loaded with a payload. Examples of DNA nanocleu are described in Sun W et al, J Am Chem Soc. 2014 Oct 22; 136(42): 14722-5; and Sun Wet al, Angew Chem Int Ed Engl. 2015 Oct 5; 54(41): 12029-33. The DNA nanocleu can have a palindromic sequence that is partially complementary to the gRNA in the Cas:gRNA ribonucleoprotein complex. The DNA nanocleu can be coated, for example, with PEI, to induce endosomal escape.

[0365]

[0338] Gold nanoparticles

[0366] In some embodiments, the delivery vehicle comprises gold nanoparticles (AuNPs, also called colloidal gold). The gold nanoparticles can be complexed with the cargo, e.g., Cas:gRNA RNP. The gold nanoparticles can be coated, e.g., coated with silicate and endosome-disrupting polymer PAsp(DET). An example of a gold nanoparticle is AuraSense Therapeutics' spherical nucleic acid (SNA TM ) constructs, and those described in Mout R, et al. (2017). ACS Nano 11:2452-8; Lee K, et al. (2017). Nat Biomed Eng 1:889-901.

[0367]

[0340] iTOP

[0368] In some embodiments, the delivery vehicle comprises iTOP. iTOP refers to a combination of small molecules that drive highly efficient intracellular delivery of native proteins, independent of any transduction peptide. iTOP can be used for inducible transduction with NaCl-mediated hyperosmolality and propane betaine together with the transduction compound (propane betaine) to trigger macropinocytosis uptake of extracellular macromolecules into cells. Examples of iTOP methods and reagents include those described in D'Astolfo DS, Pagliero RJ, Pras A, et al. (2015). Cell 161:674-690.

[0369] Polymer-Based Particles

[0370] In some embodiments, the delivery vehicle can include a polymer-based particle (e.g., a nanoparticle). In some embodiments, the polymer-based particle can mimic the viral mechanism of membrane fusion. The polymer-based particles can be synthetic copies of influenza virus machinery and can form transfection complexes with various types of nucleic acids (siRNA, miRNA, plasmid DNA or shRNA, mRNA) that are taken up by cells through the endocytic pathway, a process that involves the formation of acidic compartments. The low pH in late endosomes serves as a chemical switch, imparting hydrophobicity to the particle surface and facilitating membrane crossing. Upon entering the cytosol, the particle releases its payload for cellular action. This active endosomal escape technique is safe as it uses a natural uptake pathway and maximizes transfection efficiency. In some embodiments, the polymer-based particles can include alkylated and carboxyalkylated branched polyethyleneimines. In some examples, the polymer-based particles are VIROMER, e.g., VIROMER RNAi, VIROMER RED, VIROMER mRNA, VIROMER CRISPR. Exemplary methods of delivery of the systems and compositions herein include those described in Bawage SS et al., Synthetic mRNA expressed Casl3a mitigates RNA virus infections, www.biorxiv.org / content / l0.1 l01 / 370460v1.full doi:doi.org / 10.1101 / 370460, Viromer(R) RED, a powerful tool for transfection of keratinocytes. doi:10.13140 / RG.2.2.16993.61281, Viromer(R) Transfection-Factbook 2018: technology, product overview, users'data., doi:10.13140 / RG.2.2.23912.16642.

[0371]

[0344] Streptolysin O (SLO)

[0372] The delivery vehicle can be streptolysin O (SLO). SLO is a toxin produced by group A streptococci that acts by creating pores in mammalian cell membranes. SLO can act reversibly, allowing proteins (e.g., up to 100 kDa) to be delivered to the cytosol of cells without compromising overall viability. Examples of SLO include those described in Sierig G, et al. (2003). Infect Immun 71:446-55; Walev I, et al. (2001). Proc Natl Acad Sci US A 98:3185-90; Teng KW, et al. (2017). Elife 6:e25460.

[0373]

[0346] Multifunctional Envelope-Type Nanodevice (MEND)

[0374] The delivery vehicle can include a multifunctional enveloped nanodevice (MEND). The MEND can include a condensed plasmid DNA, a PLL core, and a lipid film shell. The MEND can further include a cell membrane penetrating peptide (e.g., stearyl octaarginine). The cell membrane penetrating peptide can be present in the lipid shell. The lipid envelope can be modified with one or more functional components, such as polyethylene glycol (e.g., for increasing vascular circulation time), a ligand for targeting specific tissues / cells, an additional cell membrane penetrating peptide (e.g., for larger cell delivery), a lipid for improving endosomal escape, and a nuclear delivery tag. In some examples, the MEND can be a tetralamellar MEND (T-MEND), which can target the cell nucleus and mitochondria. In certain examples, the MEND can be a PEG-peptide-DOPE conjugated MEND (PPD-MEND), which can target bladder cancer cells. Examples of MENDs include those described in Kogure K, et al. (2004). J Control Release 98:317-23; Nakamura T, et al. (2012). Ace Chem Res 45:1113-21.

[0375]

[0348] Lipid-coated mesoporous silica particles

[0376] The delivery vehicle can include lipid-coated mesoporous silica particles. The lipid-coated mesoporous silica particles can include a mesoporous silica nanoparticle core and a lipid membrane shell. The silica core can have a large internal surface area, which allows for high cargo loading capacity. In some embodiments, the pore size, pore chemistry, and overall particle size can be modified to load different types of cargo. The lipid coating of the particles can also be modified to maximize cargo loading, increase circulation time, and provide precise targeting and cargo release. Examples of lipid-coated mesoporous silica particles include those described in Du X, et al. (2014). Biomaterials 35:5580-90; Durfee PN, et al. (2016). ACS Nano 10:8325-45.

[0377]

[0350] Inorganic nanoparticles

[0378]

[0351] Delivery vehicle can include inorganic nanoparticles.Examples of inorganic nanoparticles include carbon nanotubes (CNTs) (e.g., as described in Bates Kand Kostarelos K. (2013). Adv Drug Deliv Rev 65: 2023-33), bare mesoporous silica nanoparticles (MSNPs) (e.g., as described in Luo GF, et al. (2014). Sci Rep 4: 6064), and dense silica nanoparticles (SiNPs) (e.g., as described in Luo D and Saltzman WM. (2000). Nat Biotechnol 18: 893-5).

[0379]

[0352] How to use

[0380]

[0353] The compositions and systems herein can be used in a variety of applications, including the modification of non-animal organisms, such as plants and fungi, and the modification of animals, and the treatment and diagnosis of diseases in plants, animals, and humans. In general, the compositions and systems can be introduced into a cell, tissue, organ, or organism, where the expression and / or activity of one or more genes can be modified.

[0381]

[0354] Cells and Living Organisms

[0382] The present disclosure provides recombinant Cas proteins, CRISPR-Cas systems, constructs, polynucleotides encoding one or more components of the CRISPR-Cas system, and / or cells, tissues, and organisms comprising vectors comprising the polynucleotides. The present disclosure also provides nucleotide sequences encoding effector proteins that are codon-optimized for expression in eukaryotic organisms or eukaryotic cells in any of the methods or compositions described herein. In one embodiment of the present disclosure, the codon-optimized effector protein is any Cas protein discussed herein that is codon-optimized for operability in eukaryotic cells or organisms, such as cells or organisms as mentioned elsewhere herein, including, but not limited to, yeast cells, or mammalian cells or organisms, including mouse cells, rat cells, and human cells or non-human eukaryotic organisms, such as plants.

[0383]

[0356] In certain embodiments, modification of the targeted locus of interest may result in a eukaryotic cell comprising altered expression of at least one gene product, a eukaryotic cell comprising altered expression of at least one gene product, where expression of at least one gene product is increased, a eukaryotic cell comprising altered expression of at least one gene product, where expression of at least one gene product is decreased, or a eukaryotic cell comprising an edited genome.

[0384]

[0357] In certain embodiments, the eukaryotic cell can be a mammalian cell or a human cell.

[0385]

[0358] In further embodiments, the non-naturally occurring or recombinant compositions, vector systems, or delivery systems described herein can be used for site-specific gene knockout, site-specific genome editing, RNA sequence-specific interference, or multiplexed genome modification.

[0386] Also provided are gene products of the cells, cell lines, or organisms described herein. In certain embodiments, the amount of expressed gene product may be greater or less than the amount of gene product from a cell that does not have altered expression or an edited genome. In certain embodiments, the gene product may be altered compared to the gene product from a cell that does not have altered expression or an edited genome.

[0387] Also provided herein is a composition comprising the cells provided herein. In some embodiments, provided herein is a pharmaceutical composition comprising the cells provided herein and a pharma- ceutically acceptable carrier.

[0388] Method for modifying gene expression

[0389] In another aspect, provided herein are methods for modifying gene expression in a cell or in vivo in a subject. In particular, the methods provided herein can be used to modify expression of a gene product in a cell while minimizing off-target modifications.

[0390]

[0363] In some embodiments, provided herein are methods of modifying expression of a gene product and minimizing off-target modifications in vivo in a population of cells or in a subject, the methods comprising introducing into the population of cells or cells of the subject (i) a construct described herein, or a polypeptide expressed by the construct, and (ii) at least one sgRNA, wherein the KRAB and / or a modulator of gene expression provides a modification of at least one nucleotide near the gene and / or within a control element of the gene, thereby modifying expression of the gene product.

[0391] In some embodiments, the construct of step (i) has a construct of formula I: 5'-(Am1-Bm2)n1-CasN-(Am3-Bm4)n2-CasC-Tp-E-3'(I), where one of A and B is a polynucleotide encoding DNMT3A or a portion thereof and the other of A and B is a polynucleotide encoding DNMT3L or a portion thereof, CasN is a polynucleotide encoding the N-terminal portion of dCas9, CasC is a polynucleotide encoding the C-terminal portion of dCas9, E is 5'-(Am5-Bm6)n3-Kr-Dq-3' or 5'-Kr-Dq-(Am5-Bm6)n3-3', K is a polynucleotide encoding KRAB or a portion thereof, and D is a polynucleotide encoding a model of gene expression. and m1 is an integer selected from 0 to 20; q is an integer selected from 0 to 5; and r is an integer selected from 0 to 5, wherein when p is 0, at least one of m1, m2, m3, and m4 is not 0, at least one of n1 and n2 is not 0, and at least one of q and r is not 0.

[0392]

[0365] In some embodiments, the construct has a structure of formula II: 5'-CasN-(Am3-Bm4)n2-CasC-Kr-Dq-3'(II), where n2 is an integer selected from 1 and 2, r is an integer selected from 1 to 5, q is an integer selected from 0 to 5, and at least one of m3 and m4 is not 0.

[0393] In some embodiments, the construct has a structure of formula IIa: 5'-CasN-(AB)-CasC-Kr-Dq-3' (IIa). In some embodiments, the construct has a structure of formula III: 5'-(Am1-Bm2)n1-CasN-(Am3-Bm4)n2-CasC-Tp-E-3' (III), where p is an integer selected from 1 to 20.

[0394] In some embodiments, the construct has a structure of formula IIIa: 5'-CasN-CasC-Tp-3' (IIIa). In some embodiments, the construct has a structure of formula IIIb: 5'-(Am1-Bm2)-CasN-CasC-Tp-E-3' (IIIb), where at least one of m1 and m2 is not 0. In some embodiments, the construct has a structure of formula IIIb-1: 5'-(AB)-CasN-CasC-Tp-Kr-Dq-3' (IIIb-1), where r is an integer selected from 1 to 5 and q is an integer selected from 0 to 5.

[0395] In some embodiments, the construct has a structure of formula IIIc: 5'-CasN-CasC-Tp-E-3' (IIIc), where n3 is an integer selected from 1 and 2, r is an integer selected from 1 to 5, q is an integer selected from 0 to 5, and at least one of m5 and m6 is not 0. In some embodiments, the construct has a structure of formula IIIc-1: 5'-CasN-CasC-Tp-(Am5-Bm6)n3-Kr-Dq-3' (IIIc-1). In some embodiments, the construct has a structure of formula IIIc-2: 5'-CasN-CasC-Tp-Kr-Dq-(Am5-Bm6)n3-3' (IIIc-2).

[0396]

[0369] Without wishing to be bound by theory, when T comprises a polynucleotide encoding an epitope capable of binding to an antibody or antigen-binding fragment thereof, the 5'-(Am1-Bm2)n1-CasN-(Am3-Bm4)n2-CasC-Tp-3' portion of Formula I, the 5'-(Am1-Bm2)n1-CasN-(Am3-Bm4)n2-CasC-Tp-3' portion of Formula III, -3' portion of the formula IIIb, 5'-(Am1-Bm2)-CasN-CasC-Tp-3' portion of the formula IIIb, 5'-(AB)-CasN-CasC-Tp-3' portion of the formula IIIb-1, 5'-CasN-CasC-Tp-3' portion of the formula IIIc, 5'-CasN-CasC-Tp-3' portion of the formula IIIc-1, or 5'-CasN-CasC-Tp-3' portion of the formula IIIc-2 It is hypothesized that the polypeptide of (i) comprising a 5'-E-3' portion, the sgRNA of (ii), and multiple copies of the polypeptide of (i) comprising a 5'-E-3' portion of Formula I, a 5'-E-3' portion of Formula III, a 5'-E-3' portion of Formula IIIb, a 5'-Kr-Dq-3' portion of Formula IIIb-1, a 5'-E-3' portion of Formula IIIc, a 5'-(Am5-Bm6)n3-Kr-Dq-3' portion of Formula IIIc-1, or a 5'-Kr-Dq-(Am5-Bm6)n3-3' portion of Formula IIIc-2, are recruited to a genomic locus by binding of the antibody or antigen-binding fragment thereof to the epitope, thereby recruiting the polypeptide expressed by the construct to the genomic locus and altering expression of a gene product in a population of cells.

[0397] In some embodiments, the method further comprises introducing into the (iii) cell a second construct comprising 5'-(Am5-Bm6)n3-Kr-Dq-3' or 5'-Kr-Dq-(Am5-Bm6)n3-3' of E, or a polypeptide expressed by the second construct. Without wishing to be bound by theory, it is believed that the multiple copies of the polypeptide of (i), the sgRNA, and the polypeptide of (iii), comprising the 5'-CasN-CasC-Tp-3' portion of Formula IIIa, are recruited to a genomic locus by binding of the antibody or antigen-binding fragment thereof to the epitope, thereby recruiting the polypeptide expressed by the construct of (i) and the construct of (iii) to the genomic locus and altering expression of the gene product in the population of cells.

[0398]

[0371] Without wishing to be bound by theory, when T comprises a polypeptide sequence capable of binding to a nucleic acid structural element, the 5'-(Am1-Bm2)n1-CasN-(Am3-Bm4)n2-CasC-Tp-3' portion of formula I, the 5'-(Am1-Bm2)n1-CasN-(Am3-Bm4)n2-CasC-Tp-3' portion of formula III, the 5'-(Am1-Bm2)n1-CasN-(Am3-Bm4)n2-CasC-Tp-3' portion of formula IIIb, The 5'-(Am1-Bm2)-CasN-CasC-Tp-3' portion of formula IIIb-1, the 5'-(AB)-CasN-CasC-Tp-3' portion of formula IIIc, the 5'-CasN-CasC-Tp-3' portion of formula IIIc-1, or the 5'-CasN-CasC-Tp-3' portion of formula IIIc-2. It is believed that the polypeptide of (i), the sgRNA of (ii), and multiple copies of the polypeptide of (i), comprising a 5'-E-3' portion of Formula I, a 5'-E-3' portion of Formula III, a 5'-E-3' portion of Formula IIIb, a 5'-Kr-Dq-3' portion of Formula IIIb-1, a 5'-E-3' portion of Formula IIIc, a 5'-(Am5-Bm6)n3-Kr-Dq-3' portion of Formula IIIc-1, or a 5'-Kr-Dq-(Am5-Bm6)n3-3' portion of Formula IIIc-2, are recruited to a genomic locus by binding of the polypeptide sequence capable of binding to a nucleic acid structural element to the nucleic acid structural element, thereby recruiting a polypeptide expressed by the construct to the genomic locus and altering expression of a gene product in a population of cells.

[0399] In some embodiments, the method further comprises (iii) introducing into the cell a second construct comprising 5'-(Am5-Bm6)n3-Kr-Dq-3' or 5'-Kr-Dq-(Am5-Bm6)n3-3' of E, or a polypeptide expressed by the second construct. Without wishing to be bound by theory, it is believed that the multiple copies of the (i) polypeptide comprising the 5'-CasN-CasC-Tp-3' portion of Formula IIIa, the (ii) sgRNA, and the (iii) polypeptide are recruited to a genomic locus by binding of the polypeptide sequence capable of binding to a nucleic acid structural element to the nucleic acid structural element, thereby recruiting the polypeptide expressed by the construct to the genomic locus and altering expression of the gene product in the population of cells.

[0400] The methods provided herein can be used to modify gene expression in cells in culture, isolated cells, or in vivo, e.g., in cells within a subject. In vivo methods of modifying gene expression can be used to treat disease, as further described below.

[0401] In some embodiments, the methods provided herein reduce expression of a target gene. For example, the reduction in expression of the target gene can be measured relative to gene expression of the target gene product in the cells prior to exposure to the constructs provided herein, or compared to unmodified cells. In some embodiments, the methods provided herein reduce expression of the target gene by 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% in a plurality of modified cells compared to a wild-type cell population. In some embodiments, the ratio of on-site modifications of a gene product to off-site modifications of a gene product is about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, or 2: 1. In some embodiments, the ratio of on-site modifications to off-site modifications is 10: 1 or less.

[0402] The at least one nucleotide modification introduced by KRAB and / or a modulator of gene expression can be, for example, DNA methylation or a histone modification. The modification can be located in any region of the target gene where it achieves the desired effect (e.g., reduced gene expression), including, for example, the coding sequence of the gene or a regulatory element. In some embodiments, the modification occurs in the core promoter, proximal promoter, distal enhancer, silencer, insulator element, border element, or locus control region.

[0403] The modification may occur at a nucleotide located upstream or downstream of the transcription start site of the target gene. In some embodiments, the modification occurs at a nucleotide located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp, or about 1500bp upstream of the transcription start site of the gene. In some embodiments, the modification occurs at a nucleotide located within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp downstream of the transcription start site of the gene.

[0404] Exemplary Treatment Methods

[0405]

[0378] In another aspect, provided herein is a method for treating or alleviating a symptom of a gene product associated disorder in a subject, comprising the step of introducing into a cell of the subject (i) a construct provided herein, or a polypeptide expressed by the construct, and (ii) at least one sgRNA, wherein a KRAB and / or modulator of gene expression provides an alteration of at least one nucleotide near the gene and / or within a control element of the gene, thereby altering expression of the gene product and treating or alleviating a symptom of the gene product associated disorder in the subject.

[0406] The present disclosure provides the use of CRISPR-Cas systems for the treatment of various diseases and disorders. In some embodiments, the disclosure described herein relates to a method for treatment in which a cell is edited ex vivo by CRISPR or a base editor to regulate at least one gene, and then the edited cell is administered to a patient in need thereof. In some embodiments, the editing involves knocking in, knocking out, or knocking down the expression of at least one target gene in the cell. In certain embodiments, the editing inserts an exogenous gene, minigene, or sequence, which may include one or more exons in trans, or natural or synthetic in trans, at the target gene locus, hotspot locus, safe harbor locus, of the gene genomic location, where a new gene or genetic element can be introduced without disrupting the expression or regulation of adjacent genes or correcting, by insertion or deletion, one or more mutations in the DNA sequence encoding the target gene's regulatory element. In some embodiments, the editing involves introducing one or more point mutations into the nucleic acid (e.g., genomic DNA) of the target cell.

[0407] In some embodiments, the treatment is for an organ disease / disorder, including liver disease, eye disease, muscle disease, heart disease, blood disease, brain disease, kidney disease, or may include treatment for autoimmune diseases, central nervous system diseases, cancer and other proliferative diseases, neurodegenerative diseases, inflammatory diseases, metabolic disorders, musculoskeletal disorders, and the like. In some embodiments, the disease is a liver disease. In some embodiments, the disease is an eye disease. In some embodiments, the disease is a CNS disease. In some embodiments, the disease is cancer. In some embodiments, the disease is selected from the group consisting of familial hypercholesterolemia (FH), nonalcoholic steatohepatitis (NASH), Parkinson's disease, hepatic fibrosis (HF), age-related macular disease (AMD), Angelman syndrome (AS), type 2 diabetes, β-thalassemia, and hepatocellular carcinoma.

[0408] In some embodiments, regulation is affected by alterations in the target genes VEGFA, PCSK9, ANGPTL3, PTBP1, TTR, Ube3a-ATS, Ptp1b, APOC3, hsd17b13, bcl11a, or TGF-β. VEGFA. VEGFA plays an important role in angiogenesis and is overexpressed in some cancers. PCSK9 plays a key role in cholesterol management. ANGPTL3 plays a role in triglyceride metabolism. PTBP1 encodes a nuclear ribonucleoprotein and is involved in the regulation of transcription. TTR encodes the transporter protein transthyretin. Ube3a-ATS encodes a ubiquitin ligase and mutations in Ube3a-ATS are associated with Angelman syndrome. Ptp1b encodes protein tyrosine phosphatase 1B, which regulates insulin signaling. APOC3 encodes apolipoprotein C3, which plays a key role in lipid transport and metabolism. hsd17b13 encodes liver-specific hydroxysteroid 17β-dehydrogenase 13. BCL11A is a transcriptional repressor. TGFβ regulates several cellular pathways that are critically associated with proliferation and differentiation.

[0409]

[0382] The terms "subject" and "patient" are used interchangeably herein. In some embodiments, the subject is a human.

[0410]

[0383] Example

[0411] Design and construction of plasmids containing constructs of the present disclosure

[0412]

[0385] Epigenetic modification editors (e.g., including Dnmt3a CD, Dnmt3l CD, dSpCas9, KRAB and / or other histone modifiers) containing the HA epitope, P2A, and BFP were optimized by Genscript into nucleic acid sequences suitable for mammalian expression and synthesis, and then cloned into the pLV-CAG vector containing the CAG promoter and WPRE to express the complete epigenetic modification editor and the self-sheared BFP.

[0413] In optimizing the different functional elements and the order of the different functional elements, the different functional elements were optimized by Genscript into a nucleic acid sequence suitable for mammalian expression and synthesis. The vector except for the element to be replaced was first amplified by PCR, then the element to be replaced was amplified from the company synthesized sequence while introducing the homology arm sequence, and finally the different elements were recombined into the vector by NEBuilder reagent to construct the final expression plasmid.

[0414] Example 1: Constructs for epigenetic modification of CD151 and CD81 genes

[0415] According to the above-mentioned method, different sets of versions of constructs targeting CD151 gene and / or CD81 were generated, and the structures of these constructs are shown in Figures 1B, 2B, 4B, and 5B. The constructs shown in Figure 2E were generated as described: (1) Dnmt-dCas9+scFv-Krab generated by fusing Dnmt3A-Dnmt3L-dCas9-10xGCN4 (SEQ ID NOs: 151 and 179), T2A (SEQ ID NOs: 99 and 103), and scFv-Krab (SEQ ID NOs: 152 and 180); (2) dCas9-10xGCN4 (SEQ ID NOs: 153 and 181), T2A (SEQ ID NOs: 99 and 103), and scFv-Dnmt3 (3) dCas9+scFv-Dnmt+scFv-Krab generated by fusing dCas9-10×GCN4 (sequence numbers 153 and 181), T2A (sequence numbers 99 and 103), scFv-Dnmt3A-Dnmt3L (sequence numbers 200 and 201), and scFv-Krab (sequence numbers 202 and 203).

[0416] Repression of constructs at CD151 and / or CD81 using different targeting sgRNAs (Table 4) was measured by FACS. Results are shown in Figure 1C, Figures 2C-2E, Figures 4C-4F, and Figures 5C-5D. Editors with different effectors or conformations showed wide variation in repression of gene expression, strongly suggesting that various modifications will need to be made when optimizing epigenetic editors.

[0417] Example 2: EPICAS constructs for VEGFA knockdown

[0418] Three versions of EPICAS specific for VEGFA were generated (EPICAS-V1, -V2, and -V3). The structures of these constructs are shown in FIG. 6A. EPICAS-V1 contains the KRAB domain, while V2 and V3 contain the ZIM3-KRAB domain, and the orientation of DNMT3L and DNMT3A was reversed in V2 (3A-3L instead of 3L-3A as in V1 and V3).

[0419] The editing efficiency of constructs at VEGFA using two different sgRNAs targeting human VEGFA (Table 4) was measured by qPCR. The results are shown in Figure 6B. The reduction of mRNA expression by sgRNA1 was similar between constructs.

[0420] Example 3: Constructs for in vivo epigenetic modification of mouse PCSK9

[0421]

[0394] The following four versions of the construct were For testing of editing efficiency in vivo experiments, the following constructs were generated: (1) DNMT3A-DNMT3L-dCas9-KRAB (SEQ ID NOs: 138 and 166), (2) DNMT3A-DNMT3L-dCas9-EZH2 (SEQ ID NOs: 139 and 167), (3) DNMT3A-DNMT3L-dCas9-G9A (SEQ ID NOs: 140 and 168), (4) DNMT3A-DNMT3L-dCas9-HDAC3 (SEQ ID NOs: 144 and 172), (5) dCas9N-7-Dnmt3A-Dnmt3L-dCas9C-7-Krab (this construct is generated by fusing dCas9N-7 (SEQ ID NO: 14), Dnmt3A-Dnmt3L (SEQ ID NO: 71), dCas9C-7 (SEQ ID NO: 15), and Krab (SEQ ID NO: 51)).

[0422]

[0395] The DNA sequence corresponding to each version was in vitro transcribed to obtain mRNA, which was then mixed with two gRNAs targeting PCSK9 (SEQ ID NO: 198-199) in a ratio of 2:1:1 to prepare LNPs (molar ratio of LNP elements: MC3: cholesterol: DSPC: DMG-PEG2000 = 50: 38.5: 10: 1.5), which were then injected into wild-type C57 mice via tail vein injection. The injected dose was 4.5 mg (RNA mass) per kilogram of mouse body weight. Blood samples were collected from the mice on days 7, 14, and 21 of injection, and the expression of PCSK9 protein in the blood was measured by Elisa and compared with mice not injected with LNP and mice injected with mRNA + control gRNA (sequence GAAGAGCCTGAGGCTCTTCT).

[0423]

[0396] The results show that protein expression of PCSK9 in the serum of mice in the experimental group was significantly lower than that in the control group at all three time points, with a decrease of approximately more than 50%.

Claims

1. Constructs of Formula I: 5’-(A ｍ１ -B ｍ２ ) ｎ１ -CasN-(A ｍ３ -B ｍ４ ) ｎ２ -CasC-T ｐ -E-3’(I)、 [In the formula, One of A and B is a polynucleotide encoding DNMT3A or a part thereof, and the other of A and B is a polynucleotide encoding DNMT3L or a part thereof; CasN is a polynucleotide encoding the N-terminal portion of dCas9; CasC is a polynucleotide encoding the C-terminal portion of dCas9; E is 5'-(A ｍ５ -B ｍ６ ) ｎ３ -K ｒ -D ｑ -3' or 5'-K ｒ -D ｑ - (A ｍ５ -B ｍ６ ) ｎ３ -3', K is a polynucleotide encoding KRAB or a portion thereof, D is a polynucleotide encoding a modulator of gene expression; T comprises a polynucleotide encoding (i) an epitope capable of binding to an antibody or antigen-binding fragment thereof, or (ii) a polypeptide sequence capable of binding to a nucleic acid structural element; m1, m2, m3, m4, m5, and m6 each independently represent an integer selected from 0 to 3; n1, n2, and n3 each independently represent an integer selected from 0 to 2; p is an integer selected from 0 to 20; q is an integer selected from 0 to 5; r is an integer selected from 0 to 5; and When p is 0, at least one of m1, m2, m3, and m4 is not 0, at least one of n1 and n2 is not 0, and at least one of q and r is not 0.

2. The construct of claim 1 of formula II: 5'-CasN-(A ｍ３ -B ｍ４ ) ｎ２ -E-E ｒ -D ｑ -3'(II), [In the formula, n2 is an integer selected from 1 and 2; r is an integer selected from 1 to 5; q is an integer selected from 0 to 5; and At least one of m3 and m4 is not 0.

3. The construct according to claim 2 of formula IIa: 5'-CasN--A-B-Cass-- ｒ --D ｑ -3'(99a)。

4. The construct of claim 1 of formula III: 5'(A ｍ１  ｍ２ ) ｎ１ _____________________ ｍ３  ｍ４ ) ｎ２ _________ ｐ !3'(|)、 [wherein p is an integer selected from 1 to 20].

5. The construct according to claim 4 of formula IIIa: 5'-CasN-CasC-T ｐ -3'(IIIa)。

6. The construct according to claim 4 of formula IIIb: 5'-(A) ｍ１ -B ｍ２ )-CasN-CasC-T ｐ -E-3' (IIIa), [In the formula, at least one of m1 and m2 is not 0].

7. The construct according to claim 6, of formula IIIb-1: 5'-(A-B)-CasN-CasC-T ｐ -K ｒ -D ｑ -3'(IIIb-1)、 [In the formula, r is an integer selected from 1 to 5; and and q is an integer selected from 0 to 5.

8. The construct according to claim 4 of formula IIIc: ______________________ ｐ !3'(|Bc)、 [In the formula, n3 is an integer selected from 1 and 2; r is an integer selected from 1 to 5; and q is an integer selected from 0 to 5; and At least one of m5 and m6 is not 0.

9. The construct according to claim 8, of formula IIIc-1: 5-CasNC!as--T ｐ \(A ｍ５ -B ｍ６ ) ) ｎ３ -: ｒ --D ｑ -3'(99919-1)。

10. The construct according to claim 8, of formula IIIc-2: 5-CasNC!as--T ｐ -: ｒ --D ｑ \(A ｍ５ -B ｍ６ ) ) ｎ３ -3'(999c-2)。

11. The construct of claim 1 of formula IV: 5'-(A-B)-CasN-CasC-K ｒ -D ｑ -3', [In the formula, r is an integer selected from 0 to 5; q is an integer selected from 0 to 5; and At least one of q and r is not 0.

12. The construct of any one of claims 1 to 11, wherein T comprises (a) a polynucleotide encoding an epitope capable of binding to an antibody or an antigen-binding fragment thereof, and further comprises (b) a polynucleotide encoding a self-cleaving peptide at the 3' end of the polynucleotide of (a).

13. The construct of claim 12 , wherein the 3′ end of the nucleotide sequence encoding the self-cleaving peptide further comprises a polynucleotide encoding (c) an antibody or antigen-binding fragment thereof capable of binding to the epitope.

14. The construct of any one of claims 1 to 11, wherein the T comprises (a) a polynucleotide encoding a polypeptide sequence capable of binding to a nucleotide sequence element, and further comprises, at the 3' end of the polynucleotide of (a), a polynucleotide encoding a self-cleaving peptide.

15. The construct of any one of claims 1 to 14, wherein the self-cleaving peptide is selected from the group consisting of T2A, P2A, E2A and F2A self-cleaving peptides.

16. The construct of claim 15, wherein the self-cleaving peptide is T2A.

17. The construct of any one of claims 1 to 16, wherein the DNMT3A comprises the amino acid sequence of SEQ ID NO:

69.

18. The construct of any one of claims 1 to 17, wherein the polynucleotide encoding DNMT3A comprises the nucleic acid sequence of SEQ ID NO:

83.

19. The DNMT3L is Homo sapiens DNMT3L, Mus musculus DNMT3L, Mus caroli DNMT3L, Mus Pahari DNMT3L, Rattus norvegicus DNMT3L, Rattus rattus DNMT3L, Albicantis niloticus DNMT3L, Grammomys surdaster DNMT3L, or Mastomys 19. The construct of any one of claims 1 to 18, comprising Mastomys coucha DNTM3L.

20. The construct of any one of claims 1 to 19, wherein the DNMT3L comprises an amino acid sequence of any one of SEQ ID NOs: 74 to 82.

21. The construct according to any one of claims 1 to 20, wherein the polynucleotide encoding DNMT3L comprises a nucleic acid sequence of any one of SEQ ID NOs: 84 to 92.

22. The construct of any one of claims 1 to 21, wherein the KRAB comprises the amino acid sequence of SEQ ID NO: 51, 53, or 230-241.

23. The construct of any one of claims 1 to 22, wherein the polynucleotide encoding KRAB comprises the nucleic acid sequence of SEQ ID NO: 52, 54, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, or 228.

24. 24. The construct of any one of claims 1 to 23, wherein the modulator of gene expression comprises Kruppel-associated repression box (KRAB), enhancer of zete homolog 2 (EZH2), G9A, lysine-specific histone demethylase 1A (LSD1), heterochromatin protein 1 (HP1), friend of GATA protein 1 (FOG1), histone deacetylase (HDAC3), and / or DOT1L.

25. The method of any one of claims 1 to 24, wherein the modulator of gene expression comprises the amino acid sequence of SEQ ID NO: 51, 53, 55, 57, 59, 61, 63, 65, or 67.

26. The method of any one of claims 1 to 25, wherein the polynucleotide encoding the modulator of gene expression comprises the nucleic acid sequence of SEQ ID NO: 52, 54, 56, 58, 60, 62, 64, 66, or 68.

27. The dCas9 includes Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, and Streptococcus pasteurianus dCas9. dCas9, Lactobacillus farciminis dCas9, Spirochaeta globus dCas9, Azospirillum (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvivacrum 27. The construct of any one of claims 1 to 26, comprising Parvibaculum lavamentivorans dCas9, Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9.

28. The construct of any one of claims 1 to 27, wherein the dCas9 comprises an amino acid sequence of any one of SEQ ID NOs: 106 to 122.

29. 29. The construct of any one of claims 1 to 28, wherein the dCas9 comprises Staphylococcus pyogenes dCas9.

30. The construct of any one of claims 1 to 29, wherein the dCas9 comprises the amino acid sequence of SEQ ID NO:

1.

31. The construct of any one of claims 1 to 30, wherein the N-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24.

32. The construct of any one of claims 1 to 31, wherein the C-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25.

33. The construct of any one of claims 1 to 32, wherein the polynucleotide encoding dCas9 comprises the nucleic acid sequence of SEQ ID NO:

26.

34. The construct of any one of claims 1 to 33, wherein the polynucleotide encoding the N-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

35. The construct of any one of claims 1 to 34, wherein the polynucleotide encoding the C-terminal portion of dCas9 comprises the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

36. 36. The construct of any one of claims 1 to 35, wherein the epitope capable of binding to an antibody or antigen-binding fragment thereof is selected from the group consisting of GCN4, T2A, 10x GCN4, and 10XGFP-11.

37. The construct of claim 36, wherein the GCN4 comprises the amino acid sequence of SEQ ID NO:

97.

38. The construct of claim 37, wherein the polynucleotide encoding GCN4 comprises the nucleic acid sequence of SEQ ID NO:

101.

39. The construct of any one of claims 36 to 38, wherein the antibody or antigen-binding fragment is a single domain antibody, a scFv, a Fab, a VH, a VHH, or an antibody mimetic.

40. The construct of claim 39, wherein the antigen-binding fragment is an scFv.

41. 41. The construct of any one of claims 1 to 40, wherein the polypeptide sequence capable of binding to a nucleic acid structural element is selected from the group consisting of MS2 bacteriophage coat protein (MCP), PP7, and PCP.

42. 42. The construct of claim 41 , wherein the polypeptide sequence capable of binding to a nucleic acid structural element is an MCP.

43. 43. The construct of claim 42, wherein the MCP comprises the amino acid sequence of SEQ ID NO:

100.

44. The construct according to claims 42 to 43, wherein the polynucleotide encoding MCP comprises the nucleic acid sequence of SEQ ID NO:

105.

45. The construct of any one of claims 41 to 44, wherein the nucleic acid structural element is an RNA hairpin motif.

46. 46. ​​The construct of claim 45, wherein the RNA hairpin motif is selected from the group consisting of MS2 and PP7.

47. The construct of claims 45 to 46, wherein the RNA hairpin motif is an MS2 RNA hairpin motif.

48. 48. The construct of claim 47, wherein the MS2 RNA hairpin motif comprises the nucleic acid sequence of SEQ ID NO:

104.

49. A construct according to any one of claims 1 to 48, comprising a nucleic acid sequence according to any one of SEQ ID NOs: 162 to 189.

50. A polypeptide expressed by a construct according to any one of claims 1 to 49.

51. A vector comprising the construct of any one of claims 1 to 49.

52. 52. The vector of Claim 51, further comprising a polynucleotide encoding a single guide RNA (sgRNA).

53. The vector of claims 51 to 52, wherein the polynucleotide encoding the sgRNA further comprises 2 to 20 copies of a nucleic acid structural element.

54. A cell comprising a construct according to any one of claims 1 to 49.

55. A cell comprising the polypeptide of claim 50.

56. A cell comprising the vector according to any one of claims 51 to 53.

57. The cell of any one of claims 54 to 56, further comprising at least one sgRNA.

58. 58. The cell of claim 57, wherein the at least one sgRNA further comprises 2 to 20 copies of the nucleic acid structural element.

59. A composition comprising a construct according to any one of claims 1 to 49.

60. A composition comprising the polypeptide of claim 50.

61. A composition comprising the vector according to any one of claims 51 to 53.

62. 62. The composition of any one of claims 59 to 61, further comprising at least one sgRNA.

63. 63. The composition of claim 62, wherein the sgRNA further comprises 2 to 20 copies of the nucleic acid structural element.

64. The composition of any one of claims 59 to 63, further comprising a pharma- ceutically acceptable carrier.

65. 1. A method of modifying expression of a gene product and minimizing off-target modifications in a population of cells, comprising administering to the population of cells: i) a construct according to any one of claims 1 to 48 or a polypeptide expressed by said construct; ii) at least one sgRNA; and introducing wherein the modulator of KRAB and / or gene expression provides an alteration of at least one nucleotide near the gene and / or within a regulatory element of the gene, thereby altering expression of the gene product. method.

66. (a) 5'-(A ｍ１ -B ｍ２ ) ｎ１ -CasN-(A ｍ３ -B ｍ４ ) ｎ２ -CasC-T ｐ -3' part, 5'-(A ｍ１ -B ｍ２ ) ｎ１ -CasN-(A ｍ３ -B ｍ４ ) ｎ２ -CasC-T ｐ -3' part, 5'-(A ｍ１ -B ｍ２ )-CasN-CasC-T ｐ -3' part, 5'-(AB)-CasN-CasC-T of formula IIIb-1 ｐ -3' part, 5'-CasN-CasC-T of formula IIIc ｐ -3' part, 5'-CasN-CasC-T of formula IIIc-1 ｐ -3' portion, or 5'-CasN-CasC-T of formula IIIc-2 ｐ -3' part The polypeptide of i) and the sgRNA of ii) are recruited to a genomic locus; and (b) The 5'-E-3' portion of formula I, The 5'-E-3' portion of formula III: The 5'-E-3' moiety of formula IIIb, 5'-K of Formula IIIb-1 ｒ -D ｑ -3' part, The 5'-E-3' portion of formula IIIc, 5'-(A ｍ５ -B ｍ６ ) ｎ３ -K ｒ -D ｑ -3' portion, or 5'-K of Formula IIIc-2 ｒ -D ｑ - (A ｍ５ -B ｍ６ ) ｎ３ -3' part i) multiple copies of the polypeptide of is recruited to a genomic locus by binding of the antibody or antigen-binding fragment thereof to the epitope, thereby recruiting the polypeptide expressed by the construct to the genomic locus and altering expression of a gene product in a population of cells.

66. The method of claim 65.

67. The cells, iii) 5'-(A ｍ５ -B ｍ６ ) ｎ３ -K ｒ -D ｑ -3' or 5'-K ｒ -D ｑ - (A ｍ５ -B ｍ６ ) ｎ３ -3', or a polypeptide expressed by said second construct, into the cell; where 5'-CasN-CasC-T of formula IIIa ｐ i) the polypeptide comprising the 3' portion and the sgRNA are recruited to a genomic locus; wherein multiple copies of the polypeptide of iii) are recruited to a genomic locus upon binding of the antibody or antigen-binding fragment thereof to the epitope, thereby recruiting the polypeptide expressed by the construct of i) and the construct of iii) to the genomic locus and altering expression of a gene product in a population of cells.

67. The method of claim 66.

68. (a) 5'-(A ｍ１ -B ｍ２ ) ｎ１ -CasN-(A ｍ３ -B ｍ４ ) ｎ２ -CasC-T ｐ -3' part, 5'-(A ｍ１ -B ｍ２ ) ｎ１ -CasN-(A ｍ３ -B ｍ４ ) ｎ２ -CasC-T ｐ -3' part, 5'-(A ｍ１ -B ｍ２ )-CasN-CasC-T ｐ -3' part, 5'-(AB)-CasN-CasC-T of formula IIIb-1 ｐ -3' part, 5'-CasN-CasC-T of formula IIIc ｐ -3' part, 5'-CasN-CasC-T of formula IIIc-1 ｐ -3' portion, or 5'-CasN-CasC-T of formula IIIc-2 ｐ -3' part The polypeptide of i) and the sgRNA of ii) are recruited to a genomic locus; and (b) The 5'-E-3' portion of formula I, The 5'-E-3' portion of formula III, The 5'-E-3' moiety of formula IIIb, 5'-K of Formula IIIb-1 ｒ -D ｑ -3' part, The 5'-E-3' portion of formula IIIc, 5'-(A ｍ５ -B ｍ６ ) ｎ３ -K ｒ -D ｑ -3' portion, or 5'-K of Formula IIIc-2 ｒ -D ｑ - (A ｍ５ -B ｍ６ ) ｎ３ -3' part i) multiple copies of the polypeptide of is recruited to a genomic locus by binding of a polypeptide sequence capable of binding to a nucleic acid structural element to said nucleic acid structural element, thereby recruiting said polypeptide expressed by said construct to said genomic locus and modifying expression of a gene product in a population of cells.

66. The method of claim 65.

69. The cells, iii) 5'-(A ｍ５ -B ｍ６ ) ｎ３ -K ｒ -D ｑ -3' or 5'-K ｒ -D ｑ - (A ｍ５ -B ｍ６ ) ｎ３ -3', or a polypeptide expressed by said second construct; where 5'-CasN-CasC-T of formula IIIa ｐ i) the polypeptide comprising the 3' portion and the sgRNA are recruited to a genomic locus; wherein multiple copies of the polypeptide of iii) are recruited to a genomic locus by binding of said polypeptide sequence capable of binding to a nucleic acid structural element to said nucleic acid structural element, thereby recruiting said polypeptide expressed by said construct to said genomic locus and modifying expression of a gene product in a population of cells.

69. The method of claim 68.

70. 1. An in vivo method of reducing or eliminating expression of a gene product in a subject, comprising administering to a cell of the subject: i) a construct according to any one of claims 1 to 48 or a polypeptide expressed by said construct; ii) at least one sgRNA; and introducing wherein the modulator of KRAB and / or gene expression provides an alteration of at least one nucleotide near the gene and / or within a regulatory element of the gene, thereby altering the expression of the gene product in the subject. method.

71. 1. A method for treating or alleviating a symptom of a gene product associated disorder in a subject, comprising administering to a cell of the subject: i) a construct according to any one of claims 1 to 49 or a polypeptide expressed by said construct; ii) at least one sgRNA; and introducing wherein the modulator of KRAB and / or gene expression provides an alteration of at least one nucleotide near said gene and / or within a regulatory element of said gene, thereby altering said expression of said gene product and treating or alleviating a symptom of said gene product-associated disorder in said subject. method.

72. 70. The method of any one of claims 65-69, wherein the expression of the gene product is reduced by 50-100% in the plurality of engineered cells compared to a wild-type cell population.

73. 70. The method of any one of claims 65 to 69, wherein the ratio of on-site modifications of the gene product to off-site modifications of the gene product is about 10:

1.

74. The method according to any one of claims 65 to 73, wherein the modification of at least one nucleotide is DNA methylation or a histone modification.

75. 75. The method of claim 74, wherein the modification of at least one nucleotide is DNA methylation.

76. 76. The method of any one of claims 65 to 75, wherein the control element of the gene is a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a border element, or a locus control region.

77. 77. The method of any one of claims 65 to 76, wherein the at least one nucleotide modification near the gene and / or within the control element of the gene is located within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp upstream of the transcription start site of the gene.

78. 77. The method of any one of claims 65 to 76, wherein the at least one nucleotide modification near the gene and / or within the control element of the gene is located within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, or about 1500 bp downstream of the transcription start site of the gene.

79. The method of any one of claims 65 to 78, wherein the construct is a deoxyribonucleic acid (DNA).

80. 79. The method of any one of claims 65 to 78, wherein the construct is a messenger ribonucleic acid (mRNA).

81. The method of any one of claims 65 to 80, wherein the construct is formulated in a liposome or lipid nanoparticle.

82. 81. The method of any one of claims 65 to 80, wherein the construct and the sgRNA are formulated in a liposome or lipid nanoparticle.

83. 83. The method of Claim 82, wherein the construct and the sgRNA are formulated in the same liposome or lipid nanoparticle.

84. 83. The method of Claim 82, wherein the construct and the sgRNA are formulated in different liposomes or lipid nanoparticles.

85. 85. The method of any one of claims 81 to 84, wherein the liposome or lipid nanoparticle comprises an ionized lipid (20% to 70%, molar ratio), a PEGylated lipid (0% to 30%, molar ratio), a supporting lipid (30% to 50%, molar ratio), and cholesterol (10% to 50%, molar ratio).

86. 86. The method of claim 85, wherein the ionizable lipid is selected from the group consisting of a pH-responsive ionizable lipid, a thermally responsive ionizable lipid, and a light-responsive ionizable lipid.

87. The method of any one of claims 65 to 80, wherein the construct is formulated in an AAV vector.

88. 81. The method of any one of claims 65 to 80, wherein the construct and the sgRNA are formulated in an AAV vector.

89. 89. The method of Claim 88, wherein the construct and the sgRNA are formulated in the same AAV vector.

90. 89. The method of Claim 88, wherein the construct and the sgRNA are formulated in different AAV vectors.

91. 91. The method of any one of claims 65 to 90, wherein the construct or the polypeptide expressed by the construct is delivered to cells by local injection, systemic injection, or a combination thereof.

92. The method of any one of claims 65 to 91, wherein the gene product is selected from the group consisting of a VEGFA gene product, a PCSK9 gene product, an ANGPTL3 gene product, a PTBP1 gene product, a TTR gene product, a Ube3a-ATS gene product, a Ptp1b gene product, an APOC3 gene product, a hsd17b13 gene product, a bcl11a gene product, and a TGF-β gene product.

93. The method of any one of claims 70 to 92, wherein the subject is a human.

94. 93. The method of any one of claims 67 to 92, wherein the method is for the treatment or prevention of a disease or disorder selected from the group consisting of familial hypercholesterolemia (FH), non-alcoholic steatohepatitis (NASH), Parkinson's disease, liver fibrosis (HF), age-related macular disease (AMD), Angelman syndrome (AS), type 2 diabetes, β-thalassemia, and hepatocellular carcinoma.

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