RNA-guided gene editing and gene regulation

Abstract

To provide: clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-based (Cas) 9-associated system related compositions; methods of using the CRISPR / Cas9-associated system related compositions for gene expression and genome engineering; compositions and methods of using the compositions for gene expression and genetic engineering in muscle, such as skeletal muscle and cardiac muscle.SOLUTION: A fusion protein comprises two heterologous polypeptide domains. The first polypeptide domain comprises a clustered regularly interspaced short palindromic repeats associated (Cas) protein. The second polypeptide domain has activity selected from the group consisting of transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, and demethylase activity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-citation of related applications This application claims priority over U.S. Provisional Patent Application No. 61 / 831,481 (filed June 5, 2013), U.S. Provisional Patent Application No. 61 / 839,127 (filed June 25, 2013), U.S. Provisional Patent Application No. 61 / 904,911 (filed November 15, 2013), U.S. Provisional Patent Application No. 61 / 967,466 (filed March 19, 2014), and U.S. Provisional Patent Application No. 61 / 981,575 (filed April 18, 2014) (all of which are incorporated herein by reference in their entirety). Description of government ownership This invention was made possible with government support through federal grant numbers DP2-OD008586 and R01DA036865 provided by the NIH and CBET-1151035 provided by the National Science Foundation. The U.S. Government has certain rights to this invention.

[0002] Technical field This disclosure relates to the fields of gene expression modification, genomic manipulation, and gene genome modification using clustered, equidistant short palindromic repeat (CRISPR) / CRISPR-associated (Cas)9-dependent systems and viral delivery systems. This disclosure also relates to the fields of genomic manipulation and gene genome modification in muscle (e.g., skeletal muscle and cardiac muscle). [Background technology]

[0003] In mammalian systems, synthetic transcription factors have been manipulated to control gene expression for a wide range of medical and scientific applications, including stimulating tissue regeneration, drug screening, compensating for gene deficiencies, activating deactivated tumor suppressors, regulating stem cell differentiation, performing gene screening, and creating synthetic gene circuits. These transcription factors may target promoters or enhancers of endogenous genes, or they may be intentionally designed to recognize sequences independently of the mammalian genome for transgene regulation. The most common approach to manipulating novel transcription factors targeting user-specific sequences is based on the DNA-binding domains of zinc finger proteins and transcriptional activator-like effectors (TALFs). Both approaches require manipulating novel proteins with unique DNA-binding specificity by utilizing the principles of protein-DNA interactions in these domains. While these methods have achieved widespread success for many applications, the protein manipulation required to manipulate protein-DNA interactions is labor-intensive and demands specialized expertise.

[0004] Furthermore, these novel proteins are not always effective. The reason for this is still unclear, but it may be related to epigenetic modifications and the influence of chromatin state on protein-to-genomic target site binding. In addition, ensuring that these novel proteins are delivered to each cell, as with other components, is extremely difficult. Existing methods for delivering these novel proteins and their diverse components involve delivery to cells via separate plasmids or vectors, which result in expression levels that can vary greatly due to differences in copy number. Moreover, gene activation after transfection is transient due to plasmid DNA dilution, and transient gene expression is not sufficient to produce a therapeutic effect. Furthermore, this approach is unsuitable for cell types where transfection is not easily performed. Therefore, another limitation of these novel proteins is the effectiveness of their transcriptional activation.

[0005] Site-specific nucleases can be used to introduce site-specific double-strand breaks at target genomic loci. These DNA breaks stimulate the innate DNA repair mechanism, resulting in one of two possible repair pathways. In the absence of a donor template, the break is repaired by non-homologous end joining (NHEJ), an error-prone repair pathway resulting in small DNA insertions or deletions. This method allows for the intentional disruption, deletion, or modification of the target gene sequence's reading frame. However, if a donor template is provided along with the nuclease, the cellular mechanism will repair the break by homologous recombination (homologous recombination is several orders of magnitude enhanced in the presence of DNA breaks). This method allows for the introduction of changes specific to the DNA sequence at the target site. Manipulated nucleases have been used for gene editing in various human stem cells and cell lines, or in mouse liver. However, a major hurdle to implementing these techniques is in vivo delivery to specific tissues in a manner that facilitates effective, efficient, and successful genome modification.

[0006] Genetic disorders have a devastating impact on children in the United States. These diseases have so far been incurable, and patients can only manage to alleviate symptoms. Over decades, the field of gene therapy has offered the prospect of a cure for these diseases. However, technological hurdles regarding the safe and efficient delivery of therapeutic genes to cells and patients limit this approach. Duchenne muscular dystrophy (DMD) is the most common single-gene disorder, occurring in 1 in 3,500 men. DMD results from hereditary or accidental mutations in the dystrophin gene. Dystrophin is an essential component of a protein complex necessary for the maintenance and regulation of muscle cell function. DMD patients typically lose the ability to physically support themselves in childhood, and their fragility progresses during adolescence, leading to death in their twenties. Experimental gene therapy approaches for DMD to date either require repeated administration of transient gene delivery vehicles or rely on the permanent integration of exogenous genetic material into genomic DNA. Both of these methods raise significant safety concerns. Furthermore, these strategies have been limited by the inability to deliver the large and complex dystrophin gene sequence. [Overview of the project] [Means for solving the problem]

[0007] The present invention aims to provide a fusion protein comprising two heterologous polypeptide domains. The first polypeptide domain comprises an aggregated, equidistant short palindromic repeat-associated (Cas) protein, and the second polypeptide domain has an activity selected from the group consisting of: transcriptional activation activity, transcriptional repression activity, transcriptional deactivation activity, histone modification activity, nuclease activity, nucleic acid binding activity, methylase activity, and demethylase activity. The Cas protein may contain Cas9. The Cas9 may contain at least one amino acid mutation that knocks out the nuclease activity of Cas9. The at least one amino acid mutation may be at least one of D10A and H840A. The Cas protein may contain iCas9 (amino acids 36-1403 of SEQ ID NO: 1). The second polypeptide domain may have transcriptional activation activity. The second polypeptide domain may contain at least one VP16 transcriptional activation domain repeat. The second polypeptide domain may contain a VP16 tetramer ("VP64") or a p65 activation domain. The fusion protein may further include a linker that connects the first polypeptide domain to the second polypeptide domain. The fusion protein may also include iCas9-VP64.

[0008] The present invention aims to provide a DNA targeting system comprising the fusion protein and at least one guide RNA (gRNA). The at least one gRNA may include a 12-22 base pair complementary polynucleotide sequence of the target DNA sequence followed by a protospacer flanking motif. The at least one gRNA may target the promoter region of a gene, the enhancer region of a gene, or the transcription region of a gene. The at least one gRNA may target the intron of a gene. The at least one gRNA may target the exon of a gene. The at least one gRNA may target the promoter region of a gene selected from the group consisting of ASCL1, BRN2, MYT1L, NANOG, VEGFA, TERT, IL1B, IL1R2, IL1RN, HBG1, HBG2, and MYOD1. The at least one gRNA may include at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625. The present invention aims to provide a DNA targeting system comprising Cas9 and at least one guide RNA (gRNA) that binds to the dystrophin gene. At least one gRNA can target the introns of the dystrophin gene. At least one gRNA can target the exons of the dystrophin gene. The at least one guide RNA can include at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625. The DNA targeting system can include one to ten different gRNAs. The present invention aims to provide isolated polynucleotides encoding the fusion protein or the DNA targeting system. The present invention aims to provide a vector containing the aforementioned isolated polynucleotide. The present invention aims to provide cells containing the isolated polynucleotide or the vector.

[0009] The present invention aims to provide a method for regulating the expression of mammalian genes in cells. The method comprises contacting cells with the fusion protein, the DNA targeting system, the isolated polynucleotide, or the vector. Gene expression can be induced. The present invention aims to provide a method for transdifferentiating cells or inducing cell differentiation. The method comprises contacting cells with the fusion protein, the DNA targeting system, the isolated polynucleotide, or the vector. The cells may be fibroblasts or induced pluripotent stem cells. Fibroblasts may be transdifferentiated into neuronal cells or myogenic cells. The DNA targeting system may be contacted with cells, and at least one gRNA may target the promoter region of at least one gene selected from the group consisting of ASCL1, BRN2, MYOD1, and MYT1L. The DNA targeting system may include at least one gRNA targeting the promoter region of the ASCL1 gene and at least one gRNA targeting the promoter region of the BRN2 gene. The DNA targeting system may include one to 20 different gRNAs. The DNA targeting system may include 8 to 16 different gRNAs. The DNA targeting system may include dCas9-VP64. The DNA targeting system may be delivered to cells virally or nonvirally.

[0010] The present invention aims to provide a method for modifying a mutant gene in cells. The method comprises administering the DNA targeting system, the isolated polynucleotide, or the vector to cells. The modification of the mutant gene may include homology-directed repair. The method may further include administering donor DNA to cells. The mutant gene may include frameshift mutations that produce immature stop codons and truncated gene products. The modification of the mutant gene may include nuclease-mediated non-homological end junctions. The modification of the mutant gene may include deletion of an immature stop codon, disruption of a splice acceptor site, deletion of one or more exons, or disruption of a splice donor sequence. Deletion of one or more exons may result in modification of the leading frame. The present invention aims to provide a method for treating target animals carrying a mutant dystrophin gene in need of treatment. The method comprises administering the DNA targeting system, the isolated polynucleotide, or the vector to the target animal. The target animal may suffer from Duchenne muscular dystrophy.

[0011] The present invention aims to provide a method for modifying a mutant dystrophin gene in cells. The method comprises administering the DNA targeting system, the isolated polynucleotide, the vector, or the cells to cells containing the mutant dystrophin gene. The mutant dystrophin gene may include an immature stop codon, a disrupted leading frame due to gene deletion, an abnormal splice acceptor site, or an abnormal splice donor site, where the target region is located upstream or downstream of the immature stop codon, disrupted leading frame, abnormal splice acceptor site, or abnormal splice donor site. Modification of the mutant dystrophin gene may include homology-directed repair. The method may further include administering donor DNA to the cells. The mutant dystrophin gene may include frameshift mutations that produce immature stop codons and truncated gene products. Modification of the mutant dystrophin gene may include nuclease-mediated non-homologous end junctions. Modification of the mutant dystrophin gene may include deletion of immature stop codons, correction of disrupted leading frames, or modification of splicing by disrupting splice acceptor sites or splice donor sequences. Modification of the mutant dystrophin gene may include deletion of exons 45-55 or exon 51. The present invention aims to provide a kit comprising the fusion protein, the DNA targeting system, the isolated polynucleotide, the vector, or the cells.

[0012] The present invention aims to provide a method for regulating mammalian gene expression in cells. The method comprises the step of contacting cells with a polynucleotide encoding a DNA targeting system. The DNA targeting system comprises the fusion protein and at least one guide RNA (gRNA). The DNA targeting system may contain 1 to 10 different gRNAs. Different gRNAs can bind to different regions within a target gene. The target region may be separated by at least one nucleotide. The target region may be separated by about 15 to about 700 base pairs. Each of the different gRNAs can bind to at least one different target gene. The different target genes may be located on the same chromosome. The different target genes may be located on different chromosomes. The at least one target region may be in a non-open chromatin region, an open chromatin region, a promoter region of the target gene, an enhancer region of the target gene, a transcription region of the target gene, or an upstream region of the transcription start site of the target gene. The at least one target region may be located about 1 to about 1000 base pairs upstream of the transcription start site of the target gene. The at least one target region may be located approximately 1 to 600 base pairs upstream of the transcription start site of the target gene. Gene expression can be induced. The DNA targeting system may include two, three, four, five, six, seven, eight, nine, or ten different gRNAs. The at least one guide RNA may target the promoter region of a gene selected from the group consisting of ASCL1, BRN2, MYT1L, NANOG, VEGFA, TERT, IL1B, IL1R2, IL1RN, HBG1, HBG2, and MYOD1. The at least one guide RNA may include at least one of sequence numbers: 5-40, 65-144, 492-515, 540-563, and 585-625. The at least one target region may be located within an intron or exon of the target gene.

[0013] The present invention aims to provide a composition for inducing mammalian gene expression in cells. The composition comprises the fusion protein and at least one guide RNA (gRNA). The present invention aims to provide a composition for inducing mammalian gene expression in cells. The composition comprises an isolated polynucleotide sequence encoding the fusion protein and at least one guide RNA (gRNA). The at least one guide RNA can target the promoter region of a gene selected from the group consisting of ASCL1, BRN2, MYT1L, NANOG, VEGFA, TERT, IL1B, IL1R2, IL1RN, HBG1, HBG2, and MYOD1. The at least one guide RNA may include at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625. The present invention aims to provide cells containing the composition that induces mammalian gene expression in cells. The present invention aims to provide a composition for inducing mammalian gene expression in cells, or a kit comprising cells containing the composition for inducing mammalian gene expression in cells. The present invention aims to provide a kit for inducing mammalian gene expression in cells. The kit comprises a composition for inducing mammalian gene expression in cells, or cells containing the composition for inducing mammalian gene expression in cells.

[0014] The present invention aims to provide a composition for genome editing in the muscle of a target animal. The composition comprises a modified adeno-associated virus (AAV) vector and a nucleotide sequence encoding a site-specific nuclease. The muscle can be skeletal muscle or cardiac muscle. The modified AAV vector may have enhanced tissue tropism for cardiac and skeletal muscle. The site-specific nuclease may include a zinc finger nuclease, a TAL effector nuclease, or a CRISPR / Cas9 system. The site-specific nuclease can bind to a gene or locus within the muscle cell. The gene or locus may be a dystrophin gene. The composition may further contain donor DNA or a transgene. The present invention aims to provide a kit comprising the composition for genome editing in the muscle of a target animal. The present invention aims to provide a method for genome editing in the muscle of a target animal. The method comprises the step of administering a composition for genome editing in the muscle of a target animal to the muscle, where the muscle is skeletal muscle or cardiac muscle. Genome editing may include modification of a mutant gene or insertion of a transgene. Modification of a mutant gene may include deletion, rearrangement, or replacement of a mutant gene. Modification of a mutant gene may include nuclease-mediated non-homologous terminal joining or homology-directed repair. The present invention aims to provide a method for treating a target animal. The method comprises the step of administering a composition for genome editing in the muscle of the target animal to the muscle of the target animal, where the muscle is skeletal muscle or cardiac muscle. The target animal may suffer from skeletal muscle symptoms or genetic disorders. The target animal may suffer from Duchenne muscular dystrophy. The present invention aims to provide a method for modifying a mutated gene in a target animal, the method comprising the step of administering a genome-editing composition to the muscle of the target animal. The muscle can be skeletal muscle or cardiac muscle. The composition can be injected into the skeletal muscle of the target animal. The composition can be injected systemically into the target animal. The skeletal muscle may be the tibialis anterior muscle.

[0015] The present invention aims to provide a modified lentiviral vector for genome editing in a target animal, wherein the vector comprises a first polynucleotide sequence encoding the fusion protein and a second polynucleotide sequence encoding at least one sgRNA. The first polynucleotide sequence can be operably ligated to a first promoter. The first promoter may be a constitutive promoter, an inducible promoter, an inhibitory promoter, or a modulo promoter. The second polynucleotide sequence can encode 1 to 10 different sgRNAs. The second polynucleotide sequence may encode 2 different sgRNAs, 3 different sgRNAs, 4 different sgRNAs, 5 different sgRNAs, 6 different sgRNAs, 7 different sgRNAs, 8 different sgRNAs, 9 different sgRNAs, or 10 different sgRNAs. Each of the polynucleotide sequences encoding different sgRNAs can be operably ligated to a promoter. Each of the promoters operably ligated to different sgRNAs may be the same promoter. Each of the promoters operably ligated to different sgRNAs may be a different promoter. The promoter may be a constitutive promoter, an inducible promoter, an inhibitory promoter, or a modulo promoter. sgRNAs can bind to target genes. Each sgRNA can bind to a different target region within a single target locus. Each sgRNA can bind to a different target within a different locus. The fusion protein may contain the Cas9 protein or the iCas9-VP64 protein. The fusion protein may contain the VP64 domain, the p300 domain, or the KRAB domain. Two or more endogenous genes may be transcriptionally activated. Two or more endogenous genes may be repressed.

[0016] The present invention aims at a method for activating an endogenous gene in a cell. The method includes a step of contacting the cell with the modified lentiviral vector. The endogenous gene can be transiently activated. The endogenous gene can be stably activated. The endogenous gene can be transiently suppressed. The endogenous gene can be stably suppressed. The fusion protein can be expressed at a level similar to that of the sgRNA. The fusion protein can be expressed at a level different from that of the sgRNA. The cell can be a primary human cell. The present invention aims at a method for multiplex gene editing in a cell. The method includes a step of contacting the cell with the modified lentiviral vector. The multiplex gene editing can include correction of at least one mutant gene or insertion of a transgene. The correction of the mutant gene can include deletion, rearrangement or replacement of at least one mutant gene. The correction of at least one mutant gene can include nuclease-mediated non-homologous end joining or homology-directed repair. The multiplex gene editing can include deletion of at least one gene, where the gene is an endogenous normal gene or a mutant gene. The multiplex gene editing can include deletion of at least two genes. The multiplex gene editing can include deletion of two to ten genes. The present invention aims at a method for regulating gene expression of at least one target gene in a cell. The method includes a step of contacting the cell with the modified lentiviral vector. The gene expression of at least two genes can be regulated. The gene expression of two to ten genes can be regulated. When the gene expression level of the at least one target gene increases or decreases compared to the normal gene expression level of the at least one target gene, the gene expression of the at least one target gene can be regulated. Brief Description of the Drawings

[0017] [Figure 1]This study demonstrates activation of the human IL1RN gene by the RNA guide iCas9-VP64. (a, b) An RNA guide transcription activator was constructed by fusing an inactivated Cas9 (iCas9, D10A / H840A) to the VP64 transactivation domain. iCas9-VP64 recognizes a target site in the genome via hybridization of a guide RNA (gRNA) with a 20 bp target sequence. (c) Expression plasmids for four gRNAs or crRNA / tracrRNAs targeting sequences in the IL1RN promoter were co-transfected into HEK293T cells with an iCas9-VP64 expression plasmid. Activation of IL1RN expression was determined by qRT-PCR. (d) The four gRNA expression plasmids were co-transfected with iCas9-VP64 individually or together. Vigorous gene activation was observed by qRT-PCR only in response to the combined gRNA. (e) Activation of IL1RN expression was confirmed by determining the secretion of IL-1ra gene product into the culture medium by ELISA. IL-1ra was detected in only 3 of the 6 samples treated with combined gRNA. For (ce), data are shown as mean ± sem (n=3 independent experiments). Treatment with combined gRNA was statistically different from all other treatments by Tukey's test (*P <0.02). (f) RNA-seq was performed on samples treated with blank expression vectors (n=2) or samples cotransfected with iCas9-VP64 targeting IL1RN and expression plasmids for the four gRNAs (n=2). The only statistically significant changes in gene expression between these treatments were increases in the four IL1RN isoforms (false detection rate <3 x 10⁻⁴) and decreases in IL32 (false detection rate = 0.03). [Figure 2]Shows RNA-guided activation of human genes related to cell and gene therapy, gene reprogramming, and regenerative medicine. HEK293T cells were transfected with the iCas9-VP64 expression plasmid and four gRNAs individually or together. Target gene expression was measured by qRT-PCR and normalized to GAPDH mRNA levels. Data are shown as mean ± s.e.m. (n = 3 independent experiments). Treatment with gRNAs together was statistically different from all other treatments by Tukey's test (*P < 0.05). [Figure 3] Shows the expression of iCas9-VP64. Expression of iCas9-VP64 in transfected HEK293 cells was confirmed by Western blot against the N-terminal Flag epitope tag. The wtCas9 expression plasmid does not contain an epitope tag. [Figure 4-1] Shows the gRNA target sites and positions of DNase hypersensitivity of human target genes. Four gRNA target sites for each locus are shown as custom tracks for each gene above, and DNase-seq data showing DNase hypersensitive open chromatin regions are shown for each gene below. DNase-seq was performed in HEK293T cells to identify DNase hypersensitive regions as previously described (Song et al., Cold Spring Harbor protocols 2010, pdb prot5384, 2010; Song et al. Genome Res 21, 1757-1767, 2011). The results indicate that open chromatin is not a requirement for gene activation by the combination of gRNA and iCas9-VP64. [Figure 4-2]This shows the gRNA target sites and DNase-sensitive locations of human target genes. Four gRNA target sites for each gene locus are shown as custom tracks for each gene, and DNase-seq data indicating DNase-sensitive open chromatin regions are shown for each gene below. DNase-seq was performed in HEK293T cells to identify DNase-sensitive regions, as previously described (Song et al., Cold Spring Harbor protocols 2010, pdb prot5384, 2010; Song et al. Genome Res 21, 1757-1767, 2011). The results indicate that open chromatin is not a requirement for gene activation by the combination of gRNA and iCas9-VP64. [Figure 4-3] This shows the gRNA target sites and DNase-sensitive locations of human target genes. Four gRNA target sites for each gene locus are shown as custom tracks for each gene, and DNase-seq data indicating DNase-sensitive open chromatin regions are shown for each gene below. DNase-seq was performed in HEK293T cells to identify DNase-sensitive regions, as previously described (Song et al., Cold Spring Harbor protocols 2010, pdb prot5384, 2010; Song et al. Genome Res 21, 1757-1767, 2011). The results indicate that open chromatin is not a requirement for gene activation by the combination of gRNA and iCas9-VP64. [Figure 4-4]This shows the gRNA target sites and DNase-sensitive locations of human target genes. Four gRNA target sites for each gene locus are shown as custom tracks for each gene, and DNase-seq data indicating DNase-sensitive open chromatin regions are shown for each gene below. DNase-seq was performed in HEK293T cells to identify DNase-sensitive regions, as previously described (Song et al., Cold Spring Harbor protocols 2010, pdb prot5384, 2010; Song et al. Genome Res 21, 1757-1767, 2011). The results indicate that open chromatin is not a requirement for gene activation by the combination of gRNA and iCas9-VP64. [Figure 5] This study demonstrates the absence of nuclease activity by iCas9-VP64. Wild-type Cas9 or inactivated (D10A, H840A) iCas9-VP64 expression plasmids were cotransfected with expression plasmids for four different guide RNAs targeting the IL1RN promoter. Nuclease activity was determined by surveyor assay (Guschin et al., Methods Mol Biol 649, 247-256, 2010). Low molecular weight bands and DNA repair via non-homologous end joining, indicators of nuclease activity, were present only after treatment with wild-type Cas9, supporting the cessation of nuclease activity by iCas9-VP64. [Figure 6]RNA-seq results for samples treated with gRNAs targeting HBG1 and HBG2 are shown. RNA-seq was performed on samples treated with a control empty expression vector (n=3) or on samples cotransfected with expression plasmids for iCas9-VP64 and four gRNAs targeting HBG1 (n=2). Three of these gRNAs also targeted HBG2. Increases were observed in both HBG1 and HBG2 compared to the control, but these were not statistically significant due to low expression levels. The only statistically significant changes in gene expression between these treatments were decreases in IL32 (false detection rate = 0.0007) and TNFRS9 (false detection rate = 0.002). [Figure 7] This study demonstrates the upregulation of Ascl1 and γ-globin by iCas9-VP64. HEK293T cells were transfected with iCas9-VP64 and four gRNAs targeting the ASCL1 or HBG1 promoter. Corresponding Ascl1 and γ-globin protein levels were determined by Western blotting. Low levels of these proteins were detected in HEK293T cells, and increased expression was detected after iCas9-VP64 treatment in two separate experiments. [Figure 8-1] This shows the activation of downstream targets of Ascl1 in mouse embryonic fibroblasts treated with iCas9-VP64. Mouse embryonic fibroblasts (MEFs) were transfected with a control GFP expression plasmid or an iCas9-VP64 expression plasmid and four gRNA expression plasmids targeting ASCL1 in a 50:50 or 75:25 ratio. (a) The gRNA target site of the human ASCL1 promoter (SEQ ID NO: 3) is conserved in the mouse ASCL1 promoter (SEQ ID NO: 4). The target site is shown by a solid line, and the transcription region is shown by a dotted line. [Figure 8-2]This study demonstrates the activation of downstream targets of Ascl1 in mouse embryonic fibroblasts treated with iCas9-VP64. Mouse embryonic fibroblasts (MEFs) were transfected with either a control GFP expression plasmid or an iCas9-VP64 expression plasmid and four gRNA expression plasmids targeting ASCL1 in a 50:50 or 75:25 ratio. (b) ASCL1 expression in MEFs increased 2 days after iCas9-VP64 / gRNA treatment, as determined by qRT-PCR. (ch) After 10 days in neuronal induction medium, cells were stained for Ascl1 and Tuj1 (an early marker of neuronal differentiation) (cd), or for Tuj1 and MAP2 (a marker of more mature neuronal differentiation) (df). Some Tuj1-positive cells adopted neuronal morphology (fg), while single cells were found to be Tuj1 and MAP2-positive (g). (h) Tuj1-positive cells were readily identified in iCas9-VP64 / gRNA-treated cultures (~0.05%) but were absent in controls. Data are presented as mean ± standard error of the mean for n=3 independent experiments. gRNA 75 / 25 was significantly different from gRNA 50 / 50 and controls (*P<0.01, Tukey test). [Figure 9A] (a) Shows the iCas9-VP64 protein sequence (SEQ ID NO: 1). [Figure 9B] (b) The sequence of the gRNA expression cassette having the U6 promoter (Sequence ID: 2) is shown. [Figure 10-1] Standard curves for qRT-PCR are shown. For each gene, the experimental sample with the highest expression level was diluted to create the standard curve. The standard curves were assayed by qRT-PCR to ensure efficient amplification over an appropriate dynamic range. The efficiency of the overall amplification reaction was 90-115%. [Figure 10-2] Standard curves for qRT-PCR are shown. For each gene, the experimental sample with the highest expression level was diluted to create the standard curve. The standard curves were assayed by qRT-PCR to ensure efficient amplification over an appropriate dynamic range. The efficiency of the overall amplification reaction was 90-115%. [Figure 11]Figures 11(a) and 11(b) demonstrate the effectiveness of RNA-guided repair. Figure 11(a) shows the results of a surveyor assay of genomic DNA collected from HEK293T cells two days after co-transfection with Cas9 along with an empty vector (negative control) or gRNA. Figure 11(b) shows the location of the gRNA target. Figure 11(c) shows the expected cleavage size for each gRNA. [Figure 12] This shows RNA-guided repair in DMD8036(del48-50) cells as demonstrated by a surveyor assay. [Figure 13] This shows RNA-guided repair in DMD8036(del48-50) cells, as demonstrated by PCR across all gene loci. PCR of the wild-type dystrophin gene produces a fragment of size 1447 bp, while PCR of the mutant gene in the DMD8036 cell line shows a deletion of approximately 817 bp. The deletion band after introduction of the CRISPR / Cas9-dependent system was approximately 630 bp. [Figure 14] This shows RNA-guided repair in DMD8036 (del48-50) cells as demonstrated by Western blotting (using MANDYS8 (anti-dystrophin antibody) and GAPDH antibody (positive control)). [Figure 15] ChIP sequencing data showing specific binding of iCas9-VP64 to the IL1RN promoter is shown. HEK293T cells were transfected with iCas9-VP64 targeting the IL1RN promoter. [Figure 16]This shows CRISPR / Cas9 targeting the dystrophin gene. (A) To enable gene editing to repair dystrophin expression resulting from a wide variety of patient-specific mutations, sgRNA sequences were designed to bind to sequences in the mutation hotspot region of exons 45-55 of the dystrophin gene. Arrows within introns indicate sgRNA targets designed to delete entire exons from the genome. Arrows within exons indicate sgRNA targets designed to create targeted frameshifts within the dystrophin gene. (B) An example of frame correction following a small insertion or deletion by NHEJ DNA repair of exon 51 using CR3 sgRNA. (C) Schematic diagram of a composite sgRNA target designed to delete exon 51 and repair the dystrophin reading frame in a patient mutation with a deletion of exons 48-50. (D) Schematic diagram of a composite sgRNA target designed to delete the entire exon 45-55 region to accommodate a wide variety of DMD patient mutations. [Figure 17] Table 7 shows the TBE-PAGE gel image used to quantify the results of the surveyor assay measuring gene modification on day 3. The asterisk indicates the expected band size, which is an indicator of nuclease activity. [Figure 18] Table 7 shows the TBE-PAGE gel image used to quantify the results of the surveyor assay measuring gene modification on day 10. The asterisk indicates the expected band size, which is an indicator of nuclease activity. [Figure 19]This shows fluorescence-activated flow sorting to enrich genetically modified DMD myoblasts. (A) A plasmid expressing human codon-optimized SpCas9 protein linked to a GFP marker using a T2A ribosome skipping peptide sequence was co-electroporated into human DMD myoblasts along with one or more plasmids containing an sgRNA expression cassette. (B) The indicated sgRNA was separately co-transfected into HEK239T cells with separate plasmids expressing SpCas9 with GFP linked to SpCas9 by a T2A ribosome skipping peptide sequence (bottom) or SpCas9 without the above (top). The frequency of genetic modification was determined by a surveyor assay 3 days after transfection. (C) DMD myoblasts with deletions of exons 48-45 of the dystrophin gene were treated with sgRNA that corrects the dystrophin reading frame in these patient cells. Genetic modification was determined in unsorted (bulk) or GFP+ sorted cells 20 days after electroporation. (D) GFP expression in DMD myoblasts 3 days after electroporation with the expression plasmid shown. Transfection efficiency and selected cell population are indicated by the gate region. [Figure 20]This shows the frameshift of the target for repairing the dystrophin reading frame using CRISPR / Cas9. (A) The 5' region of exon 51 was targeted using sgRNA CR3 (which binds immediately upstream of the first out-of-frame stop codon). PAM: Protospacer adjacent motif. (B) The exon 51 locus was PCR amplified from HEK293T cells treated with SpCas9 and CR3 expression cassettes. The sequences of individual clones were determined by Sanger sequencing. The top sequence (bold, exons in red) is the natural, unmodified sequence. The number of clones for each sequence is shown in parentheses. (C) Summary of overall gene editing efficiency and the reading frame shift resulting from the gene modification shown in (B). (D) Western blot of dystrophin expression in human DMD myoblasts treated with SpCas9 and CR3 sgRNA expression cassettes (Figure 19C) that induce a target-induced frameshift to repair the dystrophin reading frame. Dystrophin expression was examined at 6 days of differentiation using an antibody against the rod domain of the dystrophin protein. [Figure 21]This shows the deletion of exon 51 of the human genome using complex CRISPR / Cas9 gene editing. (A) Endpoint genomic PCR of the entire exon 51 in human DMD myoblasts with deletions of exons 48-50. The top arrow indicates the expected location of the full-length PCR amplicon, and the two arrows below indicate the expected locations of the PCR amplicon with the deletion resulting from the indicated sgRNA combination. (B) The PCR product from (A) was cloned, and the sequence of each clone was determined to identify insertions and deletions at the target locus. The top row shows the wild-type unmodified sequence, and the triangles indicate SpCas9 cleavage sites. A representative chromatographic diagram showing the expected deletion ligation sequence is shown on the right. (C) Endpoint RT-PCR analysis of dystrophin mRNA transcripts in CRISPR / Cas9 modified human Δ48-50 DMD myoblasts treated with the indicated sgRNA. A representative chromatographic diagram of the expected deletion PCR product is shown on the right. Asterisk: Band resulting from hybridization between the deletion product chain and the unmodified chain. (D) Restoration of dystrophin protein expression by CRISPR / Cas9 genome editing was determined by Western blotting against dystrophin protein (using GAPDH as a loading control). Arrows indicate the expected repaired dystrophin protein band. [Figure 22]This shows the complete deletion of the exon 45-55 region in human DMD myoblasts by complex CRISPR / Cas9 gene editing. (A) Endpoint genomic PCR of genomic DNA detecting the region between intron 44 and intron 55 after treatment of HEK293T or DMD myoblasts with the indicated sgRNA. (B) Individual clones of PCR products with the expected size due to the deletion from DMD myoblasts in (A) were analyzed by Sanger sequencing to determine the sequence with the genomic deletion at the target locus. The bottom shows a representative chromatographic diagram showing the expected deletion ligature sequence. (C) Endpoint RT-PCR analysis of dystrophin mRNA transcripts from CRISPR / Cas9-modified human Δ48-50 DMD myoblasts treated with the indicated sgRNA. A representative chromatographic diagram of the expected deletion PCR product is shown on the right. (D) Analysis of repair dystrophin protein expression by Western blotting after electroporation of DMD myoblasts with sgRNA targeting intron 44 and / or intron 55. [Figure 23] This study demonstrates the enrichment of genetically modified DMD myoblasts used in in vivo cell transplantation experiments by flow cytometry. DMD myoblasts were treated with Cas9 in or without sgRNA expression vectors for CR1 and CR5, and GFP+ cells were selected by flow cytometry. Deletions at exon 51 were detected by endpoint PCR using primers that flanked the locus. Neg ctrl: DMD myoblasts were treated with Cas9 alone, and GFP+ cells were selected. [Figure 24]This figure shows the restored human dystrophin expression after transplantation of CRISPR / Cas9-treated human DMD myoblasts into immunodeficient mice. Human Δ48-50 DMD myoblasts were treated with SpCas9, CR1, and CR5 to delete exon 51, and then sorted for GFP expression as shown in Figure 19. These sorted cells and untreated control cells were injected into the hind limbs of immunodeficient mice, and human-specific protein expression in muscle fibers was assessed 4 weeks after transplantation. Frozen sections were stained with anti-human spectrin (spectrin is expressed in both uncorrected and corrected myoblasts fused with mouse muscle fibers) or anti-human dystrophin antibody as shown. White arrows indicate human dystrophin-positive muscle fibers. [Figure 25] Further immunofluorescence images examining human dystrophin expression are shown. Serial sections stained with anti-human spectrin are shown in the upper left. (AC) Section of muscle injected with untreated human DMD myoblasts. (DF) Section of muscle injected with CR1 / 5-treated human DMD myoblasts, enriched by flow cytometry. White arrows indicate dystrophin-positive fibers. [Figure 26]This paper evaluates the off-target effects of CRISPR / Cas9 toxicity and CR1 / CR5-mediated deletion of exon 51 in human cells. (A) Results of cytotoxicity assays in HEK293T cells treated with human-optimized SpCas9 and the indicated sgRNA construct. Cytotoxicity is measured against the viability of GFP-positive cells co-transfected with the indicated nuclease. I-SceI is a well-characterized non-toxic meganuclease, and GZF3 is a known toxic zinc finger nuclease. (B) Surveyor analysis of off-target sites in selected hDMD cells treated with expression cassettes encoding Cas9 and the indicated sgRNA. These three off-target sites tested in hDMD cells were identified from a panel of 50 predicted sites tested in HEK293T cells (Figure 27 and Table 4). TGT: On-target locus of the indicated sgRNA. OT: Off-target locus. (C,D) Endpoint nested PCR for detecting chromosomal translocations in HEK293T cells treated with Ca9 and CR1 (C) or selected hDMD cells treated with Cas9, CR1, and CR5 (D). The schematic diagram shows the relative positions of nested primer pairs customized for each translocation event. The expected size of each band was estimated based on the primer size and the expected location of the sgRNA cleavage site at each locus. Asterisks indicate bands detected at the expected size. Identification of the bands in (C) was confirmed by Sanger sequencing of each end (Figure 30). A representative chromatographic diagram of P2 / P5 translocation in HEK293T cells is shown. [Figure 27] Table 4 shows TBE-PAGE gel images used to quantify the results of surveyor assays to measure on-target and off-target gene modifications. The asterisks indicate the expected size of the band, which is an indicator of nuclease activity. [Figure 28]This document presents an endpoint nested PCR for detecting chromosomal translocations induced by CRISPR / Cas9 off-target activity against CR3 and CR6 / CR36 in human cells. Nested endpoint PCR analysis was used to detect translocations in HEK239T or selected hDMD cells treated with Cas9 and CR3 (A) as shown, HEK293T cells treated with Cas9 and CR36 alone (B), or selected hDMD cells treated with Cas9, CR6, and CR36 expression cassettes (C). A second nested PCR reaction for translocations was amplified using custom primers for each expected translocation locus to maximize specificity (see Table 4). Schematic diagrams show the relative positions of nested primer pairs used to examine the presence of translocations. Each possible translocation event was first amplified from genomic DNA isolated from cells treated in the presence or absence of the indicated sgRNA. The second nested PCR reaction was performed using primers within the PCR amplicon expected to result from the translocation. The predicted size was estimated based on the primer binding sites shown and the expected sgRNA cleavage sites at each locus. * indicates a band detected at the predicted size and validated by Sanger sequencing at each end. # indicates an amplicon showing a sequence other than the translocation predicted by Sanger sequencing (likely a result of mispriming during nested PCR). [Figure 29] Figure 28 shows Sanger sequencing chromatography diagrams of bands detected in HEK239T cells treated with Cas9 and CR3 gene cassettes, resulting from translocations between CR3 and CR3-OT1 (located on chromosomes X and 1, respectively). Arrows indicate regions homologous to the chromosomes shown near the expected breakpoints brought about by appropriate sgRNAs. Note that sequencing readings may not coincide near breakpoints due to the error-prone DNA repair characteristics of non-homologous end joining. [Figure 30]Figure 26C shows a Sanger sequencing chromatography diagram for the band detected (these bands are the result of translocations between CR1 and CR1-OT1 (located on chromosomes X and 16, respectively) in HEK293T cells treated with Cas9 and CR1 gene cassettes). Arrows indicate phase regions relative to the display chromosome near the expected breakpoint mediated by the appropriate sgRNA. Note that sequencing readings may not coincide near breakpoints due to the error-prone DNA repair characteristics of non-homologous end joining. [Figure 31] This outlines the in vivo procedures for AAV injection and tissue sampling. [Figure 32] Surveyor analysis of Rosa26 ZFN activity in vitro and in vivo in skeletal muscle after AAV-SASTG-ROSA delivery is shown. Arrows indicate expected bands resulting from surveyor cuts. nd = not detected. (a) The indicated amount of virus was transduced into proliferating C2C12 cells, and samples were collected 4 days after infection. Arrows indicate expected band sizes resulting from surveyor cuts. (b) C2C12 cells were incubated in differentiation medium for 5 days, followed by transduction of the indicated amount of AAV-SASTG-ROSA virus in 24-well plates. Samples were collected 10 days after transduction. (c) The indicated amount of AAV-SASTG-ROSA was directly injected into the tibialis anterior muscle of C57BL / 6J mice, and muscle was collected 4 weeks post-infection. The collected TA muscle was divided into eight separate fragments for genomic DNA analysis. Each is shown in a separate lane. [Figure 33] The Rosa T2A opt DNA sequence (SEQ ID NO: 434) and Rosa T2A opt protein sequence (SEQ ID NO: 435) are shown. [Figure 34-1] This shows the SASTG capsid DNA sequence (SEQ ID NO: 436). [Figure 34-2] The SASTG capsid peptide sequence (SEQ ID NO: 437) is shown. [Figure 35]The DZF16 ZFN target site sequence (SEQ ID NO: 442), the DZF16-L6 left complete amino acid sequence (SEQ ID NO: 443), and the DZF16-R6 right complete amino acid sequence (SEQ ID NO: 444) are shown. [Figure 36] The E51C3 ZFN target site sequence (SEQ ID NO: 445), the E51C3-3L left complete amino acid sequence (SEQ ID NO: 446), and the E51C3-3R right complete amino acid sequence (SEQ ID NO: 447) are shown. [Figure 37] The following sequences are shown: DZF15 ZFN target site sequence (SEQ ID NO: 448), DZF15-L6 left complete amino acid sequence (SEQ ID NO: 449), DZF15-R6 right complete amino acid sequence (SEQ ID NO: 450), DZF15-L5 left complete amino acid sequence (SEQ ID NO: 451), and DZF15-R5 right complete amino acid sequence (SEQ ID NO: 452). [Figure 38] The E51C4 ZFN target site sequence (SEQ ID NO: 453), the E51C4-4L left complete amino acid sequence (SEQ ID NO: 454), and the E51C4-4R right complete amino acid sequence (SEQ ID NO: 455) are shown. [Figure 39] Schematic diagrams of the "single vector, complex CRISPR system," the "dual vector, complex CRISPR system," and the "single vector, single gRNA system" are shown. [Figure 40] The nucleotide sequences of SaCas9-NLS (NLS is underlined) (SEQ ID NO: 64) and SaCas9 gRNA (SEQ ID NO: 116) are shown. [Figure 41] NmCas9 (NLS1 is underlined, NLS2 is underlined and bold, and HA tags are shown in bold), NmCas9 short hairpin (Source: Thomson PNAS 2013) (Sequence ID: 118), and NmCas9 long hairpin (Source: Church Nature Biotech 2013) (Sequence ID: 119) are shown. [Figure 42]This study demonstrates the validity of sgRNA and lentiviral Cas9 expression constructs. (a) HEK293T cells were transfected with a construct encoding a unique PolIII promoter that expresses sgRNA targeting the AAVS1 locus, or a construct containing an hU6 promoter immediately followed by polythymidine ("Poly-T") for termination. The expression of the indicated promoter / sgRNA was examined 2 days after transfection using endpoint RT-PCR. -RT: No reverse transcriptase control. (b) HEK293T cells were transfected with an expression vector encoding AAVS1 zinc finger nuclease or Cas9-T2A-GFP and the indicated promoter / sgRNA expression cassette, and the level of genetic modification was determined 3 days after transfection using a surveyor assay. HEK293T cells were transduced with a lentiviral construct encoding the Cas9-T2A-GFP construct shown in (c) in the absence of sgRNA, and Cas9 expression was determined by Western blotting by examining the N-terminal FLAG epitope tag of the Cas9 protein 7 days after transduction. [Figure 43] This shows the golden gate assembly of a single lentiviral CRISPR / Cas9 expression cassette. [Figure 44] This demonstrates single lentiviral delivery of a combined CRISPR / Cas9 system. (a) Four sgRNAs targeting distinct genomic loci were cloned in a lentiviral vector expressing active Cas9 nuclease. (b) HEK293 and primary human dermal fibroblasts were transduced with lentiviruses expressing the indicated sgRNAs, and cleavage events were assayed using a surveyor assay. HEK293 cells were assayed 7 days after transduction. Human fibroblasts were assayed 10 days after transduction. [Figure 45]This study demonstrates transient gene activation in HEK293T cells that stably express dCas9-VP64. HEK293T cells were transduced with a lentivirus to stably express dCas9-VP64, followed by transfection with a plasmid expressing the indicated sgRNA combination. Adjustable endogenous gene activation of the endogenous IL1RN(a) and HBG1(b) loci was achieved 3 days after transfection by altering the number of sgRNAs delivered. Peak levels of endogenous IL1RN(c) and HBG1(d) were observed 3–6 days post-transfection, and activation levels returned to background levels between 15 and 20 days. Importantly, the cell line could be reactivated 20 days after a second transfection, although the levels were lower than previously observed. [Figure 46] This study demonstrates stable gene activation in HEK293T using a single lentivirus complex dCas9-VP64 vector. HEK293T was transduced with a lentivirus to stably express dCas9-VP64 and the indicated gRNA combination. Adjustable endogenous gene activation of the endogenous IL1RN(a) and HBG1(b) loci was achieved 7 days after transduction by altering the number of sgRNAs delivered. Peak levels of endogenous IL1RN(c) and HBG1(d) were observed 6 days after transduction, and these activation levels persisted until day 21. [Figure 47] This shows the IL1RN mRNA expression level. [Figure 48] This diagram illustrates the direct conversion of fibroblasts into neurons through ectopic expression of BAM neuronal transcription factors. [Figure 49] (A) A schematic diagram of the dCas9-VP64 construct is shown. dCas9-VP64 is a catalytically inactive form of the Cas9 protein fused to a tetramer of the VP16 transcriptional activation domain. (B) A schematic diagram showing the RNA-guided dCas9-VP64 replenishment mechanism to the genomic target. (C) A schematic diagram of an experimental protocol for generating iN by a CRISPR / Cas9 transcription factor. [Figure 50]Endogenous ASCL1 expression on day 3 is shown in MEFs transfected with dCas9-VP64 and further transfected with gRNAs targeting the ASCL1 promoter, ASCL1 cDNA, or luciferase, with total ASCL1 protein determined by (A) qRT-PCR or (B) immunofluorescence. An asterisk (*) indicates a significant (p<0.05) increase in ASCL1 expression due to co-delivery of eight gRNAs compared to four gRNAs. Ectopic expression of ASCL1 produced more protein than the protein induced by dCas9-VP64 and eight gRNAs targeting the ASCL1 promoter, but did not activate the endogenous locus by day 3 in culture. [Figure 51] (B) Shows TUJ1 and MAP2-positive cells induced by ectopic BAM factors or gRNAs targeting dCas9-VP64 and BRN2 and ASCL1 promoters, and cells with neuronal morphology expressing the hSyn-RFP reporter on day 11 in N3 medium. [Figure 52-1] (A) Cells with neuronal morphology that are positive for the GCaMP5 calcium indicator in the presence (bottom) or absence (top) of KCl in culture medium. Figure 52 shows the activation of downstream targets of Ascl1 and Brn2 (i.e., master regulatory genes) in iCas9-VP64 treated mouse embryonic fibroblasts when fibroblasts are converted to neurons using the dCas9-VP64 transcription factor. Mouse embryonic fibroblasts (MEFs) were transfected with a control GFP expression plasmid or an iCas9-VP64 expression plasmid and a combination of eight gRNA expression plasmids targeting Asc11 and BRN2. The dCas9 transcription factor was delivered via virus. After 10 days in neuronal induction medium, cells were stained for Tuj1 (an early marker of neuronal differentiation) and MAP2 (a differentiation marker of more mature neurons). Conversion to neurons was efficient. [Figure 52-2](B) Records of normalized fluorescence intensity over time, showing cell depolarization in response to KCl addition. Figure 52 shows activation of downstream targets of Ascl1 and Brn2 (i.e., master regulatory genes) in iCas9-VP64 treated mouse embryonic fibroblasts when fibroblasts were converted to neurons using the dCas9-VP64 transcription factor. Mouse embryonic fibroblasts (MEFs) were transfected with either a control GFP expression plasmid or an iCas9-VP64 expression plasmid and a combination of eight gRNA expression plasmids targeting Asc11 and BRN2. The dCas9 transcription factor was delivered via virus. After 10 days in neuronal induction medium, cells were stained for Tuj1 (an early marker of neuronal differentiation) and MAP2 (a differentiation marker of more mature neurons). Conversion to neurons was efficient. [Figure 53] This paper presents a CRISPR / Cas9 platform for the regulatory control of mammalian genes. A. Cas9-based effectors bind to genomic sequences in the presence of a chimeric gRNA molecule consisting of a constant region that forms a complex with Cas9 (preceded by an interchangeable 20 bp protospacer that confers target site specificity). B. Cas9-based synthetic transcription factors repress the transcription of target genes by interfering with RNA polymerase activity or by binding within promoters to block the binding site of endogenous transcription factors. Targeting regulatory elements (e.g., enhancers) may also block the expression of numerous distal genes. [Figure 54] This paper demonstrates targeting of the HS2 enhancer using CRISPR / dCas9-KRAB. The HS2 region is a potent enhancer capable of distally regulating the expression of globin genes downstream of 10kb. A set of single gRNAs was designed to target sites along the enhancer region. [Figure 55-1] We demonstrate that a single gRNA targeting the HS2 enhancer results in potent transcriptional repression of globin genes. A. dCas9 and the dCas9-KRAB repressor are delivered via lentiviral vectors. Single gRNAs were transiently transfected for screening. [Figure 55-2] When assayed by quantitative RT-PCR three days after transfection, K562 cells expressing dCas9-KRAB achieved up to 80% suppression of the γ-globin gene (B), ε-globin gene (C), and β-globin gene (D) compared to control cells that did not receive gRNA treatment. [Figure 55-3] When assayed by quantitative RT-PCR three days after transfection, K562 cells expressing dCas9-KRAB achieved up to 80% suppression of the γ-globin gene (B), ε-globin gene (C), and β-globin gene (D) compared to control cells that did not receive gRNA treatment. [Figure 55-4] When assayed by quantitative RT-PCR three days after transfection, K562 cells expressing dCas9-KRAB achieved up to 80% suppression of the γ-globin gene (B), ε-globin gene (C), and β-globin gene (D) compared to control cells that did not receive gRNA treatment. [Figure 55-5] Protein expression in cells expressing D.dCas9 or dCas9-KRAB and treated with Cr4 or Cr8 shows a mild suppression of γ-globin expression on day 3 compared to β-actin control. [Figure 56A] The following shows gene expression at the globin locus after delivering various doses of gRNA plasmid to cells treated with A. lentivirus. Increasing the dose of delivered Cr4 gRNA plasmid enhances repression in dCas9-KRAB treated cells, suggesting that both the dCas9-KRAB effector and the gRNA that reaches the target play a role in achieving repression. [Figure 56B] The following shows gene expression at the globin locus in cells treated with B. dCas9 lentivirus, delivered with various doses of gRNA plasmid. Increasing the dose of delivered Cr4 gRNA plasmid enhances repression in dCas9-KRAB treated cells, suggesting that both the dCas9-KRAB effector and the gRNA reaching the target play a role in achieving repression. [Figure 56C] The following shows gene expression at the globin locus in cells treated with C. dCas9-KRAB lentivirus, delivered with varying doses of gRNA plasmid. Increasing the dose of delivered Cr4 gRNA plasmid enhances repression in dCas9-KRAB-treated cells, suggesting that both the dCas9-KRAB effector and the gRNA reaching the target play a role in achieving repression. [Figure 57-1] This study demonstrates the stable delivery of a single gRNA along with dCas9-KRAB to silence globin gene expression. A. dCas9 and the dCas9-KRAB repressor were co-expressed with a single gRNA in a lentiviral vector. When assayed by quantitative RT-PCR 7 days after transduction, K562 cells expressing dCas9-KRAB achieved up to 95% suppression of γ-globin (B), ε-globin (C), and β-globin (D) compared to control cells that did not receive lentiviral treatment. [Figure 57-2] This study demonstrates the stable delivery of a single gRNA along with dCas9-KRAB to silence globin gene expression. A. dCas9 and the dCas9-KRAB repressor were co-expressed with a single gRNA in a lentiviral vector. When assayed by quantitative RT-PCR 7 days after transduction, K562 cells expressing dCas9-KRAB achieved up to 95% suppression of γ-globin (B), ε-globin (C), and β-globin (D) compared to control cells that did not receive lentiviral treatment. [Figure 58] This demonstrates the isolation of the p300HAT "core" for histone target epigenetic modification via dCas9 fusions alone. [Figure 59] Simplified schematic diagrams of the Streptococcus pyogenes (S. pyogenes) dCas9-VP64 fusion (top) and dCas9-p300 core fusion (bottom) are shown. Protospacer adjacent motifs (PAMs) are indicated by arrows at the locus of the target gene, and synthetic guide RNA (gRNA) is indicated by hatched arrows. [Figure 60A] Figures 60A-60C present representative data from three human loci that demonstrate the effectiveness of activation using dCas9-p300 in human 293T cell cultures, compared to dCas9-VP64 and dCas9 without any fusion effector domains. [Figure 60B] Figures 60A-60C present representative data from three human loci that demonstrate the effectiveness of activation using dCas9-p300 in human 293T cell cultures, compared to dCas9-VP64 and dCas9 without any fusion effector domains. [Figure 60C] Figures 60A-60C present representative data from three human loci that demonstrate the effectiveness of activation using dCas9-p300 in human 293T cell cultures, compared to dCas9-VP64 and dCas9 without any fusion effector domains. [Figure 61A] Figures 61A-61C show the amino acid sequences of the Cas9 construct. A complete explanation of Figures 61A-61C is shown in Figure 61A. [Figure 61B] Figures 61A-61C show the amino acid sequences of the Cas9 construct. A complete explanation of Figures 61A-61C is shown in Figure 61A. [Figure 61C] Figures 61A-61C show the amino acid sequences of the Cas9 construct. A complete explanation of Figures 61A-61C is shown in Figure 61A. [Figure 62] This study demonstrates that the HAT-dCas9-p300 fusion protein cannot activate gene expression. [Figure 63] The gRNA also shows synergistic action with the dCas9-p300 core. [Figure 64]dCas9-p300 and dCas9-VP64 (different molecules) have no cumulative effect on transactivation. [Figure 65] dCas9-p300 shows non-synergistic activation of the distal regulatory region of MyoD. [Figure 66] Shows TALEN-mediated integration of mini-dystrophin into the 5'UTR of the DP427m skeletal muscle isoform of dystrophin from skeletal myoblast cell lines derived from human DMD patients with various deletions in the dystrophin gene. Cells from DMD patients were electroporated with constructs encoding TALEN pairs active in the 5'UTR and donor templates containing the mini-dystrophin gene. (a) Schematic diagram showing how mini-dystrophin is integrated into the 5'UTR. (b) Hygromycin-resistant clone cell lines were isolated and screened by PCR for successful site-specific integration into the 5'UTR using the primers shown in (a). Asterisks indicate clones selected for further analysis in (c). (c) Clonal DMD myoblasts in which the integration event was detected were differentiated for 6 days, and the expression of an HA tag fused to the C-terminus of minidystrophin was determined. [Modes for carrying out the invention]

[0018] Detailed explanation As described herein, certain methods and manipulated CRISPR / CRISPR-associated (Cas)9-dependent system compositions have been found useful for modifying or mitigating the effects of gene expression alteration, genomic manipulation, and gene mutations involved in genetic diseases. A CRISPR / Cas9-dependent system comprises a Cas9 protein and at least one guide RNA (the guide RNA provides the DNA targeting specificity of the system). In particular, this disclosure describes a Cas9 fusion protein that integrates the DNA sequence targeting function of a CRISPR / Cas9-dependent system with additional activity, thus enabling alteration of gene expression and / or epigenetic circumstances. This system may also be used to modify or mitigate the effects of genomic manipulation and gene mutations.

[0019] This disclosure also provides a CRISPR / CRISPR-associated (Cas)9-dependent system and certain compositions and methods for delivering a wide variety of gRNAs targeting one or more endogenous genes. Cotransfection of a wide variety of sgRNAs targeting a single promoter enables synergistic activation; however, cotransfection of a wide variety of plasmids results in expression levels that can vary in each cell due to differences in copy number. Furthermore, gene activation following transfection is transient due to the dilution of plasmid DNA over time. Moreover, many cell types are not readily transfected, and transient gene expression may not be sufficient to induce therapeutic effects. To address these limitations, a single lentiviral system expressing Cas9 and up to four sgRNAs from separate promoters has been developed. A platform for expressing a Cas9 or dCas9 fusion protein and up to four gRNAs from a single lentiviral vector is disclosed. This lentiviral vector expresses constitutive or inducible Cas9 or dCas9-VP64 in addition to one, two, three, or four gRNAs expressed from separate promoters. This system allows for control over both the scale and timing of CRISPR / Cas9-dependent gene regulation. Furthermore, the lentiviral platform provides potent and sustained gene expression levels that facilitate the therapeutic use of the CRISPR / Cas9 system in primary cells. Finally, this system can be used to simultaneously edit a wide variety of genes (e.g., for the simultaneous knockout of several oncogenes).

[0020] This disclosure also provides certain compositions and methods for delivering site-specific nucleases to skeletal muscle and cardiac muscle using modified adeno-associated virus (AAV) vectors. Site-specific nucleases (which are manipulable) are useful for altering gene expression, genomic manipulation, modifying or mitigating the effects of mutations in genes involved in genetic diseases, or manipulating genes involved in skeletal muscle or cardiac muscle or other conditions affecting muscle regeneration. Site-specific nucleases to be manipulated may include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and / or CRISPR / Cas9 systems for genome editing. As described herein, genes in skeletal muscle tissue have been successfully edited in vivo using this unique delivery system. The disclosed invention provides means for rewriting the human genome for therapeutic use and target model species for basic scientific use. Gene editing is highly dependent on the cell cycle and complex DNA repair pathways (which vary from tissue to tissue). Skeletal muscle is a highly complex environment, consisting of large muscle fibers with over 100 nuclei per cell. Gene therapy and biologics have generally been limited for decades due to hurdles in in vivo delivery. These challenges include carrier stability in vivo, targeting to the desired tissue, obtaining gene products with sufficient gene expression and activity, and avoiding toxicity that may outweigh the activity (which is common with gene editing tools). Other delivery vehicles (e.g., direct injection of plasmid DNA) function for gene expression in skeletal and cardiac muscle in other respects, but are not sufficiently effective for achieving detectable levels of gene editing with respect to these site-specific nucleases.

[0021] While many genes are unstable and therefore undeliverable in AAV vectors, these site-specific nucleases are remarkably stable in AAV vectors. When these site-specific nucleases are delivered and expressed, they maintain their activity in skeletal muscle tissue. The protein stability and activity of site-specific nucleases are highly tissue-type and cell-type-dependent. These highly active and stable nucleases can modify gene sequences in the complex environment of skeletal muscle. This invention provides a method for delivering the active form of this class of therapeutic agents to skeletal muscle or cardiac muscle. The method is effective, efficient, and promotes successful genome modification. This disclosure also provides a certain type of epigenetic effector molecular fusion, the dCas9-p300 fusion protein. Compared to the dCas9-VP64 fusion, this protein provides a potent and potentially more broadly applicable tool for synthetic transcriptional regulation. Target genes were activated substantially much more strongly at all loci tested than with the dCas9-VP64 fusion protein. In addition, p300 has intrinsic endogenous activity in enhancers within the human genome. The dCas9-p300 fusion protein may be able to activate endogenous target gene promoters and enhancer regions. The dCas9-p300 fusion protein can be used to activate gene expression in human tissue culture cell lines. This fusion protein can be used to accurately and predictively control the epigenetic state of target gene loci within human cells, leading to applications in differentiation control, cellular regulation, and innovative and potentially therapeutic methods. Conventional techniques have limitations in the intensity of activation and the scope and duration of epigenetic regulation (i.e., these limitations can be overcome through the use of this novel fusion protein). The headings used in this section and the entire disclosures in this section are for structural purposes only and are not intended to be restrictive.

[0022] 1.Definition Unless otherwise specified, all technical and academic terms used herein have the same meaning as those generally understood by those skilled in the art. Any inconsistencies shall be governed by this document (including definitions). Preferred methods and materials are described below, but similar or equivalent methods and materials may also be used in carrying out or testing the present invention. All publications, patent applications, patents and other references cited herein are incorporated in their entirety by reference. The materials, methods and examples disclosed herein are illustrative and not intended to be limiting. The terms “comprise,” “include,” “having,” “has,” “can,” and “contain,” as used herein, and their variations thereof, are terminally open transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The singular forms “a,” “and,” and “the” include plural corresponding words unless the context clearly indicates otherwise. Whether expressly described or not, this disclosure also intends other embodiments that “comprising,” “consisting of,” and “consisting essentially of” the embodiments or components presented herein. In the numerical ranges described herein, each intervening number is intended to be clearly defined with the same degree of precision. For example, in the range 6-9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are clearly intended.

[0023] "Adeno-associated virus" or "AAV" is used interchangeably herein and refers to a small virus belonging to the genus Dependovirus of the family Parvoviridae that infects humans and several other primate species. AAV has not been known to cause disease, and as a result, this virus produces a very mild immune response. As used herein, the term "binding region" refers to a region within the nuclease target region that the nuclease recognizes and binds to. As used interchangeably in this specification, “cardiac muscle” refers to cardiomyocytes, a type of involuntary striated muscle found in the heart wall and histological foundation. Cardiac muscle is composed of cardiomyocytes (myocardiocytes). Cardiomyocytes exhibit striations similar to those of skeletal muscle cells, but unlike skeletal muscle cells which have multiple nuclei, they contain only one proper nucleus. As used herein, “symptoms of the heart muscle” refers to symptoms related to the myocardium, such as cardiomyopathy, heart failure, arrhythmias, and inflammatory heart disease. As used herein, “coding sequence” or “coding nucleic acid” means a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence that codes for a protein. The coding sequence may further include start and end signals, which are operably linked to regulatory elements (promoters and polyadenylation signals) that can direct expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized. As used herein, “complementary” or “complementarity” means nucleic acids that can form Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairings between nucleotides or nucleotide analogues of a nucleic acid molecule. “Complementarity” refers to a property shared between two nucleic acid sequences such that, when the two nucleic acid sequences are placed antiparallel to each other, the nucleotide bases at each position are complementary.

[0024] As used herein, “modification,” “genome editing,” and “repair” refer to altering a mutant gene that codes for a truncated protein or does not code for any protein at all, so that it can express a full-length functional or partially full-length functional protein. Modification or repair of a mutant gene may include replacing the region of the mutated gene or replacing the entire mutant gene with a non-mutant copy using a repair mechanism (e.g., homology-directed repair (HDR)). Modification or repair of a mutant gene may also include repairing frameshift mutations that result in immature stop codons, abnormal splice acceptor sites, or abnormal splice donor sites by generating a double-strand break in the gene followed by repair using a non-homologous end junction (NHEJ). The NHEJ can add or delete at least one base pair during repair, which can repair the appropriate reading frame and remove the immature stop codon. Modification or repair of a mutant gene may also include disruption of abnormal splice acceptor sites or splice donor sites. Correction or restoration of mutated genes may also include deletion of non-essential gene segments by simultaneously acting two nucleases on the same DNA strand to remove DNA between the two nuclease target sites, repairing the resulting DNA break with an NHEJ, and restoring the proper reading frame. As used interchangeably herein, “donor DNA,” “donor template,” and “repair template” refer to a double-stranded DNA fragment or molecule containing at least a portion of the gene in question. Donor DNA may encode a fully functional protein or a partially functional protein.

[0025] In this specification, “Duchenne muscular dystrophy” or “DMD” are interchangeable terms referring to a recessive, lethal X-linked anomaly that leads to muscle degeneration and ultimately death. DMD is a well-known hereditary monogenic disorder that occurs in 1 in 3,500 men. DMD is the result of hereditary or accidental mutations that produce nonsense or frameshift mutations in the dystrophin gene. The majority of dystrophin mutations that cause DMD are exon deletions, which disrupt the leading frame of the dystrophin gene and cause premature translation termination. Typically, DMD patients lose the ability to physically support themselves in childhood, and their fragility progresses during adolescence, leading to death in their twenties. As used herein, “dystrophin” refers to a rod-shaped cytoplasmic protein that is part of a protein complex that spans the cell membrane and attaches the cytoskeleton of muscle fibers to the surrounding extracellular matrix. Dystrophin provides structural stability to the dystroglycan complex of the cell membrane, which is important for regulating the preservation and function of muscle cells. The dystrophin gene or “DMD gene,” as used interchangeably herein, occupies 2.2 megabases at Xp21. The primary transcript is approximately 2,400 kb, and the mature mRNA is approximately 14 kb. 79 exons encode a protein with more than 3,500 amino acids.

[0026] As used herein, “exon 51” refers to exon 51 of the dystrophin gene. Exon 51 is often adjacent to frame disruption deletions in DMD patients and has been a target for clinical trials of exon skipping with oligonucleotides. A recent clinical trial of the exon 51 skipping compound eteplirsen reported a significant functional benefit over 48 weeks, with an average of 47% of fibers being dystrophin-positive compared to the baseline. Ideally, mutations in exon 51 should be permanently corrected by genome editing based on NHEJ. As used interchangeably herein, “frameshift” or “frameshift mutation” refers to a type of genetic mutation in which the addition or deletion of one or more nucleotides results in a shift in the reading frame of a codon in mRNA. A shift in the reading frame can lead to changes in the amino acid sequence during protein translation, such as missense mutations or immature stop codons. As used herein, “functional” and “fully functional” refer to proteins that possess biological activity. A “functional gene” refers to a gene that is transcribed into mRNA, and the mRNA is translated into a functional protein.

[0027] As used herein, “fusion protein” refers to a chimeric protein created through the fusion of two or more genes that originally encode separate proteins. Translation of the fusion gene produces a single polypeptide with functional properties derived from each of the original proteins. As used herein, “genetic construct” refers to a DNA or RNA molecule containing a nucleotide sequence that codes for a protein. The coding sequence includes start or end signals operably ligated to a regulatory element (the regulatory element includes a promoter and a polyadenylation signal that can direct expression in the cells of an individual to which the nucleic acid molecule is administered). As used herein, “expressible form” refers to a gene construct containing the necessary regulatory elements operably ligated to the coding sequence such that the coding sequence is expressed when present in the cells of an individual. As used herein, “genetic disorder” refers to a disorder, particularly a condition present since birth, caused by one or more abnormalities in the genome, either partially or completely, directly or indirectly. Such abnormalities may affect the coding sequence or regulatory sequence of a gene. Genetic disorders may include, but are not limited to, DMD, hemophilia, cystic fibrosis, Huntington’s disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson’s disease, congenital hepatic porphyria, hereditary disorders of hepatic metabolism, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom’s syndrome, retinoblastoma, and Tay-Sachs disease.

[0028] As used interchangeably herein, “homologous recombination” or “HDR” refers to an intracellular mechanism that repairs double-stranded DNA damage when homologous DNA fragments are present in the nucleus (mostly in the G2 and S phases of the cell cycle). HDR induces recombination using a donor DNA template, and further HDR can be used to create genome-specific sequence changes (including the addition of entire genes by targeted induction). If the donor template is supplied with a site-specific nuclease (e.g., a CRISPR / Cas9-dependent system), the cellular mechanism will repair the break by homologous recombination (which is enhanced by several orders of magnitude in the presence of DNA breaks). When homologous DNA fragments are not present, non-homologous end junctions may occur instead. As used interchangeably in this specification, “genome editing” refers to the modification of a gene. Genome editing may include the correction or restoration of a mutated gene. Genome editing may include the knockout of a gene (e.g., a mutated gene or a normal gene). Genome editing can be used to treat diseases or enhance muscle repair by modifying the gene in question. As used herein with respect to two or more nucleic acid or polypeptide sequences, “identical” or “sameness” means that the sequences have the same residues at a specified percentage across a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing them across the specified 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 specified 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 results in one or more misaligned ends and the specified comparison region contains only single sequences, the residues of the single sequences are included in the denominator but not in 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 a computer sequencing algorithm (e.g., BLAST or BLAST 2.0).

[0029] In this specification, the terms “mutated gene” or “mutated gene” are interchangeable and refer to a gene that has undergone a detectable mutation. A mutated gene has undergone a change (e.g., loss, acquisition, or exchange of genetic material) (such changes affect the normal transmission and expression of the gene). In this specification, the term “disrupted gene” refers to a mutated gene having a mutation that produces an immature stop codon. The disrupted gene product is truncated compared to the full-length, non-disrupted gene product. As used herein, the “non-homologous end joining (NHEJ) pathway” refers to a pathway that repairs DNA double-strand breaks by direct joining of the broken ends without requiring a homologous template. Template-independent rejoining of DNA ends by NHEJ is a probabilistic, error-prone repair process that introduces random microinsertions and microdeletions (indels) at the DNA break site. This method can be used to intentionally disrupt, delete, or alter the reading frame of a targeted gene sequence. NHEJ typically utilizes short homologous DNA sequences called micro-homologies to induce repair. These micro-homologies are often located in single-strand overhangs at the ends of double-strand breaks. When the overhang is preferably compatible, NHEJ usually repairs the break accurately, but inaccurate repairs resulting in nucleotide loss can also occur (although such inaccurate repairs are far more common when the overhang is not compatible).

[0030] As used herein, a “normal gene” refers to a gene that has not undergone any alteration (e.g., loss, acquisition, or exchange of genetic material). Normal genes undergo normal gene transmission and gene expression. As used herein, “nuclease-mediated NHEJ” refers to an NHEJ initiated after a nuclease (e.g., Cas9) has cleaved double-stranded DNA. As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides linked together by a covalent bond. A single-stranded description also defines the sequence of a complementary strand. Therefore, a nucleic acid also encompasses the complementary strand of the described single-stranded nucleic acid. Many nucleic acid variants can be used for the same purpose as a given nucleic acid. Therefore, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single-stranded description provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Therefore, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions. Nucleic acids may be single-stranded or double-stranded, and may contain both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic DNA and cDNA), RNA, or hybrids, where the above may include combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0031] As used herein, “operably linked” means that the expression of a gene is under the control of a promoter to which the gene is spatially linked. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene is approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is well known in the art, this variation in distance can be tolerated without impairing the function of the promoter. As used herein, "partially functional" refers to a protein encoded by a mutated gene that has lower biological activity than a functional protein but higher biological activity than a non-functional protein. In this specification, the terms “immature stop codon” or “out-frame stop codon” are interchangeable and refer to nonsense mutations in a DNA sequence that produce a stop codon at a location not typically found in wild-type genes. Immature stop codons produce truncated or shortened proteins compared to the full-length version of the protein.

[0032] As used herein, “promoter” means a synthetic or naturally derived molecule that can confer, activate, or enhance the expression of a nucleic acid in a cell. A promoter comprises one or more specific transcriptional regulatory sequences that can further enhance expression and / or alter its spatial and / or transient expression. A promoter may also comprise distal enhancer or repressor elements, which may be located several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate the expression of gene components constitutively, or discriminatively with respect to the cell, tissue, or organ in which the expression occurs, or to the developmental stage in which the expression occurs, or in response to external stimuli (e.g., physiological stress, pathogens, metal ions, or inducing agents). Typical examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

[0033] As used interchangeably herein, “repeat variable diresidue” or “RVD” refers to a pair of adjacent amino acid residues within the DNA recognition motif of the TALE DNA-binding domain (also known as an “RVD module,” which comprises 33-35 amino acids). The RVD determines the nucleotide specificity of the RVD module. The RVD modules together form an RVD sequence. As used herein, “RVD sequence length” refers to the number of RVD modules that correspond to the length of the nucleotide sequence within the TALEN target region (i.e., binding region) recognized by the TALEN. As used herein, “position-specific nuclease” refers to an enzyme that can specifically recognize and cleave a DNA sequence. Position-specific nucleases can be manipulated. Examples of manipulated position-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR / Cas9-dependent systems. As used herein, “skeletal muscle” refers to a type of striated muscle, which is under the control of the somatic nervous system and is attached to bone by bundles of collagen fibers known as tendons. Skeletal muscle is composed of individual components known as myocytes or “muscle cells” (sometimes colloquially called “muscle fibers”). Myocytes are formed by the fusion of developing myoblasts (a type of embryonic primordial cell that gives rise to muscle cells) during a process known as myogenesis. These long, tubular, multinucleated cells are also called muscle fibers.

[0034] As used herein, “skeletal muscle symptoms” refers to symptoms related to skeletal muscle, such as muscular dystrophy, aging, muscle degeneration, wound healing, muscle weakness, or atrophy. As used interchangeably in this specification, “spacer” and “spacer region” refer to a region within a TALEN or ZFN target region, which is located between, but not part of, the binding regions for two TALENs or ZFNs. As used interchangeably herein, “subject animal” and “patient” refer to any vertebrate, which includes, but is not limited to, the following: mammals, e.g., dairy cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice; non-human primates, e.g., monkeys (e.g., crab-eating macaques or rhesus macaques, chimpanzees, etc.) and humans. In some embodiments, the subject animal may be human or non-human. The subject animal or patient may receive other forms of treatment. As used herein, “target gene” refers to any nucleotide that codes for a known or hypothetical gene product. The target gene may also be a mutated gene included in a genetic disorder. As used herein, the term “target region” refers to a region of a target gene that is designed to be cleaved by a site-specific nuclease.

[0035] As used herein, “transcriptional activator-like effector” or “TALE” refers to a protein structure that recognizes and binds to a specific DNA sequence. A “TALE DNA-binding domain” refers to a DNA-binding domain (also known as an RVD module, each recognizing a single base pair of DNA) containing a tandem sequence of 33-35 amino acid repeats. The RVDs can be arranged in any order to assemble a sequence that recognizes a specified sequence. The binding specificity of the TALE DNA-binding domain is determined by a 20-amino acid single-tear repeat followed by a series of RVDs. The TALE DNA-binding domain can have 12 to 27 RVD modules, each containing an RVD that recognizes a single base pair of DNA. Specific RVDs recognizing each of the four possible DNA nucleotides (A, T, C, and G) have been identified. Because the TALE DNA-binding domain is modular, repeats recognizing four different DNA nucleotides can be linked together to recognize any individual DNA sequence. These target-induced DNA-binding domains can then be combined with catalytic domains to create functional enzymes (including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases).

[0036] As used interchangeably herein, “transcriptional activator-like effector nuclease” or “TALEN” refers to an engineered fusion protein having a catalytic domain of a nuclease (e.g., endonuclease FokI) and a TALE DNA-binding domain designed to target a custom DNA sequence. “TALEN monomer” refers to an engineered fusion protein having a catalytic nuclease domain and a designed TALE DNA-binding domain. Two TALEN monomers can be designed to target and cleave a single TALEN target region. As used herein, “transgene” refers to a gene or genetic material containing a gene sequence that is isolated from one organism and introduced into another organism. This non-natural DNA segment may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the genetic code of the transgenic organism. The introduction of a transgene has the potential to alter the phenotype of the organism. With respect to nucleic acids, the term “variant” as used herein means: (i) a portion or fragment of the nucleotide sequence mentioned; (ii) a complement of the nucleotide sequence mentioned or a portion thereof; (iii) a nucleic acid substantially identical to the nucleic acid mentioned or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions with the nucleic acid mentioned, its complement, or a sequence substantially identical thereto.

[0037] A “variant” of a peptide or polypeptide is one in which the amino acid sequence differs due to the insertion, deletion, or conservation substitution of amino acids, but retains at least one biological activity. A variant also means a protein having a substantially identical amino acid sequence to a mentioned protein that has an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids (i.e., replacement of an amino acid with an amino acid having similar properties (e.g., hydrophobicity, degree and distribution of charged regions)) are typically recognized in the art as involving minute changes. These minute changes can be identified by considering the hydroxyl index of amino acids, as understood in the art (Kyte et al., J. Mol. Biol. 157:105-132, 1982). The hydroxyl index of amino acids is determined by considering their hydrophobicity and charge. It is well known in the art that protein function can still be maintained even when substituting amino acids with similar hydroxyl indices. For certain characteristics, amino acids with hydroxyl indices of ±2 can be substituted. The hydrophilicity of amino acids can also be used to indicate substitutions that result in proteins that retain biological function. In peptide relationships, considering the hydrophilicity of amino acids allows for the calculation of the maximum local mean hydrophilicity of the peptide. Substitutions can be performed with amino acids that have hydrophilicity within ±2 of each other. Both the hydrophobic index and hydrophilicity value of amino acids are influenced by the individual side chains of those amino acids. Consistent with such observations, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids and, in particular, their side chains, as indicated by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0038] As used herein, “vector” means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be either a DNA vector or an RNA vector. A vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. For example, a vector may encode an iCas9-VP64 fusion protein containing the amino acid sequence of SEQ ID NO: 1, or at least one gRNA nucleotide sequence from among SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625. Alternatively, a vector may encode Cas9 and at least one gRNA nucleotide sequence from among SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625.

[0039] As used herein, “zinc finger” refers to a protein structure that recognizes and binds to a DNA sequence. The zinc finger domain is the most common DNA-binding motif in the human proteome. A single zinc finger contains approximately 30 amino acids, and typically the domain functions by binding to three consecutive base pairs of DNA via the interaction of just one amino acid side chain per base pair. As used interchangeably herein, “zinc finger nuclease” or “ZFN” refers to a chimeric protein molecule comprising at least one nuclease or portion of a nuclease that can cleave DNA when fully assembled, and at least one zinc finger DNA-binding domain effectively linked to it. Unless otherwise specified, the academic and technical terms used in connection with this disclosure have the meanings that are ordinarily understood by those skilled in the art. For example, the terminology and techniques used herein with respect to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well known and commonly used in the art. The meaning and scope of terms should be clear, but where there is any implicit ambiguity, the definitions provided herein shall take precedence over any dictionaries or non-essential definitions. Furthermore, unless the context specifically requires otherwise, singular terms include plural terms, and plural terms include singular terms.

[0040] 2. Compositions for genome editing The present invention aims to provide compositions for genome editing, genome modification, or alteration of gene expression of a target gene. The composition may contain a viral vector and a fusion protein (e.g., a site-specific nuclease or a CRISPR / Cas9 system) together with at least one gRNA. a. Compositions for genome editing in muscle The present invention aims to provide compositions for genome editing target genes in the skeletal muscle or cardiac muscle of a target animal. The compositions comprise a modified AAV vector and a nucleotide sequence encoding a site-specific nuclease. The compositions deliver the active form of the site-specific nuclease to the skeletal muscle or cardiac muscle. The compositions may further contain donor DNA or transgenes. These compositions can be used in genome editing, genomic manipulation, and modification or mitigation of the effects of mutations in genes involved in genetic diseases and / or symptoms of skeletal muscle or cardiac muscle. The target gene may be involved in cell differentiation or any other process in which gene activation, repression, or disruption is desired, or the target gene may have mutations (e.g., deletions, non-rem shift mutations, or nonsense mutations). If the target gene has mutations that result in immature stop codons, abnormal splice acceptor sites, or abnormal splice donor sites, a site-specific nuclease can be designed to recognize and bind to the nucleotide sequence upstream or downstream of the immature stop codon, abnormal splice acceptor site, or abnormal splice donor site. Site-specific nucleases can also be used to disrupt normal gene splicing by introducing skipping of immature stop codons targeting splice acceptors and donors, or to restore a disrupted reading frame. Site-specific nucleases may or may not mediate off-target changes to protein-coding regions of the genome.

[0041] 3. CRISPR system In this specification, the terms “assembled, equidistant short palindromic repeats” and “CRISPR” are interchangeable and refer to loci containing a wide variety of short direct repeats found in the genomes of approximately 40% of sequenced bacteria and approximately 90% of sequenced archaea. The CRISPR system is a microbial nuclease system required for defense against invading phages and plasmids that provide acquired immunity. CRISPR loci in microbial hosts contain combinations of CRISPR-associated (Cas) genes as well as non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage. Short segments of foreign genes (referred to as spacers) are incorporated into the genome between CRISPR repeats and act as a 'memory' of past exposure. Cas9 forms a complex with the 3' end of sgRNA, and this protein-RNA pair recognizes its genomic target by complementary base pairing between the 5' end of the sgRNA sequence and a predefined 20 bp DNA sequence (known as a protospacer). This complex is induced at homologous loci in pathogen DNA via a region encoded within the crRNA (i.e., a protospacer) and a protospacer-adjacent motif (PAM) in the pathogen genome. The non-coding CRISPR sequence is transcribed and cleaved into short crRNAs containing individual spacer sequences within direct repeats (which induce the Cas nuclease at the target site (protospacer)). By simply replacing a 20 bp recognition sequence in the expressed sgRNA, the Cas9 nuclease can be induced to a new genomic target. Using CRISPR spacers, exogenous genetic elements can be recognized and silenced in a manner similar to RNAi in eukaryotes.

[0042] Three types of CRISPR systems (Type I, II, and III effector systems) are known. The Type II effector system performs targeted DNA double-strand breaks in four sequential steps using a single effector enzyme, Cas9, for dsDNA cleavage. Compared to the Type I and Type III effector systems (which require a wide variety of distinct effectors working as a complex), the Type II effector system can also function in other environments (e.g., eukaryotic cells). The Type II effector system consists of a long pre-crRNA (transcribed from a spacer-containing CRISPR locus), the Cas9 protein, and tracrRNA (required for pre-crRNA processing). The tracrRNA hybridizes with a repeat region that separates the spacer of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. Following this cleavage, a second cleavage event by Cas9 occurs within each spacer, producing tracrRNA and mature crRNA that remains associated with Cas9, forming the Cas9:crRNA-tracrRNA complex.

[0043] The Cas9:crRNA-tracrRNA complex unwinds the DNA double helix and searches for and cleaves sequences that match the crRNA. Target recognition occurs when complementarity is detected between the “protospacer” sequence in the target DNA and the residual spacer sequence in the crRNA. Cas9 mediates the cleavage of the target DNA if the correct protospacer adjacency motif (PAM) is also present at the 3' end of the protospacer. To target a protospacer, the sequence must be immediately followed by the protospacer adjacency motif (PAM) (a short sequence recognized by the Cas9 nuclease necessary for DNA cleavage). Different type II systems require different PAMs. The Streptococcus pyogenes CRISPR system may have a PAM sequence for this Cas9 (SpCas9) as 5'-NRG-3' (where R is A or G, characterizing the specificity of this system in human cells). The unique capability of the CRISPR / Cas9 system lies in its direct ability to simultaneously target a wide variety of distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, while the Streptococcus pyogenes type II system naturally prefers the use of the “NGG” sequence (where “N” can be any nucleotide), the manipulated system also tolerates other PAM sequences (e.g., “NAG”) (Hsu et al., Nature Biotechnology (2013) doi:10.1038 / nbt.2647). Similarly, Cas9 derived from Neisseria meningitidis (NmCas9) typically has the natural PAM NNNNGATT, but is active against a variety of RAMs (including the highly degenerate NNNNGNNN PAM) (Esvelt et al. Nature Methods (2013) doi:10.1038 / nmeth.2681).

[0044] 4. CRISPR / Cas9-based systems The operational form of the Streptococcus pyogenes type II effector system has been shown to perform genome editing functions in human cells. In this system, the Cas9 protein is guided to a target site in the genome by a synthetically reconstituted “guide RNA” (“gRNA,” also used interchangeably herein as chimeric single guide RNA “sgRNA”) (the guide RNA is generally a crRNA-tracrRNA fusion that eliminates the need for RNase III and crRNA processing (see Figure 53A)). This specification provides CRISPR / Cas-dependent operational systems used for genome editing and the treatment of genetic diseases. The CRISPR / Cas9-dependent operational system can be designed to target any gene. Such genes include those necessary for genetic diseases, aging, tissue regeneration, or wound healing. The CRISPR / Cas9-dependent system may include the Cas9 protein or a Cas9 fusion protein and at least one gRNA. The Cas9 fusion protein may include domains with different activities (for example, activities endogenous to Cas9), such as a transactivation domain. The target gene may be required for cell differentiation or any other process in which gene activation is desired, or it may have mutations (e.g., frameshift mutations or nonsense mutations). If the target gene has mutations that result in an immature stop codon, an abnormal splice activator site, or an abnormal splice donor site, the CRISPR / Cas9-dependent system can be designed to recognize and bind to the nucleotide sequence upstream or downstream of the immature stop codon, abnormal splice activator site, or abnormal splice donor site. The CRISPR / Cas9-dependent system can also be used to target splice acceptors and donors to introduce skipping of the immature stop codon, disrupt normal gene splicing, or restore a disrupted reading frame. The CRISPR / Cas9-dependent system may or may not mediate off-target changes to the protein-coding region of the genome.

[0045] a.Cas9 The CRISPR / Cas9-dependent system may include the Cas9 protein or a Cas9 fusion protein. The Cas9 protein is an endonuclease that cleaves nucleic acids, is encoded by the CRISPR locus, and is required by the type II CRISPR system. The Cas9 protein may originate from any bacterium or archaeal species (e.g., Streptococcus pyogenes). The Cas9 protein can be mutated to inactivate its nuclease activity. Inactivated Cas9 protein from Streptococcus pyogenes (iCas9, also referred to as “dCas9”), lacking endonuclease activity, has recently been used via gRNA to target genes in bacteria, yeast, and human cells, silencing gene expression by bypassing steric interference. As used herein, “iCas9” and “dCas9” both refer to Cas9 proteins with the amino acid substitutions D10A and H840A, in which their nuclease activity is inactivated. For example, the CRISPR / Cas9-dependent system may include the Cas9 of SEQ ID NO: 459 or 461.

[0046] b.Cas9 fusion protein The CRISPR / Cas9-dependent system may include a fusion protein. The fusion protein may contain two heterologous polypeptide domains, the first polypeptide domain containing a Cas protein, and the second polypeptide domain having activity (e.g., transcriptional activating activity, transcriptional repressing activity, transcriptional deactivating activity, histone modifying activity, nuclease activity, nucleic acid binding activity, methylase activity, or demethylase activity). The fusion protein may contain a Cas9 protein or the above-described mutant Cas9 protein, the Cas9 protein being fused to a second polypeptide domain having, for example, transcriptional activating activity, transcriptional repressing activity, transcriptional deactivating activity, histone modifying activity, nuclease activity, nucleic acid binding activity, methylase activity, or demethylase activity. (1) Transcriptional activation activity The second polypeptide domain can have transcriptional activation activity, i.e., it can be a transactivation domain. For example, gene expression of endogenous mammalian genes (e.g., human genes) can be achieved by inducing a fusion protein of iCas9 and the transactivation domain, along with gRNA, to a mammalian promoter target. The transactivation domain can contain one VP16 protein, a variety of VP16 proteins (e.g., VP48 domain or VP64 domain), or the p65 domain with NF kappa B transcriptional activator activity. For example, the fusion protein could be iCas9-VP64. (2) Transcriptional repression activity The second polypeptide domain may possess transcriptional repressive activity. The second polypeptide domain may also have Kruppel-associated box activity (e.g., KRAB domain), ERF repressor domain activity, Mxi1 repressor domain activity, SID4X repressor domain activity, Mad-SID repressor domain activity, or TATA box-binding protein activity. For example, the fusion protein may be dCas9-KRAB.

[0047] (3) Transcriptional deactivation factor activity The second polypeptide domain may have transcription de-transcriptional activity. The second polypeptide domain may have eukaryotic cell de-transcriptional activity 1 (ERF1) or eukaryotic cell de-transcriptional activity 3 (ERF3). (4) Histone modification activity The second polypeptide domain may have histone modification activity. The second polypeptide domain may have histone deacetylase, histone acetyltransferase, histone demethylase, or histone methyltransferase activity. The histone acetyltransferase may be p300 or CREB-binding protein (CBP), or a fragment thereof. For example, the fusion protein may be dCas9-p300. (5) Nuclease activity The second polypeptide domain may have nuclease activity different from that of the Cas9 protein. A nuclease, or a protein with nuclease activity, is an enzyme that can cleave phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases can usually be further divided into endonucleases and exonucleases, although some enzymes may fall into both categories. Well-known nucleases are deoxyribonucleases and ribonucleases.

[0048] (6) Nucleic acid binding activity The second polypeptide domain may have nucleic acid-binding activity or a nucleic acid-binding protein. The DNA-binding domain (DBD) is an independently folding protein domain that includes at least one motif that recognizes double-stranded or single-stranded DNA. The DBD may recognize a specific DNA sequence (recognition sequence) or have a general affinity for DNA. The nucleic acid-binding region is selected from the group consisting of: helix-turn-helix region, leucine zipper region, winged helix region, winged helix-turn-helix region, helix-loop-helix region, immunoglobulin fold, B3 domain, zinc finger, HMG-box, Wor3 domain, and TAL effector DNA-binding domain. (7) Methylase activity The second polypeptide domain may have methylase activity, which is involved in the transfer of methyl groups to DNA, RNA, proteins, small molecules, cytosine, or adenine. The second polypeptide domain may contain DNA methyltransferase. (8) Demethylase activity The second polypeptide domain may possess demethylase activity. The second polypeptide domain may include enzymes that remove methyl (CH3-) groups from nucleic acids, proteins (especially histones), and other molecules. Alternatively, the second polypeptide may convert methyl groups to hydroxymethylcytosine in the DNA demethylation mechanism. The second polypeptide can catalyze this reaction. For example, Tet1 may be a second polypeptide capable of catalyzing this reaction.

[0049] c.gRNA gRNA provides targeting for the CRISPR / Cas9-dependent system. A gRNA is a fusion of two non-coding RNAs (crRNA and tracrRNA). sgRNA can target any desired DNA sequence by swapping sequences that encode a 20 bp protospacer (which confers targeting specificity through complementary base pairing with the desired DNA target). gRNA mimics the naturally occurring crRNA:tracrRNA complex (required for the type II effector system). This diluent (which may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA) acts as a guide for Cas9 to cleave the target nucleic acid. The terms “target region,” “target sequence,” or “protospacer,” used interchangeably herein, refer to the region of the target gene targeted by the CRISPR / Cas9-dependent system. The CRISPR / Cas9-dependent system may include at least one gRNA, where the gRNAs target different DNA sequences. The target DNA sequences may overlap. The target sequence or protospacer is accompanied by a PAM sequence at its 3' end. Different type II systems require different PAMs. For example, the Streptococcus pyogenes type II system uses the “NGG” sequence (where “N” can be any nucleotide).

[0050] The number of gRNAs administered to the cells may 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 18 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.The number of gRNAs administered to the cells is: at least 50 different gRNAs from at least 1 gRNA, at least 45 different gRNAs from at least 1 gRNA, at least 40 different gRNAs from at least 1 gRNA, at least 35 different gRNAs from at least 1 gRNA, at least 30 different gRNAs from at least 1 gRNA, at least 25 different gRNAs from at least 1 gRNA, at least 20 different gRNAs from at least 1 gRNA, at least 16 different gRNAs from at least 1 gRNA, at least 12 different gRNAs from at least 1 gRNA, at least 8 different gRNAs from at least 1 gRNA, at least 4 different gRNAs from at least 1 gRNA, at least 50 different gRNAs from at least 4 different gRNAs, at least 45 different gRNAs from at least 4 different gRNAs, at least 40 different gRNAs from at least 4 different gRNAs, at least 35 different gRNAs from at least 4 different gRNAs, and at least 4 This could be at least 30 different gRNAs from 1 different gRNA, at least 25 different gRNAs from at least 4 different gRNAs, at least 20 different gRNAs from at least 4 different gRNAs, at least 16 different gRNAs from at least 4 different gRNAs, at least 12 different gRNAs from at least 4 different gRNAs, at least 8 different gRNAs from at least 4 different gRNAs, at least 50 different gRNAs from at least 8 different gRNAs, at least 45 different gRNAs from at least 8 different gRNAs, at least 40 different gRNAs from at least 8 different gRNAs, at least 35 different gRNAs from at least 8 different gRNAs, at least 30 different gRNAs from at least 8 different gRNAs, at least 25 different gRNAs from at least 8 different gRNAs, at least 20 different gRNAs from 8 different gRNAs, at least 16 different gRNAs from at least 8 different gRNAs, or at least 12 different gRNAs from 8 different gRNAs.

[0051] The gRNA may include a complementary polynucleotide sequence of the target gene sequence followed by a PAM sequence. The gRNA may include a “G” at the 5' end of the complementary polynucleotide sequence. The gRNA may include a complementary polynucleotide sequence of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, or at least 35 base pairs of the target DNA sequence followed by a PAM sequence. The PAM sequence may be “NGG”, where “N” can be any nucleotide. The gRNA may target at least one of the promoter region, enhancer region, or transcription region of the target gene. The gRNA may contain at least one nucleic acid sequence of sequence numbers: 5-40, 65-144, 492-515, 540-563, 585-625, 462 (Figure 40), 464 (Figure 41), and 465 (Figure 41). gRNA can target any nucleic acid sequence. The nucleic acid sequence target may be DNA. The DNA may be any gene. For example, gRNA can target genes such as BRN2, MYT1L, ASCL1, NANOG, VEGFA, TERT, IL1B, IL1R2, IL1RN, HBG1, HBG2, MYOD1, OCT4, and DMD.

[0052] (1) Dystrophin Dystrophin is a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix and across the cell membrane. Dystrophin provides structural stability to the dystroglycan complex of the cell membrane. The dystrophin gene occupies 2.2 megabases at the Xp21 locus. The primary transcript is approximately 2,400 kb, and the mature mRNA is approximately 14 kb. 79 exons encode a protein of over 3,500 amino acids. Normal skeletal muscle tissue contains only small amounts of dystrophin, but a lack of dystrophin in abnormal expression leads to severe and incurable symptoms. Some mutations in the dystrophin gene result in the production of deficient dystrophin and a severe dystrophin phenotype in affected individuals. Some mutations in the dystrophin gene result in a partially functioning dystrophin protein and a much milder dystrophin phenotype in affected individuals. DMD is the result of hereditary or accidental mutations that produce nonsense or frameshift mutations in the dystrophin gene. Naturally occurring mutations and their consequences are relatively well understood in DMD. In-frame deletions occurring in the exon 45-55 region can produce a highly functional dystrophin protein, and many carriers are asymptomatic or exhibit mild symptoms. Furthermore, theoretically, more than 60% of patients can be treated by targeting the exons in this region of the dystrophin gene. Attempts have been made to restore the disrupted dystrophin leading frame in DMD patients by skipping non-essential exons during mRNA splicing, thereby producing a functional dystrophin protein with an internal deletion. Deletion of the internal dystrophin exon preserves the proper leading frame but causes a less severe Becker muscular dystrophy.

[0053] (2) CRISPR / Cas9-dependent systems targeting dystrophin A CRISPR / Cas9-dependent system specific to the dystrophin gene is disclosed herein. The CRISPR / Cas9-dependent system may include Cas9 and at least one gRNA that targets the dystrophin gene. The CRISPR / Cas9-dependent system can bind to and recognize the target gene. The target region can be selected immediately upstream of any existing out-frame stop codon so that an insertion or deletion during the repair process can restore the dystrophin reading frame by frame transformation. The target region may also be a splice acceptor or splice donor site so that an insertion or deletion during the repair process can disrupt splicing and restore the dystrophin reading frame by disruption of the splice site and exon removal. The target region may also be an abnormal stop codon so that an insertion or deletion during the repair process can restore the dystrophin reading frame by removal or disruption of the stop codon. By targeting the mutation hotspot in exons 45-55 and introducing either small insertions and deletions within the exon or large deletions in one or more exons, single or complex sgRNAs can be designed to restore the dystrophin reading frame. After treatment with Cas9 and one or more sgRNAs, dystrophin expression can be restored in vitro in muscle cells of Duchenne patients. Human dystrophin was detected in vivo after transplanting genetically modified patient cells into immunodeficient mice. Importantly, this unique complex gene editing capability of the CRISPR / Cas9 system enables the efficient creation of large deletions in this mutation hotspot region, which can correct up to 62% of patient mutations by general-purpose or patient-specific gene editing approaches. CRISPR / Cas9-dependent systems can utilize gRNAs of various sequences and lengths. Examples of gRNAs can be found in Table 6. CRISPR / Cas9-dependent systems can target nucleic acid sequences of SEQ ID NOs: 65-144 or their complements. The gRNAs may contain nucleotide sequences selected from the group consisting of SEQ ID NOs: 65-144 or their complements. For example, the disclosed CRISPR / Cas9-dependent systems were manipulated to mediate highly efficient gene editing at exon 51 of the dystrophin gene. These CRISPR / Cas9-dependent systems restored dystrophin protein expression in cells derived from DMD patients.

[0054] (a) Exxon 51 and 45-55 Exon 51 is often adjacent to frame disruption deletions in DMD. Nearly 15% of all DMD patients can be treated by utilizing exon skipping to remove exon 51 of the dystrophin transcript. This class of DMD mutations is ideally suited for permanent modification using NHEJ-based genome editing and HDR. For targeted induction modification of exon 51 of the human dystrophin gene, we developed the CRISPR / Cas9-dependent systems described herein. These CRISPR / Cas9-dependent systems were transfected into human DMD cells to mediate efficient gene modification and conversion to a modified reading frame. Protein restoration was associated with frame recovery and was detected in a mixed population of CRISPR / Cas9-dependent system-treated cells. Similarly, nearly 62% of all DMD patients were treated using exon 45-55 removal in the dystrophin transcript. (3) AAV / CRISPR structures AAV can be used to deliver CRISPR using a variety of construct structures (see Figure 39). For example, AAV can deliver Cas9 and gRNA expression cassettes in separate vectors. Alternatively, if a small Cas9 protein (derived from Staphylococcus aureus or Neisseria meningitidis) is used, both Cas9 and up to two gRNA cassettes can be combined into a single AAV vector within a 4.7kb package limit (see Figure 39).

[0055] 5. A hybrid CRISPR / Cas9-based system This disclosure aims to create a complex CRISPR / Cas9-dependent system comprising a CRISPR / Cas9-dependent system (e.g., Cas9 or dCas9) and a wide variety of gRNAs targeting one or more endogenous genes. This platform utilizes a convenient golden gate cloning method to rapidly incorporate up to four independent sgRNA expression cassettes into a single lentiviral vector. Each sgRNA was efficiently expressed and capable of mediating complex gene editing at dispersed loci in immortalized and primary human cell lines. Transient transcriptional activation in cell lines stably expressing dCas9-VP64 was demonstrated to be prepared by synergistic activation by one to four sgRNAs. Furthermore, the single lentiviral vector can induce sustained and long-term endogenous gene expression in immortalized and primary human cells. This system enables rapid assembly of a single lentiviral vector that allows for efficient complex gene editing in model and primary cell lines. The combined CRISPR / Cas9-dependent system offers the potential for transcriptional activation and tunable induction of transcriptional activation. When readily generated by the Golden Gate assembly, the final vector expresses constitutive Cas9 or dCas9-VP64 in addition to one, two, three, or four sgRNAs expressed from independent promoters. Each promoter can efficiently express an sgRNA, which induces similar levels of Cas9 nuclease activity. Furthermore, lentiviral delivery of a single vector expressing Cas9 and four sgRNAs targeting distinct loci results in simultaneous combined gene editing of all four loci. Tunicated transcriptional activation of two endogenous genes in both transient and stable environments was achieved using lentiviral delivery of Cas9 with or without sgRNAs. Highly efficient and long-lasting gene activation is achieved in primary human cells. This system is therefore a promising and efficient method for producing combined gene editing and long-lasting transcriptional activation in human cells.

[0056] The complex CRISPR / Cas9-dependent system enables efficient complex gene editing, simultaneously inactivating a wide variety of genes. By co-expressing just one Cas9 protein with two or more sgRNAs, the CRISPR / Cas9-dependent system can simultaneously target diverse and distinct genomic loci, making it unique for complex gene editing or synergistic activation applications. The CRISPR / Cas9-dependent system greatly facilitates molecular targeting processes to new sites by simply modifying the expressed sgRNA molecule. Combining a single lentiviral vector with methods (chemically or optogenetically) to achieve inducible control of these components can advance the elucidation of the dynamic changes in gene regulation, both temporally and spatially. A combined CRISPR / Cas9-dependent system can transcriptionally activate two or more endogenous genes. A combined CRISPR / Cas9-dependent system can transcriptionally repress two or more endogenous genes. For example, at least two endogenous genes, at least three endogenous genes, at least four endogenous genes, at least five endogenous genes, or at least ten endogenous genes can be activated or repressed by a combined CRISPR / Cas9-dependent system. Two to fifteen genes, two to ten genes, two to five genes, five to fifteen genes, or five to ten genes can be activated or repressed by a combined CRISPR / Cas9-dependent system.

[0057] (1) Modified lentiviral vector The combined CRISPR / Cas9-dependent system may include a modified lentiviral vector. The modified lentiviral vector comprises a first polynucleotide sequence encoding a fusion protein and a second polynucleotide sequence encoding at least one sgRNA. The fusion protein may be the fusion protein of the CRISPR / Cas9-dependent system described above. The first polynucleotide sequence may be operably ligated to a promoter. The promoter may be a constitutive promoter, an inducible promoter, an inhibitable promoter, or a moduloable promoter. The second polynucleotide sequence encodes at least one sgRNA. For example, the second polynucleotide sequence can encode at least one sgRNA, at least two sgRNAs, at least three sgRNAs, at least four sgRNAs, at least five sgRNAs, at least six sgRNAs, at least seven sgRNAs, at least eight sgRNAs, at least nine sgRNAs, at least ten sgRNAs, at least eleven sgRNAs, at least twelve sgRNAs, at least thirteen sgRNAs, at least fourteen sgRNAs, at least fifteen sgRNAs, at least sixteen sgRNAs, at least seventeen sgRNAs, at least eighteen sgRNAs, at least nineteen sgRNAs, at least twenty sgRNAs, at least twenty-five sgRNAs, at least thirty sgRNAs, at least thirty-five sgRNAs, at least forty sgRNAs, at least forty-five sgRNAs, or at least fifty sgRNAs.The second polynucleotide sequence consists of 50 sgRNAs from one sgRNA, 45 sgRNAs from one sgRNA, 40 sgRNAs from one sgRNA, 35 sgRNAs from one sgRNA, 30 sgRNAs from one sgRNA, 25 different sgRNAs from one sgRNA, 20 sgRNAs from one sgRNA, 16 sgRNAs from one sgRNA, 8 different sgRNAs from one sgRNA, 50 different sgRNAs from 4 different sgRNAs, 45 different sgRNAs from 4 different sgRNAs, 40 different sgRNAs from 4 different sgRNAs, 35 different sgRNAs from 4 different sgRNAs, 30 different sgRNAs from 4 different sgRNAs, 25 different sgRNAs from 4 different sgRNAs, 20 different sgRNAs from 4 different sgRNAs, 16 different sgRNAs from 4 different sgRNAs, and 4 different sgRN A can encode 8 different sgRNAs, 50 different sgRNAs from 8 different sgRNAs, 45 different sgRNAs from 8 different sgRNAs, 40 different sgRNAs from 8 different sgRNAs, 35 different sgRNAs from 8 different sgRNAs, 30 different sgRNAs from 8 different sgRNAs, 25 different sgRNAs from 8 different sgRNAs, 20 different sgRNAs from 8 different sgRNAs, 16 different sgRNAs from 8 different sgRNAs, 50 different sgRNAs from 16 different sgRNAs, 45 different sgRNAs from 16 different sgRNAs, 40 different sgRNAs from 16 different sgRNAs, 35 different sgRNAs from 16 different sgRNAs, 30 different sgRNAs from 16 different sgRNAs, 25 different sgRNAs from 16 different sgRNAs, and 20 different sgRNAs from 16 different sgRNAs. Each of the polynucleotide sequences encoding different sgRNAs can be operably ligated to a promoter. The promoter operably ligated to different sgRNAs may be the same promoter. The promoters operably ligated to different sgRNAs may be different promoters. The promoter may be a constitutive promoter, an inducible promoter, an inhibitable promoter, or a moduloable promoter. At least one sgRNA can bind to a target gene or locus. If two or more sgRNAs are present, each sgRNA can bind to a different target region within a single target locus, or each sgRNA can bind to a different target within a different gene locus. The fusion protein may contain the Cas9 protein or the iCas9-VP64 protein. The fusion protein may contain the VP64 domain, the p300 domain, or the KRAB domain.

[0058] 6. Position-specific nuclease The compositions described above include a nucleotide sequence encoding a site-specific nuclease that binds to and cleaves a target region. The site-specific nuclease can be manipulated. For example, the site-specific nuclease to be manipulated may be a CRISPR / Cas9-dependent system, a ZFN, or a TALEN. The site-specific nuclease can bind to and cleave a gene or locus in the genome of a skeletal muscle or cardiac muscle cell. For example, the gene or locus may be the Rosa26 locus or the dystrophin gene. a. CRISPR / Cas9-dependent systems Using the CRISPR / Cas9-dependent system described above, site-specific double-strand breaks can be introduced into target inducement genomic loci. b. Zinc finger nuclease (ZFN) Site-specific nucleases can be ZFNs. A single zinc finger contains approximately 30 amino acids, and its domain functions by binding to three consecutive base pairs of DNA via the interaction of one amino acid side chain per base pair. The modular structure of the zinc finger motif allows for the binding of a series of domains, enabling the recognition and targeting of extension sequences in multiples of three nucleotides. These target-inducing DNA-binding domains can be combined with a nuclease domain (e.g., FokI) to generate site-specific nucleases. These are referred to as "zinc finger nucleases" (ZFNs), which can be used to introduce site-specific double-strand breaks at targeted genomic loci. These DNA breaks stimulate the innate DNA repair mechanism, resulting in one of two possible repair pathways (NHEJ and HDR). For example, ZFNs can target the Rosa26 locus (Perez-Pinera et al. Nucleic Acids Research (2012) 40:3741-3752) or the dystrophin gene. Examples of ZFNs are shown in Table 1 and Figures 35-38. In Table 1, DNA recognition helices are underlined, and “Fok ELD-S” and “Fok KKR-S” refer to the FokI nuclease domain (which is fused with the zinc finger protein DNA binding domain). In Figures 35-38, target DNA sequences within target sites (i.e., SEQ ID NOs: 442, 445, 448, and 453) and DNA recognition helices within ZFN amino acid sequences (i.e., SEQ ID NOs: 443, 444, 446, 447, 449-452, and 455) are underlined, respectively.

[0059] [Table 1]

[0060] c.TAL Effector Nuclease (TALEN) TALENs can be used to introduce site-directed double-strand breaks into targeted genomic loci. Site-directed double-strand breaks occur when two distinct TALENs bind to nearby DNA sequences, thereby allowing for FokI dimerization and cleavage of the target DNA. TALENs have advanced gene editing due to their high success rate and efficiency in gene editing. This DNA cleavage stimulates the innate DNA repair mechanism, resulting in one of two possible repair pathways: homology-directed repair (HDR) or non-homologous end junction (NHEJ) pathways. TALENs can be designed to target any gene involved in genetic disease. A TALEN may include a nuclease and a TALEN DNA-binding domain that binds to a target gene within the TALEN target region. The target gene may have mutations (e.g., frameshift mutations or nonsense mutations). If the target gene has a mutation that produces an immature stop codon, the TALEN may be designed to recognize and bind to a nucleotide sequence upstream or downstream of the immature stop codon. The “TALEN target region” includes binding regions and a spacer region (located between the binding regions) for two TALENs. The two TALENs bind to different binding regions within the TALEN target region, after which the TALEN target region is cleaved. An example of a TALEN is described in International Patent Application No. PCT / US2013 / 038536 (the aforementioned document is incorporated herein by reference in its entirety).

[0061] 7. Transfer Activator The compositions described above include a nucleotide sequence encoding a transcriptional activator that activates a target gene. The transcriptional activator can be manipulated. For example, the manipulated transcriptional activator may be a CRISPR / Cas9-dependent system, a zinc finger fusion protein, or a TALE fusion protein. a. CRISPR / Cas9-dependent systems The CRISPR / Cas9-dependent system described above can be used to activate the transcription of target genes in conjunction with RNA. The CRISPR / Cas9-dependent system may include the fusion protein described above, where the second polypeptide domain has transcriptional activation activity or histone modification activity. For example, the second polypeptide domain may include VP-64 or p300. b. Zinc finger fusion protein The transcriptional activator may be a zinc finger fusion protein. The target-inducing DNA-binding domain of the zinc finger described above can be combined with a domain having transcriptional activation activity or histone modification activity. For example, the domain may include VP64 or p300. c.TALE fusion protein TALE fusion proteins can be used to activate the transcription of target genes. TALE fusion proteins may include a TALE DNA-binding domain and a domain having transcriptional activation or histone modification activity. For example, the domain may include VP64 or p300.

[0062] 8. Composition The present invention aims to provide compositions for altering gene expression and manipulating or modifying the genomic DNA of cells or target animals. The compositions may also include a viral delivery system. a. Compositions for genome editing in muscle The present invention aims to provide a composition for genome editing a target gene in the skeletal muscle or cardiac muscle of a target animal. The composition comprises a modified AAV vector and a nucleotide sequence encoding a site-specific nuclease. The composition delivers the active form of the site-specific nuclease to the skeletal muscle or cardiac muscle. The composition may further contain donor DNA or a transgene. These compositions can be used in genome editing, genomic manipulation, and modification or mitigation of the effects of gene mutations involved in genetic diseases and / or other skeletal muscle or cardiac muscle symptoms. The target gene may be involved in cell differentiation or any other process (in which the suppression or disruption of gene activation is desired), or it may have mutations (e.g., deletions, frameshift mutations, or nonsense mutations). If the target gene has an immature stop codon, an abnormal splice acceptor site, or an abnormal splice donor site, the site-specific nuclease may be designed to recognize and bind to an upstream or downstream nucleotide sequence from the immature stop codon, abnormal splice acceptor site, or abnormal splice donor site. Alternatively, the site-specific nuclease can be used to disrupt normal gene splicing by targeting splice acceptors and donors to introduce skipping of the immature stop codon, or by restoring a disrupted reading frame. The site-specific nuclease may or may not mediate off-target changes in the protein-coding regions of the genome.

[0063] b. Adeno-associated virus vectors The compositions described above include a modified adeno-associated virus (AAV) vector. The modified AAV vector may have enhanced myocardial and skeletal muscle tropism. The modified AAV vector can deliver and express a site-specific nuclease to mammalian cells. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). The modified AAV vector can deliver nucleases to skeletal and cardiac muscle in vivo. The modified AAV vector may be based on one or more of several capsids (including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9). The modified AAV vector may also be based on an AAV2 pseudotype containing another myocardial AAV capsid. The aforementioned vectors include, for example, AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, and AAV / SASTG vectors, which efficiently transduce skeletal muscle or cardiac muscle through systemic or local delivery (Seto et al. Current Gene Therapy (2012) 12:139-151). c. CRISPR / Cas9-dependent systems This disclosure also provides DNA targeting systems or compositions of at least one CRISPR / Cas9-dependent system described above. These compositions can be used in genome editing, genomic manipulation, and modification or mitigation of the effects of gene mutations involved in genetic diseases. The compositions comprise a CRISPR / Cas9-dependent system, the system comprising the Cas9 protein or Cas9 fusion protein described above. The CRISPR / Cas9-dependent system may also include at least one gRNA described above. d. A hybrid CRISPR / Cas9-based system This disclosure also provides the composite CRISPR / Cas9-dependent systems described above. These compositions can be used in genome editing, genomic manipulation, and modification or mitigation of the effects of gene mutations involved in genetic diseases. These compositions can be used to target two or more genes. The composition comprises a modified lentiviral vector, the vector comprising a CRISPR / Cas9-dependent system containing the Cas9 protein or Cas9 fusion protein described above, and two or more gRNAs described above.

[0064] 9.How to use The possible applications of the composition extend across many areas of science and biotechnology. The disclosed composition can be used to repair disease-causing gene mutations. The disclosed composition can be used to disrupt genes, thereby resulting in muscle regeneration or strengthening, or a reduction in muscle aging. The disclosed composition can be used to introduce therapeutic genes (e.g., coagulation factors or monoclonal antibodies) systemically expressed from skeletal muscle or cardiac muscle. The disclosed composition can be used to modulate gene expression in mammals. The disclosed composition can be used to trans-differentiate or induce differentiation in cells, or to correct mutated genes in cells. Examples of gene activation, genetic reprogramming, and regenerative medicine related to cell and gene therapy are provided. Cell lineage details can be reprogrammed using RNA-guided transcription activators. Activation of endogenous genes encoding key regulators of cell fate may potentially provide a faster, more efficient, stable, or specific method for genetic reprogramming, trans-differentiation, and / or induced differentiation than forced overexpression of these factors.

[0065] 10. How to edit the genome in muscle This disclosure aims to provide a method for genome editing in the skeletal muscle or cardiac muscle of a target animal. The method includes the step of administering the above-described genome editing composition to the skeletal muscle or cardiac muscle of a target animal. Genome editing may include modification of mutant genes or insertion of transgenes. Modification of mutant genes may include deletion, rearrangement, or replacement of the mutant gene. Modification of mutant genes may include nuclease-mediated NHEJ or HDR.

[0066] 11. How to use a CRISPR / Cas9-dependent system The potential applications of CRISPR / Cas9-dependent systems extend across many areas of science and biotechnology. The disclosed CRISPR / Cas9-dependent systems can be used to modulate mammalian gene expression. The disclosed CRISPR / Cas9-dependent systems can be used to trans-differentiate or induce differentiation in cells, or to modify mutant genes in cells. Examples of gene activation, genetic reprogramming, and regenerative medicine related to cell and gene therapy are provided. Cell lineage subdivisions can be reprogrammed using RNA-guided transcription activators. While these experiments have shown incomplete and inefficient reprogramming, many methods exist that could improve this approach. These include repeated transfection of iCas9-VP64 / gRNA combinations, stable expression of the factor, and targeted induction of a wide variety of genes (e.g., Brn2 and Mytl1) in addition to Ascl1 for trans-differentiation into neuronal phenotypes. Activation of endogenous genes encoding key regulators of cell fate may potentially provide a faster, more efficient, stable, or specific method for genetic reprogramming and trans- or induced differentiation of cells than forced overexpression of these factors. Finally, fusion of Cas9 with other domains (including repressive and epigenetic modification domains) could offer a much greater diversity of RNA-guided transcriptional regulators, complementing other RNA-based tools for mammalian cell manipulation.

[0067] a. Methods for activating gene expression This disclosure provides a mechanism for activating the expression of endogenous genes (e.g., mammalian genes). This mechanism involves targeting and inducing transcription activators to promoters via RNA using the CRISPR / Cas9-dependent system described above. This differs fundamentally from previously described methods involving the manipulation of sequence-specific DNA-binding proteins and offers an opportunity for targeted gene regulation. Since the generation of gRNA expression plasmids requires only the synthesis of two short custom oligonucleotides and a single cloning step, many novel gene activators can be generated rapidly and economically. gRNAs can also be directly transfected into cells following in vitro transcription. While a wide variety of gRNAs targeted and induced to a single promoter have been shown, simultaneous targeting and induction to multiple promoters may also be possible. Recognition of genomic target sites by RNA rather than protein also avoids the limitations of targeting epigenetically modified sites (e.g., methylated DNA). In contrast to conventional methods involving the manipulation of DNA-binding proteins, Cas9 fused with a transcriptional activation domain can be guided to a target by a guide RNA molecule, thereby inducing the expression of endogenous human genes. This straightforward and versatile approach to target-induced gene activation avoids the need to manipulate novel proteins and enables the regulation of widely dispersed synthetic genes. The method may include administering to cells or target animals a CRISPR / Cas9-dependent system described above, a polynucleotide or vector encoding the CRISPR / Cas9-dependent system, or a DNA targeting system or composition containing at least one CRISPR / Cas9-dependent system. The method may include administering the CRISPR / Cas9-dependent system, for example, a Cas9 fusion protein containing a transcriptional activation domain or a nucleotide sequence encoding the Cas9 fusion protein. The Cas9 fusion protein may include a transcriptional activation domain (e.g., VP16 protein) or a transcriptional coactivator (e.g., p300 protein).

[0068] (1) dCas9-VP16 The Cas9 fusion protein may contain a transcriptional activation domain (e.g., a VP16 protein). This transcriptional activation domain may contain at least one VP16 protein, at least two VP16 proteins, at least three VP16 proteins, at least four VP16 proteins (i.e., a VP64 activator domain), at least five VP16 proteins, at least six VP16 proteins, at least six VP16 proteins, or at least ten VP16 proteins. The Cas9 protein may be a Cas9 protein in which the nuclease activity has been inactivated. For example, the Cas9 protein of the fusion protein may be iCas9 (amino acids 36-1403 of SEQ ID NO: 1), which includes the D10A and H840A amino acid substitutions. The Cas9 fusion protein may be iCas9-VP64. (2) dCas9-p300 Cas9 fusion proteins can contain transcriptional co-activation domains (e.g., p300 proteins). The p300 protein (also known as EP300 or E1A-binding protein p300) is encoded by the EP300 gene and regulates the activity of many genes in tissues throughout the body. The p300 protein plays a role in regulating cell proliferation and division, promoting cell maturation and the presentation of specialized functions (differentiation), and preventing the growth of cancerous tumors. The p300 protein activates transcription by binding transcription factors to protein complexes (which perform transcription in the cell nucleus). The interaction of p300 with transcription factors is achieved by one or more p300 domains, namely the nuclear receptor interaction domain (RID), the CREG and MYB interaction domain (KIX), the cysteine / histidine domains (TAZ1 / CH1 and TAZ2 / CH3), and the interferon response binding domain (IBiD). The last four domains of p300 (KIX, TAZ1, TAZ2, and IBiD) tightly bind to both transactivation domains of the transcription factor p53 and to a sequence spanning the nine amino acid TAD, respectively. This protein functions as a histone acetyltransferase that regulates transcription via chromatin remodeling and is crucial in the processes of cell proliferation and differentiation. It mediates cAMP gene regulation by specifically binding to the phosphorylated CREB protein.

[0069] The p300 protein can activate the following: MAD (Mothers against decapentaplegic) homologue 7, MAF, TSG101, peroxisome proliferator-activated receptor alpha, NPAS2, PAX6, DDX5, MYBL2, MAD homologue 1, MAD homologue 2, lymphoid enhancer binding factor 1, SNIP1, TRERF1, STAT3, EID1, RAR-related orphan receptor alpha, ELK1, HIF1A, ING5, peroxisome proliferator-activated receptor gamma, SS18, TCF3, Zif268, and estrogen receptor alpha. A, GPS2, MyoD, YY1, ING4, PROX1, CITED1, HNF1A, MEF2C, MEF2D, MAML1, twist transcription factor, PTMA, IRF2, DTX1, flap structure-specific endonuclease 1, muscle cell-specific enhancer factor 2A, CDX2, BRCA1, HNRPU, STAT6, CITED2, RELA, TGS1, CEBPB, Mdm2, NCOA6, NFATC2, thyroid hormone receptor alpha, BCL3, TFAP2A, PCNA, P53, and TAL1. The transcriptional co-activation domain may contain the human p300 protein or a fragment thereof. The transcriptional co-activation domain may contain the wild-type human p300 protein, the mutant human p300 protein, or the aforementioned fragment. The transcriptional co-activation domain may contain the central lysine acetyltransferase domain of the human p300 protein, i.e., the p300HAT core (also known as the “p300WT core,” see Figure 58). The Cas9 protein may be a Cas9 protein in which the nuclease activity has been inactivated. For example, the Cas9 protein of the fusion protein may be iCas9 (amino acids 36-1403 of SEQ ID NO: 1), which includes the D10A and H840A amino acid substitutions. The Cas9 fusion protein may be the iCas9-p300WT core.

[0070] (3) gRNA The method also includes administering at least one CRISPR / Cas9-dependent system gRNA to cells or target animals, wherein the gRNA targets different DNA sequences. The target DNA sequences may overlap. The number of gRNAs administered to the cells may 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 18 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.The number of gRNAs administered to the cells is: at least 50 different gRNAs from at least 1 gRNA, at least 45 different gRNAs from at least 1 gRNA, at least 40 different gRNAs from at least 1 gRNA, at least 35 different gRNAs from at least 1 gRNA, at least 30 different gRNAs from at least 1 gRNA, at least 25 different gRNAs from at least 1 gRNA, at least 20 different gRNAs from at least 1 gRNA, at least 16 different gRNAs from at least 1 gRNA, at least 12 different gRNAs from at least 1 gRNA, at least 8 different gRNAs from at least 1 gRNA, at least 4 different gRNAs from at least 4 gRNAs, at least 50 different gRNAs from at least 4 different gRNAs, at least 45 different gRNAs from at least 4 different gRNAs, at least 40 different gRNAs from at least 4 different gRNAs, at least 35 different gRNAs from at least 4 different gRNAs, and at least 30 different gRNAs from at least 4 different gRNAs. It could be at least 25 different gRNAs from at least 4 different gRNAs, at least 20 different gRNAs from at least 4 different gRNAs, at least 16 different gRNAs from at least 4 different gRNAs, at least 12 different gRNAs from at least 4 different gRNAs, at least 8 different gRNAs from at least 4 different gRNAs, at least 50 different gRNAs from at least 8 different gRNAs, at least 45 different gRNAs from at least 8 different gRNAs, at least 40 different gRNAs from at least 8 different gRNAs, at least 35 different gRNAs from at least 8 different gRNAs, at least 30 different gRNAs from at least 8 different gRNAs, at least 25 different gRNAs from at least 8 different gRNAs, at least 20 different gRNAs from 8 different gRNAs, at least 16 different gRNAs from at least 8 different gRNAs, at least 12 different gRNAs from 8 different gRNAs, or at least 8 different gRNAs from at least 8 different gRNAs. The gRNA may contain a complementary polynucleotide sequence of the target DNA sequence followed by an NGG. The gRNA may contain a “G” at the 5' end of the complementary polynucleotide sequence. The gRNA may contain a complementary polynucleotide sequence of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, or at least 35 base pairs of the target DNA sequence followed by an NGG. The gRNA may target at least one of the promoter region, enhancer region, or transcription region of the target gene. The gRNA may contain at least one nucleic acid sequence of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625.

[0071] b. Methods for suppressing gene expression This disclosure provides a mechanism for repressing the expression of endogenous genes (e.g., mammalian genes). The mechanism involves targeting and inducing genomic regulatory elements (e.g., distal enhancers) via RNA using the CRISPR / Cas9-dependent system described above. The Cas9 fusion protein may include a transcriptional repressor (e.g., a KRAB repressor). The Cas9 fusion protein may be dCas9-KRAB. dCas9-KRAB can further influence epigenetic gene regulation by supplementing heterochromatin formation factors at the target induction locus. While the CRISPR / Cas9-KRAB system can repress gene transcription, it can also be used to target genomic regulatory elements (which have been inaccessible to traditional repression methods, e.g., RNA interference) (Figure 53B). Delivery of dCas9-KRAB, which is targeted and induced at distal enhancers along with gRNA, can disrupt the expression of a wide variety of genes regulated by the target induction enhancer (see Figure 53C). The targeted enhancer can be any enhancer of the gene (e.g., the HS2 enhancer).

[0072] a. Methods of trans-differentiation or induced differentiation This disclosure provides a mechanism for transdifferentiating cells or inducing cell differentiation by activating endogenous genes via RNA using the CRISPR / Cas9-dependent system described above. (1) Trans differentiation Cells can be transdifferentiated using a CRISPR / Cas9-dependent system. Transdifferentiation (also known as lineage reprogramming or direct conversion) is the process by which cells are converted from one differentiated cell type to another without passing through an intermediate pluripotent state or progenitor cell type. It is a type of metaplasia and includes the switching of the fate of all cells, including the interconversion of stem cells. Transdifferentiation of cells has potential applications in disease modeling, drug discovery, gene therapy, and regenerative medicine. Using the CRISPR / Cas9-dependent system described above, activation of endogenous genes (e.g., BRN2, MYT1L, ASCL1, NANOG, and / or MYOD1) can result in transdifferentiation of several cell types (e.g., fibroblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, or smooth muscle cells) to either neuronal or myogenic phenotypes.

[0073] (2) Induced differentiation The CRISPR / Cas9-dependent system can be used to induce differentiation of cells (e.g., stem cells, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, or smooth muscle cells). For example, stem cells (e.g., embryonic stem cells or pluripotent stem cells) can be induced to differentiate into muscle cells or vascular endothelial cells (i.e., to induce neuronal or myogenic differentiation). 12. Use of a combined CRISPR / Cas9-dependent system The combined CRISPR / Cas9-dependent system can be useful in accelerating progress in genome research with high processing efficiency by leveraging the simplicity and low cost of sgRNA design and utilizing CRISPR / Cas9 technology. For example, the single lentivirus described herein is useful for the expression of Cas9 and numerous sgRNAs in various cell lines (e.g., primary fibroblasts described herein) (Figure 47). The combined CRISPR / Cas9-dependent system can be used in the same manner as the CRISPR / Cas9-dependent system described above. In addition to the transcriptional activation and nuclease functionality described, this system may be useful for the expression of other novel Cas9 effectors that control epigenetic modifications for a variety of purposes, including the investigation of genome structure and endogenous gene regulatory pathways. Since endogenous gene regulation requires a delicate balance among a wide variety of enzymes, a complex Cas9 system with diverse functionalities would allow for the testing of complex interactions between various regulatory signals. The vectors described herein are compatible with aptamer-modified sgRNAs and orthogonal Cas9 fittings, and should enable separate gene manipulations using a single set of sgRNAs.

[0074] A combined CRISPR / Cas9-dependent system can be used to activate at least one endogenous gene in cells. This method involves contacting cells with a modified lentiviral vector. The endogenous gene may be transiently activated or stably activated. The endogenous gene may be transiently repressed or stably repressed. The fusion protein may be expressed at levels similar to sgRNA. The fusion protein may be expressed at levels different from sgRNA. The cells may be primary human cells. The combined CRISPR / Cas9-dependent system can be used in a method of combined gene editing of cells. This method includes contacting cells with a modified lentiviral vector. Combined gene editing may include modification of a mutant gene or insertion of a transgene. Modification of a mutant gene may include deletion, rearrangement, or replacement of the mutant gene. Modification of a mutant gene may include nuclease-mediated non-homologous end joining or homology-directed repair. Combined gene editing may include deletion or modification of at least one gene, where the gene is either an endogenous normal gene or a mutant gene. Combined gene editing may include deletion or modification of at least two genes. For example, at least two genes, at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes, at least eight genes, at least nine genes, or at least ten genes can be deleted or modified. A combined CRISPR / Cas9-dependent system can be used in a method for complexly regulating gene expression in cells. This method includes contacting cells with a modified lentiviral vector. This method may include regulating the gene expression level of at least one gene. Gene expression of at least one target gene is regulated when the gene expression level of the at least one target gene is increased or decreased compared to the normal gene expression level for the at least one target gene. The gene expression level may be at the RNA or protein level.

[0075] 13. A method for treating target animals by correcting mutated genes. This disclosure also aims to provide a method for correcting mutated genes in a target animal. The method comprises administering the above-described genome editing composition for skeletal muscle or cardiac muscle to the skeletal muscle or cardiac muscle of a target animal. The use of the composition, which delivers a site-specific nuclease to the skeletal muscle or cardiac muscle together with a repair template or donor DNA, can restore the expression of a protein with full or partial function (the template or DNA can replace an entire gene or region containing the mutation). Using a site-specific nuclease, a site-specific double-strand break can be introduced at a target genomic locus. A site-specific double-strand break occurs when the site-specific nuclease binds to a target DNA sequence, thereby allowing the target DNA to be broken. This DNA break stimulates the innate DNA repair mechanism, which can result in one of two possible repair pathways: homology-directed repair (HDR) or non-homologous end junction (NHEJ) pathways. This disclosure aims at genome editing using site-specific nucleases in the absence of a repair template (which can efficiently modify the leading frame and restore the expression of functional proteins involved in genetic disorders). The site-specific nucleases disclosed can be engaged using homology-directed repair or non-homologous end-joint (NHEJ) system modification approaches (which enable efficient modification in growth-restricted primary cell lines that are not well-suited to homologous recombination or selection-dependent gene modification). This approach integrates rapid and robust assembly of active site-specific nucleases with efficient gene editing for the treatment of genetic disorders caused by mutations in non-essential coding regions (resulting in frameshifts, immature stop codons, abnormal splice donor sites, or abnormal splice acceptor sites).

[0076] a. Nuclease-mediated non-homologous end joining Restoration of protein expression in endogenously mutated genes can be achieved through template-free NHEJ-mediated DNA repair. In contrast to transient methods targeting the RNA of the target gene, modification of the target gene reading frame within the genome by transiently expressed site-specific nucleases can result in permanent restoration of target gene expression in all modified cells and their offspring. Nuclease-mediated NHEJ gene modification can correct mutated target genes and offers several potential advantages over the HDR pathway. For example, NHEJ does not require a donor template (donor templates can lead to mutation introduction via nonspecific insertions). In contrast to HDR, NHEJ operates efficiently throughout the entire cell cycle and can therefore be efficiently utilized in both intracycle and postmittal cells (e.g., muscle fibers). This provides robust and permanent gene recovery as an alternative to oligonucleotide-dependent exon skipping or pharmacologically forced readthrough of stop codons, and may theoretically require the same number of steps as single-drug therapy. NHEJ gene modification using a CRISPR / Cas9-dependent system, as well as other manipulation nucleases including meganucleases and zinc finger nucleases, can be used in conjunction with other existing ex vivo and in vivo platforms for cell and gene system therapy, in addition to the plasmid electroporation approach described herein. For example, delivery of CRISPR / Cas9-based systems via mRNA-based gene transfer or as purified cell-permeable proteins could enable a DNA-free genome editing approach that completely avoids the possibility of introducing displacement through insertion.

[0077] b. Homology-oriented repair Repair of protein expression of an endogenous mutant gene may include homology-directed repair. The method described above further includes administration of a donor template to cells. The donor template may include nucleotides encoding a fully functional protein or a partially functional protein. For example, the donor template may include a miniaturized dystrophin construct (minidys (referred to as "minidys")), a fully functional dystrophin construct for the restoration of the mutant dystrophin gene, or a fragment of the dystrophin gene (resulting in the restoration of the mutant dystrophin gene after homology-directed repair).

[0078] c. A method for treating target animals by correcting mutant genes using CRISPR / Cas9. This disclosure also aims at genome editing using a CRISPR / Cas9-dependent system to restore fully or partially functional protein expression using a repair template or donor DNA (which may replace a complete gene or a region containing mutations). Using a CRISPR / Cas9-dependent system, site-specific double-strand breaks can be introduced into a target genomic locus. Site-specific double-strand breaks occur when the CRISPR / Cas9-dependent system binds to a target DNA sequence using gRNA, thereby allowing the target DNA to be cleaved. CRISPR / Cas9-dependent systems offer the advantages of progressive genome editing due to their successful and efficient high-rate gene modification. This DNA cleavage stimulates the innate DNA repair mechanism, resulting in one of two possible repair pathways: homology-directed repair (HDR) or non-homologous end junction (NHEJ) pathways. For example, a CRISPR / Cas9-dependent system induced in the dystrophin gene may include a gRNA having one of the nucleic acid sequences of SEQ ID NOs: 65-115. This disclosure aims to provide genome editing using a CRISPR / Cas9-based system without repair templates, which can efficiently modify the leading frame and restore the expression of functional proteins involved in genetic diseases. The disclosed CRISPR / Cas9-based system and method may include steps using homology-directed repair or nuclease-mediated non-homologous end-joint (NHEJ) system repair approaches (which enable efficient repair in growth-restricted primary cell lines that are not amenable to homologous recombination or selection-based gene repair). This approach integrates the rapid and robust assembly of an active CRISPR / Cas9-based system with efficient gene editing for the treatment of genetic diseases caused by mutations in non-essential coding regions (resulting in frameshifts, immature stop codons, abnormal splice donor sites, or abnormal splice acceptor sites).

[0079] This disclosure provides a method for treating patients suffering from genetic disorders (e.g., DMD) by correcting mutated genes in cells. The method comprises administering the CRISPR / Cas9-dependent system described above, a polynucleotide or vector encoding the CRISPR / Cas9-dependent system, or a composition of the CRISPR / Cas9-dependent system to cells or a target animal. The method may include administering the CRISPR / Cas9-dependent system, for example, a Cas9 protein or a Cas9 fusion protein comprising a second domain having nuclease activity, a nucleotide encoding the Cas9 protein or Cas9 fusion protein, and / or at least one gRNA (the gRNAs targeting different DNA sequences). The target DNA sequences may overlap. The number of gRNAs administered to the cells may be at least one gRNA, at least two different gRNAs, at least three different gRNAs, at least four different gRNAs, at least five different gRNAs, at least six different gRNAs, at least seven different gRNAs, at least eight different gRNAs, at least nine different gRNAs, at least ten different gRNAs, at least fifteen different gRNAs, at least twenty different gRNAs, at least thirty different gRNAs, or at least fifty different gRNAs, as described above. The gRNAs may contain at least one nucleic acid sequence from SEQ ID NOs: 65-115. This method may include homology-directed repair or non-homologous end joining.

[0080] 14. Methods of treating diseases This disclosure aims to provide a method for treating target animals in need of such treatment. The method includes administering a composition for altering gene expression and manipulating or altering genomic DNA to the tissues of the target animals in the genome editing of the cells or target animals described above. The method also includes administering a composition for genome editing of skeletal muscle or cardiac muscle described above to the skeletal muscle or cardiac muscle of the target animals. The target animals may suffer from skeletal muscle or cardiac muscle conditions or genetic diseases that cause degeneration or fragility. For example, the target animals may suffer from Duchenne muscular dystrophy described above. a. Duchenne muscular dystrophy Using the method described above, the dystrophin gene can be modified to restore fully or partially functional protein expression of the mutant dystrophin gene. In some features and embodiments, the disclosure provides a method for mitigating the effects of DMD (e.g., clinical symptoms / signs) in a patient. In some features and embodiments, the disclosure provides a method for treating DMD in a patient. In some features and embodiments, the disclosure provides a method for preventing DMD in a patient. In some features and embodiments, the disclosure provides a method for preventing further progression of DMD in a patient.

[0081] 15. Constructs and Plasmids The compositions described above may include a gene construct encoding the CRISPR / Cas9-dependent system described above. The gene construct (e.g., plasmid) may include nucleic acids encoding the CRISPR / Cas9-dependent system, e.g., Cas9 protein and Cas9 fusion protein, and / or at least one gRNA. The compositions described above may include a gene construct encoding a modified AAV and a nucleic acid sequence encoding the site-specific nuclease described above. The gene construct, e.g., plasmid, may include nucleic acids encoding the site-specific nuclease. The compositions described above may include a gene construct encoding the modified lentiviral vector described herein. The gene construct, e.g., plasmid, may include nucleic acids encoding the Cas9 fusion protein and at least one sgRNA. The gene construct may exist intracellularly as a functional extrachromosomal molecule. The gene construct may be a linear minichromosome containing centromeres, telomeres, plasmids, or cosmids. A gene construct may also be a portion of the genome of a recombinant viral vector (including recombinant lentiviruses, recombinant adenoviruses, and recombinant adenovirus-associated viruses). A gene construct may be a portion of the genetic material of an attenuated microorganism or a recombinant microbial vector that survives within a cell. A gene construct may contain regulatory elements for gene expression of the coding sequence of the nucleic acid. These regulatory elements may be promoters, enhancers, start codons, stop codons, or polyadenylation signals. Nucleic acid sequences can form gene constructs that can be vectors. Vectors may have the ability to express fusion proteins, such as Cas9 fusion proteins or site-specific nucleases. Vectors may be recombinant. Vectors may contain heterologous nucleic acids encoding fusion proteins, such as Cas9 fusion proteins or site-specific nucleases. Vectors are useful for transfecting cells with nucleic acids encoding Cas9 fusion proteins or site-specific nucleases, where transformed host cells are cultured and maintained under conditions that result in the expression of the Cas9 fusion protein or site-specific nuclease system. The coding sequence can be optimized for stability and high-level expression. In some cases, codons are selected to mitigate the formation of RNA secondary structures (e.g., those formed for intramolecular bonding).

[0082] The vector may contain heterologous nucleic acids encoding a CRISPR / Cas9-dependent system or a site-specific nuclease, and may also contain a start codon (which may be located downstream of the CRISPR / Cas9-dependent system or site-specific nuclease coding sequence). The start and stop codons may be present in frame with the CRISPR / Cas9-dependent system or site-specific nuclease coding sequence. The vector may also contain a promoter operably linked to the CRISPR / Cas9-dependent system or site-specific nuclease coding sequence. Promoter operably linked to a CRISPR / Cas9-dependent system or a site-specific nuclease coding sequence may be a Simian virus 40 (SV40) promoter, a mouse mammary cancer virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter (e.g., a CMV early promoter), an Epstein-Barr virus (EBV) promoter, or a Roussarcoma virus (RSV) promoter. Promoter may also be derived from human genes (e.g., human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein). Promoter may also be a tissue-specific promoter, such as a muscle or skin-specific promoter, and may be natural or synthetic. An example of such a promoter is described in U.S. Patent Application Publication No. US20040175727 (the aforementioned document is incorporated herein by reference in its entirety). The vector may also contain a polyadenylation signal, which may be located downstream of a CRISPR / Cas9-dependent system or a site-specific nucleotide. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal derived from the pCEP4 vector (Invitrogen, San Diego, CA).

[0083] The vector may also contain an enhancer upstream of a CRISPR / Cas9-dependent system, i.e., a Cas9 protein or Cas9 fusion protein or sgRNA, or a site-specific nuclease. The enhancer may be required for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer (e.g., an enhancer derived from CMV, HA, RSV, or EBV). Polynucleotide functional enhancers are described in U.S. Patents 5,593,972, 5,962,428 and WO94 / 016737 (the contents of each are incorporated herein by reference). The vector may also contain a mammalian origin of replication to maintain the vector outside of chromosomes and to generate numerous copies of the vector within cells. The vector may also contain a regulatory sequence, which may be well adapted for gene expression in mammalian or human cells to which the vector is administered. The vector may also contain a reporter gene (e.g., green fluorescent protein ("GFP")) and / or a selectable marker (e.g., hygromycin ("Hygro")). The vector may be an expression vector or expression system for protein synthesis using routine techniques and readily available starting materials (as described in the following reference: Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor, 1989 (as described in the above reference is incorporated herein by reference)). In some embodiments, the vector may include a nucleic acid sequence encoding a CRISPR / Cas9-dependent system (including a nucleic acid sequence encoding a Cas9 protein or a Cas9 fusion protein), and a nucleic acid sequence encoding at least one gRNA, including at least one nucleic acid sequence of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625.

[0084] 16. Pharmaceutical Compositions The composition may be a pharmaceutical composition. The pharmaceutical composition may contain about 1 ng to about 10 mg of DNA encoding a CRISPR / Cas9-dependent system or a CRISPR / Cas9-dependent system protein component (i.e., Cas9 protein or Cas9 fusion protein). The pharmaceutical composition may contain about 1 ng to about 10 mg of DNA of a modified AAV vector and nucleotides encoding a site-specific nuclease. The pharmaceutical composition may contain about 1 ng to about 10 mg of DNA of a modified lentiviral vector. The pharmaceutical compositions of the present invention are formulated according to the mode of administration in which they are used. If the pharmaceutical composition is an injectable pharmaceutical composition, they are sterile, pyrogenic factor-free, and granular-free. Preferably, an isotonic formulation is used. Generally, additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution (e.g., phosphate-buffered saline) is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0085] The composition may further contain pharmaceutically acceptable excipients. pharmaceutically acceptable excipients may be molecules functional as vehicles, adjuvants, carriers, or diluents. pharmaceutically acceptable excipients may include transfection promoters, which include: surfactants, e.g., immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogues (including monophosphoryl lipid A), muramyl peptides, quinone analogues, vesicles (e.g., squalane and squalene), hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyvalent anions, polyvalent cations, or nanoparticles, or other known transfection promoters. The transfection promoter is a polyvalent anion, a polyvalent cation (including poly-L-glutamate (LGS)), or a lipid. The transfection promoter is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the composition for genome editing in skeletal muscle or cardiac muscle at a concentration of less than 6 mg / mL. The transfection promoter may also include surfactants, such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogues (including monophosphoryl lipid A), muramil peptides, quinone analogues, and vesicles (e.g., squalane and squalene), and hyaluronic acid may also be used for administration together with the gene construct. In some embodiments, the DNA vector encoding the composition may also contain transfection accelerators, such as lipids, liposomes (including lecithin liposomes or other liposomes known in the art), DNA-liposome mixtures (see, e.g., W09324640), calcium ions, viral proteins, polyvalent anions, polyvalent cations, or nanoparticles, or other known transfection accelerators. Preferably, the transfection accelerator is a polyvalent anion, a polyvalent cation (including poly-L-glutamate (LGS)), or a lipid.

[0086] 17. Delivery Method Provided herein is a method for delivering a pharmaceutical formulation (preferably the composition described above) for providing a gene construct. Delivery of the composition may be by transfection or electroporation of the composition as a nucleic acid molecule expressed in cells and delivered to the surface of those cells. The nucleic acid molecule can be electroporated using a BioRad Gene Pulser Xcell or an Amaxa Nucleofector IIb instrument. Several different buffers may be used, including: BioRad electroporation solution, sigma phosphate buffered saline product number #D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (NV). Transfection may include a transfection reagent (e.g., Lipofectamine 2000). Upon delivery of the vector to tissues and subsequently to mammalian cells, the transfected cells will express a fusion protein, such as a CRISPR / Cas9-dependent system and / or a site-specific nuclease. The composition can be administered to mammals to alter gene expression or to remanipulate or modify the genome. For example, the composition can be administered to mammals to modify the dystrophin gene. The mammals may be humans, non-human primates, dairy cows, pigs, sheep, goats, antelopes, bison, buffalo, animals of the genus Bos, deer, hedgehogs, elephants, llamas, alpacas, mice, rats, or chickens. The mammals are preferably humans, dairy cows, pigs, or chickens.

[0087] a. CRISPR / Cas9-dependent systems Vectors encoding CRISPR / Cas9-dependent system protein components (i.e., Cas9 proteins or Cas9 fusion proteins) can be delivered to mammals by DNA injection (also known as DNA vaccine immunization), which may or may not utilize in vivo electroporation, liposome-mediated, nanoparticle-assisted, and / or recombinant vectors. Recombinant vectors can be delivered by any viral system, which may be recombinant lentiviruses, recombinant adenoviruses, and / or recombinant adeno-associated viruses. Nucleotides encoding CRISPR / Cas9-dependent system protein components (i.e., Cas9 protein or Cas9 fusion protein) can be introduced into cells to genetically modify target genes or alter gene expression (e.g., activate or repress endogenous genes). For example, nucleotides encoding CRISPR / Cas9-dependent system protein components (i.e., Cas9 protein or Cas9 fusion protein), which are induced into a mutant dystrophin gene by gRNA, can be introduced into myoblasts of DMD patients. Alternatively, they can be introduced into fibroblasts of DMD patients, and the genetically modified fibroblasts can be treated with MyoD to induce differentiation into myoblasts (these can then be transplanted into a target animal (e.g., damaged muscle of the target animal) to demonstrate that the modified dystrophin protein is functional and / or to treat the target animal). The cells to be modified may be stem cells (e.g., induced pluripotent stem cells), bone marrow-derived progenitor cells, skeletal muscle progenitor cells, human skeletal muscle myoblasts from DMD patients, CD133+ cells, mesoangioblasts, and MyoD- or Pax7-transduced cells, or other myogenic progenitor cells. For example, a CRISPR / Cas9-dependent system can induce neuronal or myogenic differentiation of induced pluripotent cells.

[0088] 18. Route of administration The composition can be administered to target animals via various routes. These routes include oral, parenteral, sublingual, transdermal, rectal, topical, inhalation, cheek administration, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intra-articular administration, or combinations thereof. For veterinary use, the composition can be administered as a formulation that is appropriately acceptable in accordance with normal veterinary practice. Veterinarians can easily determine the most appropriate administration regimen and route for individual animals. The composition can be administered by conventional syringes, needleless injectors, “microprojectile bombardment gone guns” or by physical methods (e.g., electroporation (“EP”), “hydrodynamic methods”, or ultrasound). The composition can be delivered to mammals by several techniques, including: DNA injection (also known as DNA vaccine immunization) (with or without in vitro electroporation), liposome-mediated, nanoparticle-assisted, and recombinant vectors (e.g., recombinant lentivirus, recombinant adenovirus, and recombinant adeno-associated virus). The composition can be injected into skeletal muscle or cardiac muscle. For example, the composition can be injected into the tibialis anterior muscle.

[0089] 19. Cell type Any of these delivery methods and / or routes of administration could be used for countless cell types (e.g., cell types currently being studied in relation to cell-based therapies). These cell types could be fibroblasts, pluripotent stem cells, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, or the K562 human erythroid leukemia cell line. a.DMD Cell types currently being studied for DMD cell therapy include immortalized myoblasts (e.g., wild-type and DMD patient-derived lines, e.g., Δ48-50DMD, DMD8036 (dell48-50), C25C14, and DMD-7796 cell lines), primary DMD dermal fibroblasts, induced pluripotent stem cells, bone marrow-derived progenitor cells, skeletal myoblasts, human skeletal myoblasts from DMD patients, CD133+ cells, mesoangioblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and MyoD- or Pax7-transduced cells, or other myogenic progenitor cells. Immortality of human myogenic cells can be utilized for clonal induction of genetically modified myogenic cells. Cells can be modified ex vivo to isolate and propagate clonal populations of immortalized DMD myoblasts (these clonal populations contain a genetically modified dystrophin gene and do not have other nuclease transmutations in the protein-coding region of their genome). Alternatively, transient in vivo delivery of nucleases by non-viral or non-integrated viral gene transfer, or by direct delivery of purified protein and gRNA containing cell entry motifs, can minimize the risk of exogenous DNA integration or enable highly specific in situ modification where such risk is completely absent.

[0090] 20. Kit Provided herein is a kit that can be used to edit the genome (e.g., correct a mutated gene) in skeletal muscle or cardiac muscle. The kit includes the composition for genome editing in skeletal muscle or cardiac muscle as described above, and instructions for using the composition. The instructions included in the kit may be attached to the packaging material or included as a package insert. The instructions are typically typed or printed, but are not limited to such forms. In this disclosure, any medium capable of storing such instructions and further communicating them to end users is intended. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term “instructions” may include the address of the site providing such instructions. A composition for genome editing in skeletal muscle or cardiac muscle may include the modified AAV vector and nucleotide sequences encoding a site-specific nuclease described above. The site-specific nuclease may include the ZFN, TALEN, or CRISPR / Cas9-dependent system described above, which specifically binds to and cleaves the mutant gene. The site-specific nuclease described above is included in the kit and can specifically bind to and target a specific region of the mutant gene. The site-specific nuclease may be specific to the mutant dystrophin gene described above. The kit may further include the donor DNA, gRNA, or transgene described above.

[0091] a. CRISPR / Cas9-dependent systems Provided herein is a kit that can be used to modify a mutated gene. The kit includes at least one component for modifying a mutated gene and instructions for using a CRISPR / Cas9-dependent system. The instructions included in the kit may be attached to the packaging material or included as a package insert. The instructions are typically typed or printed, but are not limited to such forms. In this disclosure, any medium that can store such instructions and further deliver them to end users is intended. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term “instructions” may include the address of a site that provides such instructions. At least one component may include at least one CRISPR / Cas9-dependent system described above, which specifically targets a gene. The kit may include a Cas9 protein or Cas9 fusion protein, a nucleotide sequence encoding the Cas9 or Cas9 fusion protein, and / or at least one gRNA. The CRISPR / Cas9-dependent system described above is included in the kit and can target specific target regions upstream, in the middle, or downstream of the coding region of a target gene. For example, the CRISPR / Cas9-dependent system may be specific to the promoter region of the target gene, or the CRISPR / Cas9-dependent system may be specific to a mutant gene (e.g., the mutant dystrophin gene described above). The kit may include the donor DNA described above.

[0092] 21. Examples It will be readily apparent to those skilled in the art that other suitable modifications and applications of the methods disclosed herein are readily applicable and can be evaluated, and that such modifications and applications can be readily made using appropriate equivalents without departing from the scope of this disclosure or the features and embodiments disclosed herein. While this disclosure has been described herein, it will be understood more clearly by referring to the following examples (these examples are intended merely to illustrate some features and embodiments of this disclosure and should not be construed as limiting the scope of this disclosure). All references, U.S. patents, and disclosures of publications cited herein are incorporated herein by reference in their entirety. The present invention has a wide variety of features, as illustrated by the non-limiting embodiments described below.

[0093] [Example 1] Materials and methods Cell culture and transfection HEK293T cells were obtained from the American Tissue Collection Center (ATCC) via Duke University Cancer Center Facilities and maintained at 37°C under 5% CO2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. HEK293T cells were transfected with lipofectamine 2000 according to the manufacturer's instructions. Transfection efficiency routinely exceeded 80% when determined by fluorescence microscopy following delivery of the control eGFP expression plasmid. Cas9 expression plasmids were transfected in a 3:1 mass ratio to individual gRNA expression plasmids or equal amounts of gRNA expression plasmids (consisting of a mixture of four equal amounts of gRNAs). Primary mouse embryonic fibroblasts (PMEF-HL, Millipore, Billerica, MA) were seeded into 24-well TCPS plates (BD, Franklin Lakes, NJ) (7500 / well) and maintained in a complete MEF culture medium consisting of high-glucose DMEM supplemented with the following (10% Prenia Select FBS (Atlanta Biologicals, Lawrenceville, GA), 25 μg / mL gentamicin (Invitrogen), 1x GlutaMAX, non-essential amino acids, sodium pyruvate, and β-mercaptoethanol (Invitrogen)) at 37°C and 5% CO2. MEF transfection was performed at 1 μg cm. -2 The procedure was performed using a single dose of whole plasmid DNA and delivered as a cation nanoconjugate following electrostatic concentration with poly(CBA-ABOL) in serum and antibiotic-free OptiMEM, as previously described (Adler et al. Molecular therapy. Nucleic acids 1, e32, 2012). OptiMEM was replaced with complete MEF culture medium 4 hours after transfection. 48 hours post-transfection, MEFs were treated for qRT-PCR, or the complete MEF culture medium was replaced with N3 neuronal induction culture medium containing: DMEM / F-12 (Invitrogen), 1xN-2 supplement (Invitrogen), 10 ng / mL human bFGF2 (Stemgent, Cambridge, MA), and 25 μg / mL gentamicin (Invitrogen). Transfection conditions were optimized using a GFP reporter vector (pmax-GFP, 3486 bp, Amaxa, Cologne, Germany). The Cas9 expression plasmid was transfected with an equimix of four gRNA expression plasmids in a mass ratio of 3:1 or 1:1.

[0094] plasmidPlasmids encoding wild-type and H804A Cas9 were obtained from Addgene (Plasmid #39312 and Plasmid #39316; Jinek, et al. Science 337, 816-821, 2012). H840A Cas9 was cloned in-frame with respect to the N-terminal FLAG epitope tag and nuclear localization sequence (NLS) using a primer pair introducing the D10A mutation in vector pcDNA3.1. The VP64 domain, NLS, and HA epitope were cloned in-frame with respect to the C-terminal Cas9 ORF (Figure 1a, Figure 9a). TracrRNA and crRNA expression cassettes (Cong et al. Science 339, 819-823, 2013) were ordered as gBlocks (Integrated DNA Technologies, IDT) and cloned using pZDonor plasmids (Sigma) with the KpnI and SacII sites. Chimeric guide RNA expression cassettes (Mali et al. Science 339, 823-826, 2013) were also ordered as gBlocks and modified to include a BbsI restriction site to facilitate rapid cloning of novel guide RNA spacer sequences (Figure 9b). Oligonucleotides containing the target sequences were obtained from IDT, hybridized, phosphorylated, and cloned using appropriate plasmids with the BbsI site. The target sequences are provided in Table 2.

[0095] [Table 2]

[0096] Western blotCells were lysed using 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, and 0.1% SDS. The lysates were mixed with loading buffer and boiled for 5 minutes. Equivolutes of protein were electrophoresed on NuPAGE® Novex 4-12% or 10% Bis-Tris Gel polyacrylamide gel and transferred to a nitrocellulose membrane. Nonspecific antibody binding was blocked using 50 mM Tris / 150 mM NaCl / 0.1% Tween-20 (TBS-T) with 5% skim milk for 30 minutes. The membrane was incubated with the following primary antibodies (overnight with HRP-conjugated anti-Flag (Cell Signaling, Cat#2044) diluted 1:1000 in 5% BSA in TBS-T; for 30 minutes with anti-GAPDH (Cell Signaling, clone 14C10) diluted 1:5000 in 5% milk in TBS-T; or anti-ASCL1 (Santa Cruz, clone sc-48449) diluted 1:500 in 5% BSA; or anti-g-globin (Santa Cruz, clone 51-7) diluted 1:500 in 5% milk), and the membrane was washed with TBS-T. The membrane labeled with the primary antibody was incubated with anti-rabbit HRP-conjugated antibody (Sigma-Aldrich) diluted 1:5000 for 30 minutes, or with anti-goat antibody (1:3000), or anti-mouse antibody (1:5000), and then washed with TBS-T for 30 minutes. Immun-Star WesternC TM The membrane was visualized using the kit (Bio-Rad), and ChemiDoc TM Images were captured using an XRS+System and processed using ImageLab software (Bio-Rad).

[0097] ELISASerum-free culture medium (OPTI-MEM) was collected and frozen at -80°C. Human IL-1ra secretion into the culture medium was quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (R&D Systems, Cat. No. DY280). Recombinant human IL-1ra was diluted with OPTI-MEM to prepare a standard curve, and IL-1ra in the culture medium was measured without dilution. Samples were concentrated approximately 8-fold by centrifugation for 20 minutes through a 3 kDa MWCO filter (Amicon Ultra, Cat # UFC500396). Reported values ​​were corrected for the concentration factor of each sample. Optical density was measured at 450 nm (wavelength corrected at 540 nm). Each standard and sample was assayed twice. The two readings were averaged and standardized by subtracting the optical density of the mean zero standard. A standard curve was created by log-transforming the data and performing linear regression on the optical density of IL-1ra concentration. The reported values ​​are the mean of three separate experiments (n=3) and the standard error of that mean (the three experiments were performed on different days using technically identical equipment, and the average was calculated for each experiment). qRT-PCR Total RNA was isolated using the RNeasy Plus RNA Isolation Kit (Qiagen). cDNA synthesis was performed using SuperScript® VILO. TM The procedure was performed using a cDNA synthesis kit (Invitrogen). Real-time PCR using PerfeCTa® SYBR® Green FastMix was performed using the CFX96 real-time PCR detection system (Bio-Rad) with the oligonucleotide primers reported in Table 3. The primers were designed using Primer3Plus software and purchased from IDT.

[0098] [Table 3] The specificity of the primers was confirmed by agarose gel electrophoresis and melting curves. The reaction efficiency over an appropriate dynamic range was calculated to ensure the linearity of the standard curve (Figure 10). The result was ΔΔC TThis is expressed as a multiplier increase in mRNA expression of the gene in question, standardized for GAPDH expression. The reported values ​​are the mean of three separate experiments (n=3) and the standard error of that mean (the three experiments were performed on different days using technically identical equipment, and the average was calculated for each experiment).

[0099] RNA-seq : An RNA-seq library was constructed. Briefly, the first strand of cDNA was synthesized from oligo-dT Dynabead™ captured mRNA using the SuperScript™ VILO™ cDNA synthesis kit (Invitrogen). The second strand was synthesized using DNA polymerase I (New England Biolabs). The cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter), and the cDNA was simultaneously fragmented using Nextera transporase (Illumina; 5 min at 55°C), and sequencing primers were inserted into the double-stranded cDNA. The transfer reaction was stopped using QG buffer (Qiagen), and the fragmented cDNA was purified on AMPure XP beads. An indexed sequencing library was generated by 6 cycles of PCR. Libraries were sequenced using 50bp single-end reads on a two-lane Illumina HiSeq 2000 instrument, generating 29 to 74 million reads per library. Bowtie (Langmeadet et al. Genome biology 10, R25, 2009) was used to align the reads with human RefSeq transcripts. DESeq (Anders et al. Genome biology 11, R106, 2010) was used to calculate the statistical significance of discriminative expression (including various hypothesis testing modifications). The number of reads from raw RNA-seq and the reads aligned with each RefSeq transcript were submitted to public access in the Gene Expression Omnibus (GEO) (accession numbers are currently being finalized).

[0100] Immunofluorescence staining To detect Tuj1 and MAP2 expression, transfected MEFs were fixed at room temperature (RT) for 20 minutes on day 10 of incubation in N3 culture medium containing 4% PFA (EMS, Hatfield, PA). The cells were then incubated at 4°C for 2 hours at room temperature with rabbit anti-Tuj1 (Covance, Princeton, New Jersey, clone TUJ1 1-15-79, 1:500) or mouse anti-MAP2 (BD, clone Ap20, 1:500) in block buffer (containing: 0.2% Triton X-100, 3% w / v BSA, and 10% goat serum (Sigma-Aldrich, Saint Louis, MO)) or mouse anti-MAP2 (BD, clone Ap20, 1:500), or with mouse anti-Ascl1 (BD, clone 24B72D11.1, 1:100) for a further 24 hours. The cells were then washed three times with PBS and incubated at room temperature for 1 hour with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen, 1:200) in block buffer, followed by three more washes with PBS. The stained MEFs were then scanned using a Nikon Eclipse mounted on a ProScan II motorized stage (Prior Scientific, Rockland, MA). The cells were scanned with a TE2000-U inverted fluorescence microscope to create mosaic images of each complete culture region. These mosaics were processed using FIJI macros, with thresholds automatically and homogeneously adjusted according to local contrast, small debris removed, and the number of Tuj1+ cells in each well counted. statistics Statistical analysis was performed using the Tukey test with an alpha equivalent to 0.05 in JMP 10 Pro.

[0101] [Example 2] result To create a CRISPR / Cas9-dependent transcriptional activation system, iCas9 was generated by mutating the catalytic residues of Cas9 (D10A, H840A) and then fused with the C-terminal VP64 acidic transactivation domain (Figure 1a, b). Western blotting of the N-terminal Flag epitope tag revealed active iCas9-VP64 expression from plasmids transfected in human embryonic kidney (HEK) 293T cells (Figure 3). The CRISPR system recognizes its target by base pairing a 20 bp sequence in the gRNA with a complementary DNA target (the complementary DNA target is followed by an NGG protospacer adjacent motif (PAM) sequence, where N is any base pair). The combination of synthetic transcription factors targeted by the endogenous human promoter results in synergistic and active gene expression activation. Therefore, four gRNAs following the NGG PAM sequence were identified within 500 bp of the transcription start site at the promoter of the IL1RN gene (Figure 4, Table 2). To compare crRNA- and gRNA-dependent targeting methods, the four target site sequences were subjected to crRNA and gRNA expression plasmids. 17The gRNAs were introduced and transfected into HEK293T cells along with the iCas9-VP64 expression plasmid. Substantial induction of IL1RN expression was not observed by qRT-PCR in crRNA combination-treated samples, but much higher levels were achieved with gRNA combinations (Figure 1c). No changes in gene expression were observed in cells treated with iCas9 expression plasmids that did not contain gRNA or VP64, demonstrating the decisive role of the activating domain in gene expression regulation (Figure 1c). Nuclease activity at these target sites was confirmed to be stopped in the iCas9-VP64 system by performing a surveyor assay (this assay detects DNA repair events in samples treated with iCas9-VP64 and wild-type Cas9) (Figure 5). Transfecting each of the four gRNAs individually or together resulted in a dramatic increase in gene expression by targeting a wide variety of sites in the promoter with the gRNA combinations (Figure 1d). High levels of IL1RN expression were observed only when the gRNA combination was co-transfected with iCas9-VP64, as was observed with other classes of engineered transcription factors (Figure 1d). Similarly, the production of the IL-1 receptor antagonist (IL-1ra) protein encoded by the IL1RN gene was observed in only 3 out of 6 samples treated with the gRNA combination through 3 separate experiments, but was not detected at all in samples treated with a single gRNA or control plasmid (Figure 1e). To investigate the specificity of gene activation by iCas9-VP64, comprehensive gene expression in HEK293T cells treated with a combination of four gRNAs was determined by RNA-seq (Figure 1f). Notably, only one gene showed a significant increase in expression compared to the control (false detection rate of 3 x 10⁻⁶). -4 The following are four isoforms expressed from the IL1RN gene locus (Figure 4), demonstrating high levels of gene activation specificity.

[0102] To demonstrate the versatility of this system, four gRNAs were designed to target the promoters of eight other genes relevant to medicine and biotechnology (including ASCL1, NANOG, HBG1 / 2, MYOD1, VEGFA, TERT, IL1B, and IL1R2) (Figure 4, Table 2). Overexpression of ASCL1 and MYOD1 results in transdifferentiation from several cell types to neuronal and myogenic phenotypes, respectively. NANOG is a marker of pluripotency and is also used in genetic reprogramming methods. Activation of homologous HBG1 and HBG2 (encodes γ-globin during fetal development) can be used as a therapeutic method for β-globin mutations in sickle cell disease. Upregulation of VEGFA with synthetic transcription factors has been explored as a method to enhance tissue regeneration and wound healing. Overexpression of telomerase (encoded by the TERT gene) can be used for cell line immobilization. IL1B encodes the IL-1β cytokine that mediates inflammation and autoimmunity. IL-1β signaling can be blocked by the expression of IL-1ra or a decoy receptor (encoded by IL1R2). The expression of each of these genes was enhanced by co-transfection of HEK293T cells with expression plasmids for iCas9-VP64 and four gRNAs, as determined by qRT-PCR (Figure 2). In some cases, the expression of a single gRNA was sufficient to induce gene expression, but in all cases, co-transfection of four gRNAs resulted in a synergistic effect (Figure 2a-d). Notably, accessibility to chromatin, as determined by DNase-seq, was not a predictor of successful gene activation (Figure 4). RNA-seq was performed on cells transfected with iCas9-VP64 and four gRNAs (targeting HBG1, three of which also target HBG2). This showed a specific and reproducible increase in the expression of both HBG1 and HBG2 (which cannot be distinguished by RNA-seq), although statistical significance was not obtained due to low total expression levels (Figure 6).Following treatment with iCas9-VP64 and four gRNAs, increased protein expression of Ascl1 and γ-globin was detected by Western blotting (Figure 7), supporting higher mRNA levels observed by qRT-PCR (Figure 2). Low baseline levels of Ascl1 and γ-globin proteins were detectable with an empty vector control. As preliminary evidence that activation of gene targets by iCas9-VP64 can lead to secondary changes in gene networks and cellular phenotype, expression plasmids for iCas9-VP64 and four gRNAs targeting ASCL1 were cotransfected into mouse embryonic fibroblasts (MEFs) (Figure 8). Overexpression of Ascl1 in MEFs was shown to partially activate the neuronal gene network (including the downstream target Tuj1). Since the gRNA target site is conserved in the human and mouse ASCL1 promoter (Figure 8a), activation of ASCL1 expression was also observed in MEFs treated with iCas9-VP64 and plasmids encoding four gRNAs (Figure 8b). Furthermore, cells expressing Ascl1 and the neuronal marker Tuj1 were readily detected by immunofluorescence 12 days post-transfection in iCas9-VP64 / gRNA-treated samples (Figure 8c-h). Tuj1-positive cells were not observed in control plasmid-treated cells.

[0103] To date, there have been no comprehensive studies on the specificity of Cas9 / CRISPR activity in mammalian cells. RNA-seq has shown that activation of targeted genes is highly specific, and activation of off-target genes is undetectable (Figure 1f, Figure 6). IL1RN and HBG1 / 2 were selected for this specificity analysis because their gene products (IL-1ra and γ-globin) do not have a secondary effect on gene expression in HEK293T cells. In contrast to the use of a single potent activator, the use of synergistic activity of a variety of weak transcriptional activators may enhance specific gene regulation because a wide variety of adjacent off-target sites are less likely to reside at other gene loci. Interestingly, the IL32 gene was moderately downregulated (false detection rate <0.03) in both samples treated with iCas9-VP64 and IL1RN-targeted or HBG1 / 2-targeted gRNA compared to a control sample treated with empty expression plasmids alone (Figure 1f, Figure 6). Since both IL1RN target induction and HBG1 / 2 target induction samples were similarly affected, it is unlikely that this is a result of off-target iCas9-VP64 activity related to the identity of the target sequence. To evaluate the specificity of iCas9-VP64 binding to the genome, ChIP sequencing was performed using an anti-HA antibody in cells treated with iCas9-VP64 and four gRNAs targeting the IL1RN promoter. The experiment showed that iCas9 targets the IL1RN promoter (Figure 15). Furthermore, the experiment showed an extremely high level of specificity (Figure 15). iCas9 had only 10 potential off-target binding sites (FDR < 5%). To further investigate specificity, RNA sequencing experiments were performed on iCas9 EGEM, and it was found that only the IL1RN gene isoform increased expression compared to the control (FDR < 3 x 10.4).

[0104] [Example 3] CRISPR materials and methods targeting the dystrophin gene Plasmid constructAs previously described (Perez-Pinera et al., Nat Methods 10:973-976, 2013), we used expression cassettes of Streptococcus pyogenes sgRNA and human codon-optimized Cas9 (hCas9) nuclease. To construct a fluorescent reporter system for enriching CRISPR / Cas9-modified cells, we synthesized a gene block (IDT) and subsequently cloned it using an hCas9 expression vector (the gene block contained a portion of the 3' end of the Cas9 coding sequence fused to a T2A skipping peptide immediately upstream of the multi-cloning site). Subsequently, we cloned the eGFP reporter gene using a T2A vector and translated both Cas9 and eGFP proteins together from the same expression vector (hCas9-T2A-GFP, SEQ ID NO: 116). Cell culture and transfection HEK293T cells were obtained from the ATCC (American Tissue Cell Collection Center) via the Duke Cell Culture Facility and maintained in DMEM supplemented with 10% calf serum and 1% penicillin / streptomycin. Immortalized myoblasts (Mamchaoui, K. et al. Skelet Muscle 1, 1-11, 2011) (one from a wild-type donor and two from Δ48-50 DMD patients) were maintained in skeletal muscle culture medium (PromoCell) supplemented with: 20% calf serum (Sigma), 50 μg / mL fetuin, 10 ng / mL human epidermal growth factor (Sigma), 1 ng / mL human basement membrane fibroblast growth factor (Sigma), 10 μg / mL human insulin (Sigma), 1% GlutaMAX (Invitrogen), and 1% penicillin / streptomycin (Invitrogen). Primary DMD dermal fibroblasts were obtained from the Coriell Cell Repository (GM05162A, Δ46-50) and maintained in DMEM supplemented with 10% fetal bovine serum, 1 ng / mL human basement membrane fibroblast growth factor, and 1% penicillin / streptomycin. All cell lines were maintained at 37°C and 5% CO2. HEK293T cells in 24-well plates were transfected with 400 ng of each expression vector using Lipofectamine 2000 (Invirogen) according to the manufacturer's protocol. Immortalized myoblasts and primary fibroblasts were transfected with 5 μg of each expression vector by electroporation using Gene Pulser XCell (BioRad), with optimized conditions for each cell strain and PBS used as the electroporation buffer (Figure 1) (Ousterout et al. Mol Ther 21:1718-1726, 2013). Transfection efficiency was measured using eGFP expression plasmid (pmaxGFP, Clontech) delivery and flow cytometry. These efficiencies were routinely over 95% in HEK293T cells and over 70% in primary fibroblasts and immortalized myoblasts. The expressed mass of the electroporated plasmids is consistent with the amount used for each CRISPR / Cas9-dependent system.

[0105] Quantitative analysis of Cel-I in endogenous gene modifications (surveyor assay) CRISPR / Cas9-dependent system-induced damage at endogenous target sites was quantified by a surveyor nuclease assay (Guschin, DY et al. Meth Mol Biol 649, 247-256, 2010) (this assay can detect the characteristics of nuclease-mediated NHEJ mutations). After transfection, cells were incubated at 37°C for 3 to 10 days, and genomic DNA was extracted using the DNeasy blood tissue kit (Qiagen). Target loci were amplified by 35 cycles of PCR using the AccuPrime High Fidelity PCR kit (Invitrogen). Primers specific to each locus, e.g., 5'-GAGTTTGGCTCAAATTGTTACTCTT-3' (SEQ ID NO: 60) and 5'-GGGAAATGGTCTAGGAGAGTAAAGT-3' (SEQ ID NO: 61), were used (see Table 4).

[0106] [Table 4-1]

[0107] [Table 4-2]

[0108] The obtained PCR products were randomly thawed and re-annealed in a thermal cycler using the following program: 95°C for 240 seconds, followed by 85°C for 60 seconds, 75°C for 60 seconds, 65°C for 60 seconds, 55°C for 60 seconds, 45°C for 60 seconds, 35°C for 60 seconds, and 25°C for 60 seconds (the rate between steps was -0.3°C / second). Following re-annealing, 8 μL of PCR product was mixed with 1 μL of surveyor nuclease S and 1 μL of enhancer S (Transgenomic) and incubated at 42°C for 1 hour. After incubation, 6 μL of digested product was loaded onto a 10% TBE polyacrylamide gel and electrophoresed at 200V for 30 minutes. The gel was stained with ethidium bromide and quantified by densitometry using ImageLab (Bio-Rad), as previously described (Guschin, et al. Meth Mol Biol 649, 247-256, 2010). Fluorescence-activated cell selection of myoblasts DMD myoblasts were electroporated with 5 micrograms each of hCas9-T2A-GFP and sgRNA expression vectors, and incubated at 37°C under 5% CO2. Three days after electroporation, the cells were trypsin-digested and collected for FACS sorting using a FACSvantage II sorting instrument. GFP-positive cells were collected and grown for analysis. PCR-dependent assay for detecting genomic deletionsExon 51 or exon 45-55 loci were amplified from genomic DNA by PCR (nvitrogen AccuPrime High Fidelity PCR kit) (using flanking primers for each locus). The flanking primers used were CelI-CR1 / 2-F and CelI-CR5-R for exon 51 analysis, and CelI-CR6-F and CelI-CR36-R for exon 45-55 analysis (Table 4). The PCR products were separated on a TAE agarose gel and stained with ethidium bromide for analysis.

[0109] PCR-based detection of translocations Gene loci with anticipated possible translocations were amplified by two-step nested PCR (Invitrogen AccuPrime High Fidelity PCR kit for each step) of genomic DNA from cells transfected with Cas9 alone (control) or with sgRNA. In the first step, the translocations that could occur at each on-target and off-target sgRNA target site were amplified by 35 cycles of PCR. A combination of surveyor primers was used for each locus, modified to include restriction sites to facilitate cloning and sequencing analysis (Table 4). One microliter of each PCR reaction was subjected to a second round of amplification by 35 cycles of PCR, using a custom-designed nested primer set for each individual anticipated translocation (Table 4). Each of the second nested PCR primers bound to approximately the same region within the primary amplicon, but each pair was optimized using Primer3 online bioinformatics software to ensure specific detection of each translocation. PCR amplicons that matched the expected length of the predicted translocation and were present only in cells treated with sgRNA were purified (using the QIAGEN gel extraction kit) and analyzed by Sanger sequencing.

[0110] mRNA analysisImmortalized myoblasts were differentiated into muscle fibers by changing the growth medium for 5 days with DMEM supplemented with 1% insulin-transferrin-selenium (Invitrogen #51500056) and 1% penicillin / streptomycin (Invitrogen #15140). The cells were then collected by trypsin digestion. Total RNA was isolated from the cells using the RNeasy Plus Mini kit (QIAGEN) according to the manufacturer's instructions. The RNA was reverse transcribed into cDNA using the VILO cDNA synthesis kit (Life Technologies #11754) and 1.5 micrograms of RNA at 42°C for 2 hours according to the manufacturer's instructions. The target gene locus was amplified by 35 cycles of PCR using the AccuPrime High Fidelity PCR kit (Invitrogen). For detection of exon 51 using CR1 / 5 or CR2 / 5, primers annealing to exons 44 and 52 were used, or for detection using CR6 / 36, primers annealing to exons 44 and 60 were used (Table 4). PCR products were electrophoresed on TAE agarose gel and stained with ethidium bromide for analysis. The degraded PCR bands were cloned and analyzed by Sanger sequencing to confirm the expected exon ligation. Table 5 is a list of primer sequences used in Example 4.

[0111] [Table 5-1]

[0112] [Table 5-2]

[0113] [Table 5-3]

[0114] [Table 5-4]

[0115] Western blot analysis : To determine dystrophin protein expression, incorrupted myoblasts were differentiated into muscle fibers by changing the growth medium with DMEM supplemented with 1% insulin-transferrin-selenium (Invitrogen) and 1% antibiotic / antifungal agent (Invitrogen) for 4–7 days (e.g., 6 or 7 days). Fibroblasts were then transdifferentiated by inducing MyoD overexpression and incubating the cells for 15 days in DMEM supplemented with 1% insulin-transferrin-selenium (Invitrogen), 1% antibiotic / antifungal agent (Invitrogen), and 3 μg / mL doxycycline. Dystrophin expression was determined 3 days after transfection of HEK293T cells. Cells were collected by trypsin digestion and lysed in RIPA buffer (Sigma) supplemented with a protease inhibitor cocktail (Sigma). Total protein content was quantified using a bicinchoninic acid assay according to the manufacturer's (Pierce) instructions. Next, the sample was mixed with NuPAGE loading buffer (Invitrogen) and 5% β-mercaptoethanol and heated at 85°C for 10 minutes. 25 micrograms of protein were separated on a 4-12% NuPAGE bis-tris gel (Invitrogen) using MES buffer (Invirogen). The protein was transferred to a nitrocellulose membrane for 1-2 hours in a transfer buffer containing 10-20% methanol (e.g., 10% methanol) and 0.01% SDS. The blot was then blocked with 5% milk-TBST at room temperature for 1 hour. The blot was examined using the following primary antibodies: MANDYS8 (1:100, Sigma D8168) and rabbit anti-GAPDH (1:5000, Cell Signaling 2118S) for dystrophin detection. Next, the blots were incubated with mouse or rabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz), and then visualized using a ChemiDoc chemiluminescence system (BioRad) and a Western-C ECL substrate (BioRad). Transplantation into immunodeficient miceAll animal experiments were conducted according to protocols approved by the Duke Institutional Animal Care & Use Committee. Cells were collected by trypsin digestion and washed with 1x Hanks buffer solution (HBBS, Sigma). Two million cells were pelleted and resuspended in 5 μL of 1x HBBS (Sigma) supplemented with cardiotoxin (Sigma #C9759) immediately before injection. The cells were transplanted into the hindlimb anterior tibialis muscle of NOD.SCID.gamma (NSG) mice (Duke CCIF Breeding Core) by intramuscular injection. Four weeks after injection, the mice were euthanized and the TA muscle was collected.

[0116] Immunofluorescence stainingCollected TA muscle tissue was incubated overnight at 4°C in 30% glycerol, then mounted in an OCT compound (Optimal Cutting Temperature compound) and frozen. Serial 10-micron sections were obtained by freeze-dividing the embedded muscle tissue at -20°C. The frozen sections were then washed with PBS to remove the OCT compound, followed by blocking at room temperature for 30-60 minutes in PBS containing 10% heat-inactivated fetal bovine serum for spectrin detection, or in PBS containing 5% heat-inactivated fetal bovine serum for dystrophin detection. The frozen sections were incubated overnight at 4°C with the following primary antibodies specific only to human epitopes: anti-spectrin (1:20, Leica NCL-SPEC1) or anti-dystrophin (1:2, Leica NCL-DYS3). After primary staining, spectrin or dystrophin expression was detected using a tyramide-dependent immunofluorescence signal amplification detection kit (Life Technologies, TSA Kit #22, catalog #T-20932). In short, frozen sections were incubated with 1:200 goat anti-mouse biotin-XX secondary antibody (Life Technologies #B2763) in block buffer at room temperature for 1 hour. The signal was then amplified with streptavidin-HRP conjugate (1:100, TSA kit) in block buffer at room temperature for 1 hour. Finally, the frozen sections were incubated with tyramide-AlexaFluor488 conjugate (1:100, TSA kit) in manufacturer-provided amplification buffer at room temperature for 10 minutes. The stained frozen sections were then mounted in ProLong AntiFade (Life Technologies #P36934) and visualized using a standard fluorescence microscope. Cytotoxic assayTo quantitatively determine potential sgRNA or SpCas9 nuclease-associated cytotoxicity, HEK293T cells were transfected with 10 ng of GFP reporter, 100 ng of SpCas9 expression vector, and 100 ng of sgRNA expression vector using lipofectamine 2000 according to the manufacturer's (Invitrogen) instructions. The percentage of GFP-positive cells was determined by flow cytometry on days 2 and 5. Survival rate was calculated as the decrease in GFP-positive cells from day 2 to day 5 and standardized against cells transfected with empty nuclease expression vectors as described (Cornu et al., Meth Mol Biol 649:237-245, 2010).

[0117] [Example 3] CRISPR targeting dystrophy genes results We designed a CRISPR / Cas9-dependent system to target the dystrophin gene. Diverse gRNAs were selected based on NNNNN NNNNN NNNNN NNNNN NGG and GNNNN NNNNN NNNNN NNNNN NGG to target various regions of the human and mouse dystrophin genes (see Tables 6, 7, and 8).

[0118] [Table 6-1]

[0119] [Table 6-2]

[0120] [Table 7]

[0121] [Table 8]

[0122] In particular, 400 ng of Cas9 was co-transfected into HEK293T cells with 400 ng of an empty vector or gRNA (targeting the region containing exon 51 (i.e., CR1, CR2, CR3, CR4, and CR5)) (see Figure 11(b)). Genomic DNA was collected two days after transfection and analyzed using a surveyor assay (see Figures 11(a) and 11(c)). The CRISPR / Cas9-dependent system was used in DMD8036(del48-50) cells to determine whether the system could repair mutant dystrophin genes. 5 μg of Cas9 was co-transfected into DMD8036(del48-50) cells with 7.5 μg of an empty vector or gRNA. Specifically, 7.5 μg of CR1 ("DCR1"), 7.5 μg of CR5 ("DCR5"), 15 μg of CR3 ("DCR3"), or a combination of 7.5 μg of CR1 and CR5 (DCR1+DCR5) were used. Genomic DNA was collected 3 days after transfection and analyzed using a surveyor assay (Figure 12) or PCR analysis across all loci (Figure 13). These loci were amplified by PCR. Using primers that flanked regions containing genomic targets for CR1 and CR5 (forward primer: 5'-gagaggttatgtggctttacca (SEQ ID NO: 457), reverse primer: 5'-ctgcgtagtgccaaaacaaa (SEQ ID NO: 458)), a band of 1447 bp was obtained for the wild-type locus, or approximately 630 bp for the deletion locus, as predicted. After 7 days of differentiation, Western blots of treated cells showed expression of dystrophin protein (see Figure 14).

[0123] [Example 5] Targeting induction of CRISPR / Cas9 to the human dystrophin gene hotspot To utilize the CRISPR / Cas9 gene editing platform for the correction of a wide range of dystrophin mutations, multiple sgRNAs targeted to the hotspot mutation regions between exons 45-55 were generated (Figure 16). A Streptococcus pyogenes system was used, which utilized a human codon-optimized SpCas9 nuclease and a chimeric single guide RNA (gRNA) that guides efficient site-specific gene editing. As in the targeting to exon 51 by TALEN in Example 4, protospacers were selected to target the 5' and 3' ends of exons 45 to 55 (the foregoing meets the 5'-NRG-3' PAM requirement of SpCas9). Small insertions or deletions resulting from NHEJ-based DNA repair within these exons can generate targeted frameshift mutations (the mutations correspond to various dystrophin mutations around each exon) (Figures 16A-16B). For example, CR3 was designed to correct dystrophin mutations or deletions around exon 51 by introducing small insertions or deletions at the 5' end of exon 51 to restore the downstream dystrophin reading frame (Figure 16B). In addition, the sgRNAs utilized the combined performance of the CRISPR / Cas9 system, similar to the method of exon skipping by oligonucleotides, and specifically deleted individual exons or a series of exons to repair the dystrophin reading frame. For this purpose, the sgRNAs targeted the intron regions around exon 51 (Figure 16C) or exons 45-55 (Figure 16D). These sgRNAs were intentionally targeted to the site closest to the downstream or upstream exons intended to be included in the generated transcript, minimizing the probability that background patient deletions would include the intron sgRNA target site.

[0124] [Example 6] Screening of sgRNAs targeting the dystrophin gene in human cells The gene editing frequency in human HEK293T cells was determined to rapidly determine the targeting efficiency of various sgRNAs. HEK293T cells were transfected with constructs encoding human codon-optimized SpCas9 and the sgRNAs of interest. Each sgRNA was designed to modify the dystrophin gene as indicated. The gene modification frequency on the 3rd or 10th day after transfection was determined by the Surveyor assay. The ratio of the measured Surveyor signals on the 3rd and 10th days was calculated to quantify the stability of gene editing for each sgRNA in human cells. As quantified by the Surveyor assay 3 days after transfection, 29 / 32 (~90%) of the sgRNAs tested were able to mediate highly efficient gene modification at the intended locus (Table 9, Figure 17). Gene editing was stable for almost all sgRNAs (signal change from day 3 to day 10 was less than 25%, Table 9, Figure 18), indicating that gene editing mediated by each individual sgRNA was well tolerated. The notable exception was CR33, which showed no detectable activity on the 10th day (however, the activity may be lower than the sensitivity of the Surveyor assay (estimated ~1%)).

[0125]

Table 9

[0126] [Example 7] Enrichment of gene-edited cells using fluorescence-dependent reporter systems sgRNAs were selected to correct specific mutations in myoblast cell lines from DMD patients. Contrary to expectations, low or undetectable gene editing activity was observed after transfection of DMD myoblasts by surveyor assay (Figure 19C, mixed population). Flow cytometry was used to select transfected cells co-expressing GFP via a 2A ribosome skipping peptide linked to the SpCas9 protein (Figure 19A). Addition of this fluorescent reporter to the SpCas9 expression vector did not appear to significantly affect gene editing activity in HEK293T cells (Figure 19B). Despite the high transfection efficiency of the control GFP expression plasmid (typically over 70%, Figure 19D, pmaxGFP), a low percentage of transfected myoblasts (~0.5-2%) expressed the fluorescent reporter 3 days after electroporation. Assuming that high levels of CRISPR / Cas9 activity are given in HEK293T cells, which are easily transfected, the inefficient transgene expression of SpCas9-T2A-GFP and sgRNA constructs in DMD cells after electroporation can explain the low gene editing efficiency observed in unselected cells. After sorting of GFP-positive DMD myoblasts, a substantial increase in detectable activity was observed at most sgRNA target loci (Figure 19C). Therefore, all subsequent experiments used cells sorted for SpCas9 expression by the expression of this fluorescent reporter.

[0127] [Example 8] Repair of the dystrophin gene by targeted-induced frameshift By using small insertions and deletions resulting from NHEJ DNA repair, it is possible to create targeted, induced frameshifts and correct abnormal reading frames. We designed sgRNA,CR3, and modified the dystrophin reading frame by introducing small insertions and deletions within exon 51 (Figure 16B, 20A). The type of insertion and deletion generated by CRISPR / Cas9 at this locus was determined by Sanger sequencing of alleles derived from genomic DNA of HEK293T cells co-transfected with expression plasmids for SpCas9 and CR3 sgRNA (Figure 20B). Notably, the insertions and deletions resulted in transformations for all three reading frames (Figure 20B, 20C). To demonstrate gene modification in relevant patient cell lines, expression plasmids for SpCas9 and CR3 sgRNA were electroporated into DMD myoblast cell lines with deletions in exons 48-50 (these deletions can be corrected by generating a frameshift at exon 51). Treated cells were selected and their gene-modifying activity was verified by a surveyor assay (CR3, selected population, Figure 19C). Further differentiation into myotubes was then tested for dystrophin expression repair. Dystrophin protein expression was observed in conjunction with detectable nuclease activity (Figure 20D). The Streptococcus pyogenes CRISPR / Cas9 system rapidly generates targeted-induced frameshifts, providing a robust method for repairing human dystrophin gene expression in response to diverse patient mutations.

[0128] [Example 9] Combined CRISPR / Cas9 gene editing mediates gene deletion at exon 51, restoring dystrophin protein expression. The combined capabilities of the CRISPR / Cas9 system provide a novel method for efficiently generating gene deletions of specific exons for targeted induced modification. Myoblasts from DMD patients with background deletions that can be modified by exon 51 skipping were treated with two combinations of sgRNAs flanking exon 51 (CR1 / CR5 or CR2 / CR5) and sorted to enrich gene-edited cells as shown in Figure 19. The expected genomic deletion was present only when both sgRNAs were electroporated into cells with SpCas9, as detected by endpoint PCR of the genomic DNA of these treated cells (Figure 21A). Sanger sequencing confirmed the expected linkage of distal chromosome segments for both deletions (Figure 21B). After differentiation of the sorted myoblasts, the exon 51 deletion was detected only in cells treated with both sgRNAs from mRNA transcripts (Figure 21C). Finally, repair of dystrophin protein expression was detected in treated cells in conjunction with the observation of genomic and mRNA-level deletions of exon 51 (Figure 21D).

[0129] [Example 10] Dystrophin recovery due to multi-exome macrogenomic deletion While addressing patient-specific mutations is a powerful application of the CRISPR / Cas9 system, developing a single method capable of addressing numerous common patient deletions would be beneficial. For example, a promising method is the removal of the entire exon region 45-55, which corrects up to 62% of known patient deletions. We tested a combined CRISPR / Cas9-dependent gene editing approach to determine its ability to efficiently induce exon 45-55 deletions in human cells. Following transfection into HEK239T cells, a predicted deletion of ~336,000 bp was detected by PCR of genomic DNA (Figure 22A). Similarly, this deletion was detected by PCR of genomic DNA in SpCas9 / sgRNA-treated cells from DMD patients carrying a background deletion of unknown length in exons 48-50 (Figure 22A). Sanger sequencing of this deletion band derived from the genomic DNA of treated DMD cells revealed the expected ligation of introns 44 and 55 adjacent to the sgRNA target site (Figure 22B). After differentiation of treated DMD cells, the expected deletion of exons 45-55 was detected in dystrophin mRNA transcripts and confirmed by Sanger sequencing to be a fusion of exons 44 and 56 (Figure 22C). Repaired protein expression was observed by Western blotting in a selected cell population (containing CRISPR / Cas9-induced deletions of exons 45-55 from the genome and the resulting mRNA transcripts) (Figure 22D). These data demonstrate that combined CRISPR / Cas9 editing provides a versatile single method for repairing the dystrophin reading frame in over 60% of DMD patients with mutations.

[0130] [Example 11] Transplantation of modified myoblasts into immunodeficient mice A promising method for treating DMD is to modify the patient's own myoblast population (these can be transplanted into the patient's skeletal muscle to restore dystrophin expression). To demonstrate the in vivo expression capability of modified cells for human dystrophin, DMD myoblast populations treated with sgRNA CR1 and CR5 (flanking at exon 51) were transplanted and screened for GFP expression, as previously done (Figures 19 and 23). After 4 weeks, human spectrin-positive muscle fibers (expressed in both modified and unmodified cells) were detected in frozen sections of injected mouse tissue (Figure 24). Many of these fibers were also positive for human dystrophin, which is localized and expressed in the myoblast membrane, indicating functional genetic modification in these cells (Figures 24 and 25). Human dystrophin-positive fibers were not observed in sections of mice injected with untreated DMD myoblasts (Figures 24 and 25), indicating that CRISPR / Cas9 modified cells are a source of human dystrophin expression.

[0131] [Example 12] Off-target and cytotoxic activity As previously described (Ousterout et al., Mol Ther 21:1718-1726, 2013), a flow cytometry-dependent GFP retention assay was applied to determine the relative cytotoxicity of the CRISPR / Cas9 system in human cells for selected sgRNAs. Minimal cytotoxicity was observed for SpCas9 expressed with or without sgRNA after transfection of human cells (Figure 26A). A publicly available tool is available to determine and rank potential off-target CRISPR / Cas9 activity based on the expected positional bias of a certain mismatch within the sgRNA protospacer sequence and the total number of mismatches with the intended target site (Hsu et al., Nat Biotechnol 31:827-826, 2013). Using this public web server, the most likely off-target sites for the sgRNA used to modify the dystrophin gene in this experiment were predicted (Table 4). The top 10 off-target sites were identified by surveyor assay in HEK293T cells treated with SPCas9 and individual sgRNA expression cassettes for CR1, CR3, CR5, CR6, or CR36. CR1, CR3, and CR36 each possessed one of these 10 expected off-target loci, and these exhibited significant levels of genetic modification (Table 4 and Figure 27). Interestingly, the CR3 off-target sequence showed substantial homology and similar modification frequencies to the intended on-target (13.3% at the intended site compared to 9.3% at OT-1) (Table 4 and Figure 27). Notably, CR3-OT1 was the only one of these three off-targets to show significant levels of activity in hDMD cells sorted by surveyor assay (Figure 26B). Nuclease activity at off-target sites can lead to unintended chromosomal rearrangement through distal recombination between the cleaved target and the off-target locus on a separate chromosome. This raises significant concerns for deletion-dependent gene modification methods due to the increased potential off-target activity when using two or more nucleases (e.g., in compound CRISPR / Cas9 gene editing). To utilize a highly sensitive nested genome PCR assay, potential translocations were investigated, and translocations were detected at proven off-target loci using both single-CRISPR / Cas9 and compound-CRISPR / Cas9 editing methods. Using this assay, translocations between on-target and off-target sites were readily detected in the model HEK293T cell line (which also exhibited high levels of off-target activity) (Figures 26C and 28A, 28B). Sanger sequencing of PCR amplicons confirmed the identity of the expected translocation events for each primer pair (Figures 29-30). The translocation subset detected in HEK293T cells was also detectable by nested PCR in selected hDMD myoblasts, although the signal was significantly weaker, and sequence identity could not be confirmed due to low product yield (Figures 26D and 28A, 28C). The translocations were not detected using this assay in HEK293T cells or selected hDMD cells treated with CR6 or CR6 / CR36, respectively (Figure 28), and the aforementioned translocations exhibited low levels of off-target activity only in HEK293T cells with CR6-OT3 (Table 4). These results highlight the importance of selecting highly specific sgRNAs (particularly for the application of complex editing), and demonstrate that this approach can benefit from ongoing research to improve the specificity of the CRISPR / Cas9 system. These data suggest that selected sgRNAs possess dystrophin gene modification capabilities with only one clearly expected off-target site exhibiting detectable levels of activity without significant toxicity.

[0132] [Example 13] Consideration Genome editing is a powerful tool for correcting genetic disorders, and recent developments in the CRISPR / Cas9 system have progressed dramatically in this field. It has presented a potential correction for DMD (the most common genetic disorder for which there are currently no approved treatment options). With many gene-dependent and cell-dependent therapies for DMD in preclinical development and clinical trials, genome editing methods can be compatible with many of these approaches. For example, genome editing can be combined with patient-specific cell-dependent therapies for DMD. The CRISPR / Cas9 system can function in human skeletal muscle myoblasts, as shown, as well as human pluripotent stem cells and other human cell lines. Importantly, gene editing with CRISPR / Cas9 does not halt the myogenic capacity of these cells, as demonstrated by efficient dystrophin expression in vitro and in vivo after transplantation into immunodeficient mice. Therefore, this approach should be compatible with cell-dependent therapies for DMD. In addition, a concentrated pool of gene-modified cells exhibited human dystrophin expression in vivo following transplantation into immunodeficient mice. CRISPR / Cas9 gene editing did not exhibit significantly harmful effects in human myoblasts, as observed by stable gene editing frequencies and minimal cytotoxicity of several sgRNAs. However, gene editing activity was confirmed at 3 out of 50 expected off-target sites across 5 sgRNAs, enabling the detection of CRISPR / Cas9-induced chromosomal translocations between on-target and off-target sites. CRISPR / Cas9 technology is an efficient and versatile method for correcting significant portions of dystrophin mutations and can be offered as a general-purpose platform for the treatment of gene disorders. In addition, in contrast to the plasmid-dependent delivery methods used herein, direct transfection of sgRNA and Cas9 mRNA can be used to shorten the Cas9 expression duration and eliminate the possibility of random plasmid integration, thereby enhancing specificity and safety. Alternatively, direct delivery of the CRISPR / Cas9 system to skeletal muscle and / or cardiac muscle by virus, plasmid, or RNA delivery vector can be used for in vivo genome editing and adaptations of this approach. The large size (~4.2 kilobases) of the Streptococcus pyogenes Cas9 gene presents a challenge to the aforementioned use with size-limited adeno-associated virus vectors. However, Cas9 genes from other species (e.g., Neisseria meningitidis and S. thermophilus) are short enough to efficiently package both Cas9 and sgRNA expression cassettes into a single AAV vector for in vivo gene editing applications.

[0133] The CRISPR / Cas9 system enables efficient modification of nearly 90% of the target gene, consistent with other reports of active system activity across diverse loci. The potency and versatility of this technology represent a significant step forward toward the flexible implementation of patient-specific gene editing. Low levels of dystrophin (including only 4% wild-type expression) may be sufficient to improve survival, motor function, and cardiomyopathy in mouse models. This level of CRISPR / Cas9 activity may be sufficient to provide therapeutic benefits. Using combined CRISPR / Cas9 for exon deletion also presents a unique combination of opportunities and challenges. In contrast to the repair of the dystrophin gene's reading frame by small indels generated by NHEJ-system DNA repair following the action of a single nuclease, complete exon deletion of the genome was performed to repair dystrophin expression. The protein product of the edited gene is predictable and already characterized in Becker muscular dystrophy patients with naturally occurring deletions, but in contrast, random indels resulting from the action of a single nuclease within the exon will lead to the generation of novel epitopes derived from each DNA repair event. Furthermore, the product resulting from exon deletion will result in repaired dystrophin for each successful gene editing, whereas gene modification by random indels within the exon will only repair the reading frame in one-third of the editing events that result in the correct reading frame. Not all of the sgRNAs tested were associated with significant cytotoxicity in human cells. Of the 50 sites examined for the five sgRNAs used, three potential off-target sites were identified as repairing dystrophin expression. Furthermore, chromosomal translocations between the intended on-target sites and these off-target sites were detectable by a highly sensitive nested PCR assay in HEK293T cells expressing high levels of Cas9 and sgRNA. Notably, the off-target activity and translocations identified in HEK293T cells (an aneuploid cell line that is immortalized and expresses very high levels of Cas9 and sgRNA) did not occur at high levels and were undetectable in some cases of hDMD myoblasts. Importantly, this level of specificity may be acceptable given the severity of DMD and the lack of apparent cytotoxicity in human cells.

[0134] [Example 14] A modified AAV capsid (referred to as SASTG (SEQ ID NOs: 436 and 437)) was developed to enhance myocardial and skeletal muscle tissue tropism (Piacentino et al. (2012) Human Gene Therapy 23:635-646). A ZFN targeting the Rosa26 locus ("Rosa26 ZFN", Figure 33; SEQ ID NOs: 434 and 435) was shown to be highly active in mouse cells (Perez-Pinera et al. Nucleic Acids Research (2012) 40:3741-3752). An AAV-SASTG vector encoding the Rosa26 ZFN protein was designed and subsequently fabricated, and then purified using a UNC viral vector core. A surveyor assay (Guschin et al., Methods Mol Biol 649, 247-256, 2010) demonstrated that AAV-SASTG Rosa26 ZFN delivery was followed by the introduction of an NHEJ mutation at the Rosa26 locus in cultured C2C12 myoblasts (undergoing active cell cycle repetition or forced differentiation by serum depletion) (data not shown). To demonstrate that adult post-mitotic skeletal muscle is targeted and induced by Rosa26 ZFN after AAV delivery, an AAV-SASTG vector encoding Rosa26 ZFN was directly injected into the tibialis anterior muscle of 6-week-old C57BL6 / J mice at a titer of 1e10 vector genomes (vg) or 2.5e10 vg / muscle. Four weeks after injection, the mice were sacrificed, the TA muscle was harvested, and divided into several fragments for genomic DNA extraction and analysis (Figure 31). Genomic DNA was PCR amplified and subjected to a Surveyor assay to detect the characteristics of NHEJ mutations of ZFN mutagenesis at the Rosa26 target site (Figure 32). Figure 32 shows the Surveyor analysis of in vitro and in vivo Rosa26 ZFN activities in skeletal muscle following delivery of AAV-SASTG-ROSA. Proliferating C2C12 was transduced with the indicated amount of virus and harvested 4 days after infection (Figure 32a). C2C12 was incubated in differentiation medium for 5 days and then transduced in a 24-well plate with the indicated amount of AAV-SASTG-ROSA virus (Figure 32b). Samples were collected 10 days after transduction. The indicated amount of AAV-SASTG-ROSA was directly injected into the tibialis anterior muscle of C57BL6 / J mice, and the muscle was harvested 4 weeks after infection. The harvested TA muscle was divided into 8 separate pieces for genomic DNA analysis (each shown in a separate lane) (Figure 32c). Notably, high levels of gene modification were detected in all fragments at the highest dose (2.5e10 vg).

[0135] [Example 15] AAV-CRISPR construct targeting the mutant dystrophin gene AAV constructs were designed for the therapeutic modification of the dystrophin gene, which causes Duchenne muscular dystrophy and degeneration of skeletal and cardiac muscle. Using AAV, a CRISPR / Cas9 system can be delivered to repair the dystrophin reading frame by deletion of exon 51, deletion of exons 45-55, disruption of splice donor or acceptor sites, or generation of a frameshift within exon 51 (Ousterout et al., Molecular Therapy 2013), thereby repairing the dystrophin reading frame and protein expression. The CRISPR / Cas9 system will contain Cas9 with sequence numbers 64 or 114 (see Figures 40 and 41). gRNAs that can be combined with these Cas9s (targeting their corresponding PAM sequences) are provided (see Figures 40 and 41, and also Tables 2 and 3).

[0136] [Example 16] Generation of induction neurons (iNs) Generating induced neurons (iNs) from other cell lineages has potential applications in regenerative medicine and neurological disease research. Direct conversion of mouse embryonic fibroblasts (MEFs) into functional neurons can be achieved by delivery of a cocktail of three neuronal transcription factors (BRN2, ASCL1, and MYT1L) (BAM factors, Figure 48). Other methods may include additional factors to induce diverse subtypes. These experiments require ectopic delivery of transcription factors and activation of endogenous loci corresponding to the maintenance of the neuronal phenotype. We manipulated the CRISPR / Cas9 system as a pluripotent transcription factor capable of targeting any promoter in the genome via an RNA-guided mechanism and activating endogenous genes in mammalian cells (Figures 49A, 49B). Materials and methods Using the CRISPR / Cas9 transcription factor, we activated the endogenous genes encoding ASCL1 and BRN2, directly reprogramming MEFs into functionally induced neurons. cell cultureMEF cells were seeded in 24-well TCPS plates or poly-D-lysine / laminin-coated coverslips. Following transduction with dCas9-VP64 and transfection with gRNA (see Tables 10 and 11 for gRNA sequences), cells were cultured for 24 hours in MEF culture medium (Adler et al. Mol Ther Nucleic Acids 1:e32, 2012), and then transferred to N3 nerve induction medium (Vierbuchen et al. Nature 463:1035-1041 (2010)) for the duration of the experiment (Figure 49B).

[0137] [Table 10]

[0138] [Table 11]

[0139] qRT-PCR and IF Endogenous ASCL1 activation was determined by qRT-PCR and immunofluorescence of MEFs (3 days after delivery of negative controls encoding dCas9-VP64 and gRNA, ASCL1 cDNA, or luciferase). The development of iNs was evaluated by co-staining of TUJ1 and MAP2 and identification of cells with neuronal morphology and elongation processes. Living Cell Reporter After 7-8 days in N3 culture medium, MEFs cultured on poly-D-lysine / laminin-coated coverslips were transduced with viruses containing hSyn-REP and MAP2-GCamP5 reporters, and the most mature iNs were identified based on their functional characteristics by calcium imaging and electrophysiology (Figure 49B). resultdCas9-VP64 and gRNA (targeting the ASCL1 promoter) activated the endogenous gene in MEF. Co-delivery of eight gRNAs activated the endogenous gene 400-fold. This is a significant increase compared to the 100-fold activation induced by co-delivery of four gRNAs (p<0.05). Nuclearly localized Ascl1 protein was detected by immunofluorescence in MEF. Ectopic Ascl1 expression produced more Ascl1 protein than dCas9-VP64 combined with either gRNA cocktail, but did not activate the endogenous gene locus by day 3 (Figure 50A, 50B). Co-positive TUJ1 and MAP2 cells with elongated processes were identified in neurogenesis culture medium at day 13 following delivery of dCas9-VP64 and ASCL1 and BRN2 promoter-targeting gRNAs (Figure 4A, first column). A similar number of TUJ1 and MAP2 co-positive cells were identified by ectopic expression of BAM factors (Figure 51A, second column). Cells expressing the hSyn-RFP reporter and possessing a neuronal morphology could be observed as early as day 11 in neurogenesis culture medium (Figure 51B). Cells expressing the MAP2-GCaMP5 calcium indicator showed KCl-induced depolarization detectable by fluorescence microscopy (Figures 52A, 52B). Direct conversion of mouse embryonic fibroblasts into TUJ1 and MAP2 co-positive cells with neuronal morphology was achieved through activation of endogenous BRN2 and ASCL1 by CRISPR / Cas9-dependent transcription factors. dCas9-VP64 produced less protein than ectopic expression of ASCL1 (Figure 50B), but neuronal cell development was similar. Activation of endogenous loci can induce a reprogramming cascade of events that are mechanistically different from those produced by ectopic expression. dCas9-VP64 was able to stably activate silent endogenous genes across heterochromatin (a feature unique to a subset of “pioneer” transcription factors). As a result, conversion of cell lineages containing CRISPR / Cas9 transcription factors overcomes epigenetic barriers to reprogramming better than ectopic expression of transcription factors, especially in cell types that are difficult to reprogram (e.g., adult cells). This could be clinically important in the field of regenerative medicine, as the use of autologous cells is often desired in cell exchange therapy.

[0140] [Example 17] Combined CRISPR / Cas9-based genome editing - Materials and methods Plasmid construct Expression cassettes for Streptococcus pyogenes sgRNA and human codon-optimized Cas9 (hCas9) were used as described above. The following additional promoters were synthesized using GeneBlock (IDT) (mU6 (Ohshima et al., Nucleic Acids Res 9:5145-5158, 1981), H1 (Myslinski et al., Nucleic Acids Res 29:2502-2509, 2001), and 7SK (Murphy et al., Cell 51:81-87, 1987) pol-III promoter) and cloned in place of the hU6 sgRNA expression cassette. GeneBlock (IDT) was cloned at the 3' end of the Cas9 coding sequence and fused to the T2A skipping peptide and eGFP gene immediately after Cas9 to monitor vector expression. The coding region for hCas9-T2A-GFP (SEQ ID NO: 145) was subsequently transferred to a lentiviral expression vector. The lentiviral vector contained a human ubiquitin C (hUbC) promoter that drives the expression of hCas9-T2A-GFP, and a restriction site immediately upstream of the hUbC promoter that facilitates the Golden Gate cloning of the sgRNA expression cassette (Figure 42A). Custom lentiviral vector assembly protocolAssembly of custom lentiviral vectors expressing up to four selected sgRNAs and active Cas9, dCas9, or dCas9-VP64 was achieved in less than 5 days. The cloning method utilized Golden Gate cloning and IIS-type restriction enzymes (the restriction enzymes cleave outside their recognition sequences, creating unique overhangs). Golden Gate assembly facilitated cloning because all four expression cassettes were ligated into the final lentiviral vector in a single step. The lentiviral vectors expressed active Cas9, cCas9, or dCas9-VP64 in addition to one, two, three, or four sgRNAs (expressed from independent promoters).

[0141] Process 1Single-stranded oligonucleotides, each containing a 20 bp protospacer, were annealed in a manner that produced sticky ends and ligated to the desired pZDonor-promoter vector. Two single-stranded oligonucleotides were ordered for each desired genomic target. To anneal the complementary oligonucleotides, the following were mixed: 8 μL sense oligonucleotide + 8 μL antisense oligonucleotide (both 10 mM) + 2 μL 10x ligase buffer. The oligonucleotides were thawed and re-annealed in a PCR instrument according to the following program: 300 seconds at 96°C, followed by 20 seconds at 85°C, 20 seconds at 75°C, 20 seconds at 65°C, 20 seconds at 55°C, 20 seconds at 45°C, 20 seconds at 35°C, and 20 seconds at 25°C (rate between steps was -0.3°C / second). For phosphorylation of the sticky ends, 1 μL of 25 mM ATP + 1 microL of T4 polynucleotide kinase (NEB) was added and incubated at 37°C for 60 minutes, followed by thermal inactivation of the enzyme at 65°C for 20 minutes. Each protospacer was ligated to the desired expression vector using T4 DNA ligase (NEB) and incubated at 16°C for 60 minutes (50 ng of vector and 1 μL of annealed oligonucleotide were used in a 10 μL reaction volume according to the manufacturer's instructions). XL1 blue chemically competent bacteria (Agilent) were transformed with 5 microL of each ligate according to the manufacturer's instructions. The transformants were plated on LB agar plates (containing 50 μg / mL kanamycin (Sigma)) and incubated overnight at 37°C. In our experience, more than 90% of the colonies will contain the desired ligation product. Sequence analysis using M13 reverse standard sequencing primers was performed to validate each of the final sgRNA constructs before proceeding to step 2.

[0142] Step 2: Construction of four promoter-gRNA cassettes into a lentiviral destination vector using the Golden Gate Assembly.After the completion of step 1, four independent plasmids will be present, each expressing a different sgRNA from a different promoter. To assemble these four different promoter-sgRNA constructs into the desired destination vector, 200 ng of each sgRNA expression plasmid and the desired lentiviral destination vector are mixed in a 20 μL reaction volume with 1 μL of T4 DNA ligase (NEB), 1 μL of BsmBI FastDigest (Fisher Scientific), and 2 μL of 10x T4 ligase buffer (NEB). The reaction mixture is incubated as follows: 10 minutes at 37°C, 15 minutes at 16°C, 30 minutes at 37°C, and 5 minutes at 80°C. SURE2 chemically competent cells (Agilent) are transformed with 5 μL of the ligated reaction mixture according to the manufacturer's instructions. The transformants are plated on LB agar plates (containing 100 μg / mL ampicillin) and incubated overnight at 37°C. In some cases, colonies may be screened by lacZ-based blue / white screening using IPTG and X-gal, but in our experience, more than 90% of transformants contain suitable ligation products. Due to the inverted repeats formed by the opposite sgRNA expression cassette, the final constructs are unstable, and therefore we recommend maintaining these plasmids in the SURE2 cell line and screening the final plasmids using sense primers (5'-TCGGGTTTATTACAGGGACAGCAG-3' (SEQ ID NO: 464)) and antisense primers (5'-TCTAAGGCCGAGTCTTATGAGCAG-3' (SEQ ID NO: 465)). These primers amplify across four promoter-gRNA regions. Due to their repeatability, different band formation patterns should be observed, along with a maximum product of approximately 1800 bp in size.

[0143] Cell culture and transfectionHEK293T cells were obtained from the Tissue Cell Collection Center (ATCC, Manassas, VA) via the Duke University Cancer Center and maintained in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. Primary human dermal fibroblasts (catalog ID: GM03348) were obtained from the Coriell Institute (Camden, New Jersey) and maintained in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were cultured at 37°C under 5% CO2. HEK293T cells were transfected with 200 ng of each sgRNA expression cassette (800 ng of total pDNA) in 24-well plates using Lipofectin 2000 (Life Technologies) according to the manufacturer's instructions. Virus production and transduction All lentiviral vectors used in this experiment were second-generation and manufactured using standard virus production methods. Briefly, 3,500,000 HEK293T cells were plated in 10 cm dishes. The following day, 20 μg of transfer vector, 6 μg of pMD2G, and 10 μg of psPAX2 were transfected into the cells using the calcium phosphate transfection method. The culture medium was changed 12-14 hours after transfection. The viral supernatant was collected at 24 and 48 hours after the culture medium change and pooled after passing through a 0.45 micron filter. For transduction, the cell culture medium was replaced with the viral supernatant (supplemented with 4 μg / mL of polyblen). The viral supernatant was replaced with fresh culture medium 12-24 hours later. Reverse transcription PCRRNA was isolated using the miRNeasy Mini RNA Isolation Kit (Qiagen). DNase digestion was performed using the DNA-free kit (Applied Biosystems). cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNA was amplified using Taq DNA polymerase (NEB), and the resulting product was electrophoresed on a TAE agarose gel. Images were captured using the ChemiDoc XRS+ System and processed using ImageLab software (Bio-Rad).

[0144] Quantitative real-time PCR RNA was isolated using the RNeasy Plus RNA Isolation Kit (Qiagen). cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR was performed using PerfeCTa SYBR Green FastMix (Quanta Biosciences) and the CFX96 Real-time PCR Detection System (Bio-Rad). Primer specificity was confirmed by agarose gel electrophoresis and melting curve analysis. Reaction efficiency over an appropriate dynamic range was calculated to ensure the linearity of the standard curve. Results are expressed as a multiple of the mRNA expression increase of the gene in question, standardized to β-actin expression using the ΔΔCt method. Reported values ​​are the average and SEM values ​​obtained from two separate experiments (n=2), where technical replicates were averaged for each experiment. Western blot: Cells were lysed with RIPA buffer (Sigma) supplemented with protease cocktail (Sigma). Protein concentration was measured using BCA protein assay reagent (Thermo Scientific) and a BioTek Synergy 2 Multi-Mode microplate reader. The lysates were mixed with loading buffer and boiled for 5 minutes, and 25 μg of protein was electrophoresed on a NuPage 10% Bis-Tris Gel polyacrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane. Non-specific antibody binding was blocked with TBST (50 mM Tris, 150 mM NaCl, and 0.1% Tween-20) containing 5% non-fat milk for 1 hour at room temperature. The membrane was incubated with the following primary antibodies: 1:250 anti-Myogenin (Santa Cruz Sc-32758) in 5% BSA overnight at 4°C in TBST; 1:1000 anti-FLAF-HRP (Cell Signaling 2044) in 5% milk for 60 minutes at room temperature in TBST; 1:5000 anti-GAPDH (Santa Cruz Sc-32758) in 5% milk for 30 minutes at room temperature in TBST. Subsequently, the membrane was washed 3 times with TBST for a total of 15 minutes. The membrane was incubated with an anti-rabbit HRP-conjugated antibody (Sigma, A6154) or an anti-mouse HRP-conjugated antibody (Santa Cruz, SC-2005) (1:5000 dilution) and washed 3 times (15 minutes each) with TBS. Immun-Star WesternC TM The membrane was visualized using an Immun-Star WesternC TM chemiluminescence kit (Bio-Rad), and images were captured using a ChemiDoc

[0145] Quantitative analysis of Cel-I in endogenous gene modificationA surveyor assay was used to quantify CRISPR / Cas9 nuclease damage at endogenous target sites (this assay can detect the characteristics of nuclease-mediated NHEJ mutations). After transfection or transduction, cells were incubated at 37°C for 3 to 10 days, and genomic DNA was extracted using the DNeasy blood tissue kit (Qiagen). Target loci were amplified by 35 cycles of PCR using the AccuPrime High Fidelity PCR kit (Invitrogen). The resulting PCR products were randomly thawed and re-annealed in a PCR instrument using the following program: 240 seconds at 95°C, followed by 60 seconds at 85°C, 60 seconds at 75°C, 60 seconds at 65°C, 60 seconds at 55°C, 60 seconds at 45°C, 60 seconds at 35°C, and 60 seconds at 25°C (rate between steps was -0.3°C / second). Following re-annealing, 8 μL of PCR product was mixed with 1 μL of surveyor nuclease S and 1 μL of enhancer S (Transgenomic), and incubated at 42°C for 1 hour. After incubation, 6 μL of digested product was loaded onto a 10% TBE polyacrylamide gel and electrophoresed at 200 V for 30 minutes. The gel was stained with ethidium bromide, and quantification was performed by densitometry using ImageLab (Bio-Rad) (Perez-Pinera et al., Nucleic Acids Res 40:3741-3751, 2012). Statistical analysis: At least two separate experiments were compiled as the mean and standard error of the mean. The effect was evaluated using multivariate ANOVA and Dunnett's post-hoc test with JMP10Pro.

[0146] [Example 18] Development of a single lentiviral vector for combined CRISPR / Cas9 application The limitations of conventional CRISPR / Cas9 gene editing systems (particularly transactivator systems) lie in the simultaneous and efficient delivery of a wide variety of sgRNAs and Cas9 proteins used in complex gene editing and synergistic gene activation, especially in cell types where transfection is difficult. To overcome this limitation, we developed a single lentiviral vector that efficiently expresses Cas9 and up to four sgRNAs. To maximize the expression efficiency of each sgRNA, this vector expresses four sgRNAs from four independent pol III promoters (human U6 promoter, mouse U6 promoter, 7SK, and H1). We detected sgRNAs targeting the AAVS1 locus using endpoint RT-PCR and confirmed that sgRNAs are expressed from each promoter (Figure 42A). To test the activity of each sgRNA expression construct, we co-transfected human HEK293T cells with each promoter construct, which independently expresses sgRNAs targeting AAVS1, together with an active Cas9 expression construct. Notably, we detected consistent and high levels of genetic modification at the target loci of each sgRNA (the modification is comparable to that of zinc finger nucleases, which exhibit high activity and are characterized in detail at the AAVS1 locus) (Figure 42B). Furthermore, lentiviral delivery of various Cas9-dependent constructs (including active Cas9 nuclease, inactive (dead) Cas9, and inactive Cas9 fused with the VP64 transactivation domain) resulted in the expression of full-length Cas9 protein in HEK293T cells, as determined by Western blotting (Figure 42C).

[0147] Using these components, we developed a Golden Gate cloning method that facilitates the rapid and efficient cloning of a wide variety of sgRNA expression cassettes in a single lentiviral vector expressing the desired Cas9 effector (Figure 43). In the first step, oligonucleotides encoding sgRNA protospacer sequences are separately cloned in various expression vectors (each having a separate promoter driving sgRNA expression). In the second step, each sgRNA expression construct is subcloned by Golden Gate assembly in a selected lentiviral Cas9 expression vector. This method enables active and rapid cloning of up to four sgRNAs in a single lentiviral vector for use in gene editing or gene activation. To express fewer than four sgRNAs, a poly-T terminator sequence is cloned downstream of the unused promoter to prevent transcription from the unused promoter. Each vector co-expresses the selected Cas9 with eGFP via a 2A skipping peptide, enabling fluorescence-activated flow sorting and enrichment of cells with high infection counts. Finally, the entire region, including the sgRNA and Cas9 expression cassette, is flanked at the loxP site, which mediates removal by Cre-lox excision.

[0148] [Example 19] Evidence of the effectiveness of a single lentiviral sgRNA / Cas9 expression vector in complex genomic manipulation. To demonstrate the independent activity of each sgRNA, we assembled a single lentiviral vector expressing active Cas9 and four sgRNAs (each targeting a distinct locus) (Figure 44A). As a control vector, we assembled a construct expressing only one sgRNA with poly-T protospacers at the other three positions. We transduced HEK293T cells and primary fibroblasts with lentiviruses expressing the indicated sgRNAs and monitored the gene modification frequency 7 or 10 days post-transduction, respectively (Figure 44b). In both cell types, the single lentiviral vector mediated highly efficient complex gene editing at all four loci (Figure 44B). Interestingly, at three of the four loci in fibroblasts, the combined expression of all four sgRNAs resulted in a higher modification frequency than the expression of a single sgRNA alone (Figure 44B). We observed efficient complex gene editing in fibroblasts (a cell type that is generally difficult to transfect). These data demonstrate that a single lentivirus efficiently expresses four active sgRNAs, and that this lentiviral platform can be used to target four distinct loci for complex CRISPR / Cas9 gene editing.

[0149] [Example 20] Transient RNA guide gene activation in cell lines stably expressing lentiviral Cas9-dependent transactivators. Next, we were interested in developing a system that enables transient gene activation by transfecting a stable Cas9-expressing model cell line with sgRNA. Different Cas9-T2A-GFP were transduced into HEK293T cells, and GFP expression was monitored using flow cytometry. After normal passage every 2-3 days, each cell line showed stable GFP expression up to 35 days post-transduction. Subsequently, transduced HEK293T cells were transfected with one to four separate sgRNA expression constructs (targeting either the IL1RN or HBG1 promoter). Transient transfection of these sgRNA constructs in stable dCas9-VP64-expressing cell lines resulted in regulatory endogenous gene activation (Figures 45A, 45B). Gene activation following transient transfection of sgRNA constructs in cell lines expressing dCas9-VP64 reached peak activation levels approximately 3–6 days post-transfection and decreased to undetectable levels by 20 days post-transfection (Figures 45C, 45D). Furthermore, we were able to reactivate each gene by a second transfection of all four sgRNAs targeting each promoter, although the activation levels were significantly lower than those observed after the first transfection (Figures 45C, 45D). The decrease in activity after the second transfection may be due to decreased vector expression or competitive proliferation of non-transduced cells. Nevertheless, these data demonstrate that lentiviral Cas9, combined with transient sgRNA delivery, can be used as a pluripotent system to regulate and transiently activate and reactivate target genes in cell lines stably transducing Cas9.

[0150] [Example 21] Stable gene activation of HEK293T cells using a single lentiviral sgRNA / Cas9 transactivator expression vector. Lentiviral delivery can enable stable and long-term gene activation via CRISPR / Cas9 transactivation. To test this, HEK293T cells were transduced using a single lentiviral vector encoding dCas9-VP64 and one to four sgRNA expression cassettes. Similar to our transient transfection results (Figure 45), we were able to regulate and vigorously activate the expression of endogenous IL1RN and HBG1 genes (Figures 46A, 46B). Gene activation induced by co-transfection of HEK293T cells with dCas9-VP64 and four sgRNAs (targeting the IL1RN and HBG1 promoters) peaked 3–5 days post-transfection, and gene expression returned to background levels 15–20 days post-transfection (Figure 4c). In contrast, lentiviral delivery of dCas9-VP64 and the same four IL1RN or HBG1 target sgRNAs induced gene activation that lasted longer than 20 days post-transduction (Figures 46C, 46D). Therefore, single lentiviral delivery of the complex dCas9-VP64 transactivator is a useful platform for efficiently and stably upregulating target endogenous genes.

[0151] [Example 22] dCas9-KRAB targeting HS2 enhancer The HS2 enhancer is a well-documented distal regulatory element necessary for the activation of the globin locus. We delivered dCas9-KRAB along with gRNAs targeting the HS2 enhancer to determine whether this system suppresses γ-, ε-, and β-globin expression in the K562 human erythroid leukemia cell line (Figure 54). We constructed a set of gRNAs targeting various sites along with the core region of the HS2 enhancer (SEQ ID NO: 467). See Table 12.

[0152] [Table 12]

[0153] Screening of single gRNAs at globin loci using CRISPR / Cas9 5-8 days prior to electroporation of a 5 μg plasmid encoding U6-sgRNA expression, dCas9 and the dCas9-KRAB effector were delivered via lentivirus (Figure 55A). Cells that were not electroporated with gRNA (no gRNA) and cells treated with gRNAs targeting different loci (IL1RN) were added as controls. A wide variety of gRNAs showed potent repression of ε-, γ-, and β-globin genes when assayed 3 days post-transfection, achieving knockdown of up to 80% (Figures 55B, 55C, 55D). Gene expression at globin loci was repressed by expressing gRNAs with dCas9 or dCas9-KRAB. In general, treatment with dCas9-KRAB resulted in stronger repression of the given gRNAs compared to dCas9 alone, suggesting a crucial role for the KRAB domain in supplementing heterochromatin factors that enhance repression. The level of suppression achieved depends on the amount of gRNA plasmid delivered by transfection only in dCas9-KRAB-treated cells (Figures 56A, 56B, 56C). Increasing the dose of Cr4 gRNA plasmid up to 10 μg increases the level of silencing of the globin gene in dCas9-KRAB-treated cells. Stable silencing of globin genes by dCas9-KRABdCas9 / dCas9-KRAB was co-expressed in K562 via lentivirus along with a single gRNA (Figure 57A). Cells not treated with lentivirus (NT), cells treated with dCas9 / dCas9-KRAB without gRNA (no gRNA), and cells treated with dCas9 / dCas9-KRAB and gRNAs targeting different loci (IL1RN) were added as controls. Lentivirus-treated cells were selected at 4 to 7 days. A wide variety of gRNAs showed potent transcriptional repression of ε-, γ-, and β-globin genes when assayed 7 days post-transduction, achieving knockdown of up to 95% (Figures 57B, 57C, 57D). ε-globin expression was most strongly silenced in response to gRNAs targeting the HS2 enhancer. Treatment with gRNA and dCas9-KRAB resulted in dramatically stronger repression than treatment with dCas9 and gRNA alone. These findings demonstrate that dCas9-KRAB, targeted and induced to the HS2 enhancer by gRNA, achieves potent repression of distal globin genes. This is the first example of epigenetic targeted induction control of distal regulatory elements by the CRISPR / Cas9 system in mammalian cells. Enhancers modulate development and disease, and this disclosure provides methods for examining and controlling enhancer function, and furthermore, using these methods, the ability of dCas9-KRAB to access chromatin at specific locations and its role in genome-wide expression can be determined.

[0154] [Example 23] dCas9-p300 A dCas9-p300 fusion protein was designed and compared to a dCas9-VP64 fusion protein (see Figure 59). The amino acid constructs of the dCas9 structure are shown in Figures 61A-61C. Human embryonic kidney tissue culture line HEK293T (ATCC;CRL-11268) was seeded in a 24-well tissue culture dish at a density of 1.5e5 cells / well one day prior to transfection (using Lipofectamine 2000 transfection reagent (Life Technologies)). After 24 hours, cells were transfected with 1 μL of Lipofectamine 2000, 375 ng of dCas9 expression constructs (dCas9, dCas9VP64, or dCas9p300, respectively), and 125 ng of pooled gRNA expression plasmids (four of each in equimolar ratios). Table 13 shows information on the gRNAs.

[0155] [Table 13]

[0156] Cells were collected 3 days after transfection and mRNA expression was assayed by RT-PCR. The RT-PCR primer sequences are shown in Table 14.

[0157] [Table 14]

[0158] RT-QPCR was standardized for GAPDH expression using the ΔΔC1 method. The results are expressed as a multiplier increase in the expression of the gene in question compared to cells treated with lipofectamine alone without DNA transfection ("NO DNA") (Figure 60A-60C). Figure 62 shows that mutant residues in the p300HAT domain cause the gene to lose its ability to activate gene expression. Figure 63 shows that a wide variety of gRNAs function synergistically with dCas9-p300, as demonstrated with dCas9-VP64.

[0159] [Example 24] Figure 66 shows that TALENs mediate the integration of the Dp427m skeletal muscle isoform of dystrophin into the 5'UTR of human DMD patients' skeletal muscle myoblasts carrying various deletions in the dystrophin gene. Constructs encoding active TALEN pairs at the 5'UTR locus and donor templates carrying the mini-dystrophin gene were transfected into DMD patient cells. Figure 66(a) is a schematic diagram showing how mini-dystrophin was integrated into the 5'UTR. Figure 66(b) shows the isolation of hygromycin-resistant clone cell lines and PCR screening for successful site-specific integration in the 5'UTR using the primers shown in Figure 66(a). Asterisks indicate clones selected for further analysis in Figure 66(c). Figure 66(c) shows that isolated DMD myoblasts with detected integration events were differentiated for 6 days, and the expression of an HA tag fused to the C-terminus of mini-dystrophin was determined.

[0160] It should be understood that the aforementioned detailed descriptions and accompanying embodiments are merely illustrative and should not be construed as limitations on the scope of the present invention (which is defined solely by the appended claims and their equivalents). Various modifications and alterations to embodiments of the disclosure will be obvious to those skilled in the art. Such modifications and alterations (including, but not limited to, those relating to chemical structure, substituents, derivatives, intermediates, synthesis, composition, formulation, or method of use of the invention) can be made without departing from the spirit and scope of the invention. Addendum

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Claims

1. A fusion protein comprising a first polypeptide domain containing a short palindromic sequence repeat-associated 9 (Cas9) protein, and a second polypeptide domain having an activity selected from transcriptional activation activity, transcriptional repression activity, transcriptional deactivation activity, histone modification activity, nuclease activity, nucleic acid binding activity, methylase activity, and demethylase activity, wherein Cas9 comprises amino acids 36-1403 of SEQ ID NO: 1, and the second polypeptide domain comprises a p300 domain, a p300HAT core domain, at least one VP16 transcriptional activation domain repeat, a VP64 domain, a p65 domain, a KRAB, a Tet1, an ERF repressor domain, an Mxi1 repressor domain, a SID4X repressor domain, or a Mad-SID repressor domain.

2. The fusion protein according to claim 1, wherein the second polypeptide domain has transcriptional activation activity.

3. A fusion protein comprising a first polypeptide domain containing a short palindromic sequence repeat-associated 9 (Cas9) protein, and a second polypeptide domain having transcriptional activation activity, wherein Cas9 comprises amino acids 36-1403 of SEQ ID NO: 1, and the second polypeptide domain comprises a p300 domain, a p300 HAT core domain, at least one VP16 transcriptional activation domain repeat, a VP64 domain, or a p65 domain.

4. The fusion protein according to any one of claims 1 to 3, wherein the second polypeptide domain comprises a p300 domain.

5. The fusion protein according to any one of claims 1 to 4, wherein the second polypeptide domain comprises a p300HAT core.

6. The fusion protein according to any one of claims 1 to 3, wherein the second polypeptide domain comprises at least one VP16 transcriptional activation domain repeat.

7. The fusion protein according to any one of claims 1 to 3 and 6, wherein the second polypeptide domain comprises a VP64 domain.

8. The fusion protein according to any one of claims 1 to 3, wherein the second polypeptide domain comprises a p65 domain.

9. The fusion protein according to any one of claims 1 to 8, further comprising a linker that connects a first polypeptide domain to a second polypeptide domain.

10. A DNA targeting system comprising a fusion protein according to any one of claims 1 to 9 and at least one guide RNA (gRNA).

11. The DNA targeting system according to claim 10, wherein the at least one gRNA comprises 12–22 nucleotides complementary to the target DNA sequence.

12. The DNA targeting system according to claim 10 or 11, wherein the at least one gRNA targets the promoter region of a gene, the enhancer region of a gene, the transcription region of a gene, the upstream region of the transcription start site of a gene, the intron of a gene, or the exon of a gene.

13. The DNA targeting system according to any one of claims 10 to 12, wherein the at least one gRNA targets an upstream region of the transcription start site of a gene, and the region is located 1 to 1000 base pairs upstream of the transcription start site.

14. The DNA targeting system according to any one of claims 10 to 13, wherein the at least one gRNA comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different gRNAs.

15. The DNA targeting system according to claim 14, wherein the different gRNAs target different regions of a gene.

16. The DNA targeting system according to claim 15, wherein the different target regions are separated by 15 to 700 base pairs.

17. The DNA targeting system according to any one of claims 14 to 16, wherein at least one of the different gRNAs binds to a target region of a different gene.

18. The DNA targeting system according to any one of claims 10 to 17, wherein the at least one gRNA targets a gene selected from ASCL1, BRN2, MYT1L, NANOG, VEGFA, TERT, IL1B, IL1R2, IL1RN, HBG1, HBG2, MYOD1, DMD, HS2, OCT4, and MYT1L.

19. The DNA targeting system according to any one of claims 10 to 18, wherein the at least one gRNA is encoded by a polynucleotide comprising at least one of SEQ ID NOs: 5-40, 65-144, 467, 492-515, 540-563, 564-584, and 585-625, or targets the said polynucleotide.

20. A polynucleotide encoding a fusion protein according to any one of claims 1 to 9.

21. A polynucleotide encoding a DNA targeting system according to any one of claims 10 to 19.

22. The polynucleotide according to claim 21, wherein the fusion protein and the at least one gRNA are operably linked to a promoter.

23. The polynucleotide according to claim 21 or 22, wherein the at least one gRNA comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 gRNAs, and each gRNA is independently operably ligated to a promoter.

24. A vector comprising a polynucleotide according to any one of claims 20 to 23.

25. The vector according to claim 24, which is a viral vector.

26. The vector according to claim 25, wherein the viral vector is a recombinant lentivirus, a recombinant adenovirus, or a recombinant adenovirus-associated virus (AAV).

27. A cell comprising a polynucleotide according to any one of claims 20 to 23 or a vector according to any one of claims 24 to 26.

28. A composition comprising a fusion protein according to any one of claims 1 to 9, a DNA targeting system according to any one of claims 10 to 19, a polynucleotide according to any one of claims 20 to 23, or a vector according to any one of claims 24 to 26.

29. A composition for modulating the gene expression of a target gene, comprising a fusion protein according to any one of claims 1 to 9, a DNA targeting system according to any one of claims 10 to 19, a polynucleotide according to any one of claims 20 to 23, or a vector according to any one of claims 24 to 26.

30. A composition for inducing mammalian gene expression in cells, comprising a fusion protein according to any one of claims 1 to 9 and at least one guide RNA (gRNA).

31. A composition for activating gene expression, comprising a fusion protein according to any one of claims 1 to 9, a DNA targeting system according to any one of claims 10 to 19, a polynucleotide according to any one of claims 20 to 23, or a vector according to any one of claims 24 to 26.

32. A composition for transdifferentiating cells or inducing cell differentiation, comprising a fusion protein according to any one of claims 1 to 9, a DNA targeting system according to any one of claims 10 to 19, a polynucleotide according to any one of claims 20 to 23, or a vector according to any one of claims 24 to 26.

33. A composition for performing complex gene editing or modifying mutated genes in cells, comprising a fusion protein according to any one of claims 1 to 9, a DNA targeting system according to any one of claims 10 to 19, a polynucleotide according to any one of claims 20 to 23, or a vector according to any one of claims 24 to 26.

34. The composition according to any one of claims 31 to 33, wherein various gRNAs target one or more endogenous genes in a cell and activate the expression of the one or more endogenous genes.

35. The composition according to any one of claims 31 to 34, wherein the DNA targeting system transcribeally activates two or more endogenous genes.

36. The composition according to claim 28 for use in regulating gene expression in cells.

37. A DNA targeting system according to any one of claims 10 to 19, for use in regulating gene expression in cells.

38. The composition according to claim 28, for use in activating gene expression in cells.

39. A DNA targeting system according to any one of claims 10 to 19, for use in activating gene expression in cells.

40. The composition according to claim 28 for use in complex gene editing or mutational gene modification in cells.

41. The DNA targeting system according to any one of claims 10 to 19, for use in complex gene editing or mutational gene modification in cells.

42. A DNA targeting system according to any one of claims 37, 39, or 41, wherein various gRNAs target one or more endogenous genes in a cell and activate the expression of the one or more endogenous genes.

43. A DNA targeting system according to any one of claims 37, 39, or 41, wherein the DNA targeting system transcribeally activates two or more endogenous genes.

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