RNA-guided gene editing and gene regulation

Abstract

Disclosed herein are system-related compositions based on clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) 9 and methods of using the system-related compositions based on CRISPR / Cas9 for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using the compositions for altering gene expression and genome engineering in muscles such as skeletal muscle and cardiac muscle.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 61 / 831,481, filed on June 5, 2013, U.S. Provisional Application No. 61 / 839,127, filed on June 25, 2013, U.S. Provisional Application No. 61 / 904,911, filed on November 15, 2013, U.S. Provisional Application No. 61 / 967,466, filed on March 19, 2014, and U.S. Provisional Application No. 61 / 981,575, filed on April 18, 2014, all of which are incorporated herein by reference in their entirety.

[0003] Government Interest Statement

[0004] This invention was made with government support under Federal Grant Nos. DP2-OD008586 and R01DA036865 awarded by the NIH and CBET-1151035 awarded by the National Science Foundation. The U.S. Government has certain rights in this invention. Technical Field

[0005] The present disclosure relates to the field of gene expression alteration, genome engineering and genome alteration of genes based on clustered regularly interspaced short palindromic repeats (Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR) / CRISPR-associated (Cas) 9 systems and viral delivery systems. The present disclosure also relates to the field of genome engineering and genome alteration of genes in muscles (e.g., skeletal muscle and cardiac muscle). Background of the Invention

[0006] Synthetic transcription factors have been modified to control gene expression for many different medical and scientific applications in mammalian systems, including stimulating tissue regeneration, drug screening, compensating for genetic defects, activating silent tumor suppressor genes, controlling stem cell differentiation, performing genetic screening, and generating synthetic gene circuits. These transcription factors can target promoters or enhancers of endogenous genes or purposefully design to identify sequences orthogonal to the mammalian genome for transgenic regulation. The most common strategy for engineering new transcription factors targeting user-defined sequences is based on the programmable DNA binding domains of zinc finger proteins and transcription-activator-like effectors (TALEs). These two methods include applying the protein-DNA interaction principles of these domains to transform new proteins with unique DNA binding specificity. Although these methods are extremely successful for many applications, it may be laborious to operate the protein engineering required for protein-DNA interactions and require specialized techniques.

[0007] In addition, these new proteins are not always effective. The reason is unknown, but it may be related to the effect of epigenetic modification and chromatin state on the binding of proteins to genomic target sites. In addition, there are challenges in ensuring that these new proteins and other components are delivered to each cell. The existing methods for delivering these new proteins and their various components include delivering them to cells with different plasmids or vectors, which, due to the difference in copy number, result in highly variable expression levels in each cell. In addition, gene activation after transfection is transient due to the dilution of plasmid DNA, and short-term gene expression may not be enough to induce therapeutic effects. In addition, this method is not suitable for cell types that are not easily transfected. Therefore, another limitation of these new proteins is the effectiveness of transcriptional activation.

[0008] Site-specific nucleases can be used to introduce site-specific double-strand breaks on the targeted genomic locus. This DNA cutting stimulates the natural DNA repair machinery, resulting in one of two possible repair pathways. In the absence of a donor template, the break can be repaired by non-homologous end joining (NHEJ) (a kind of error-prone repair pathway that causes a small amount of insertion or deletion of DNA). This method can be used to intentionally destroy, delete or change the reading frame of the targeted gene sequence. However, if the donor template is provided together with the nuclease, the cell machinery will repair the break by homologous recombination, and when DNA cutting exists, this is increased by several orders of magnitude. This method can be used to introduce specific changes into the target site of the DNA sequence. Engineered nucleases have been used for gene editing in a variety of human stem cells and cell lines and for gene editing in mouse liver. However, the main obstacle to implementing these technologies is to deliver them to specific tissues in vivo in an effective, efficient and successful genome modification-promoting manner.

[0009] Inherited genetic diseases have a devastating effect on children in the United States. These diseases are currently incurable and can only be managed by attempting to alleviate symptoms. For decades, the field of gene therapy has held promise for curing these diseases. However, technical barriers to safely and effectively delivering therapeutic genes to cells and patients have limited this approach. Duchenne Muscular Dystrophy (DMD) is the most common inherited single-gene disease, occurring in 1 in 3,500 males. DMD is the result of inherited or spontaneous mutations in the dystrophin gene. Dystrophin is a key component of a protein complex responsible for regulating muscle cell integrity and function. DMD patients typically lose the ability to physically support themselves in childhood, become increasingly frail in their teens, and die in their twenties. Existing experimental gene therapy strategies for DMD require repeated administration of transient gene delivery vectors or rely on permanent fusion of foreign genetic material into genomic DNA. Both approaches have serious safety concerns. In addition, these strategies are limited by the inability to deliver large and complex dystrophin gene sequences. SUMMARY OF THE INVENTION

[0010] The present invention relates to a fusion protein comprising two heterologous polypeptide domains. The first polypeptide domain comprises a clustered regularly spaced short palindromic repeat-related (Cas) protein, and the second polypeptide domain has an activity selected from the following: transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity and demethylase activity. The Cas protein may comprise Cas9. Cas9 may comprise at least one amino acid mutation that knocks out the nuclease activity of Cas9. At least one amino acid mutation may be at least one of D10A and H840A. The Cas protein may comprise iCas9 (SEQ ID NO: 1 amino acids 36-1403). The second polypeptide domain may have transcriptional activation activity. The second polypeptide domain may comprise at least one VP16 transcriptional activation domain repeat sequence. The second polypeptide domain may comprise a VP16 tetramer ("VP64") or a p65 activation domain. The fusion protein may also comprise a joint that connects the first polypeptide domain to the second polypeptide domain. The fusion protein may comprise iCas9-VP64.

[0011] The present invention relates to a DNA targeting system comprising the fusion protein and at least one guide RNA (gRNA). At least one gRNA may comprise a 12-22 base pair complementary polynucleotide sequence of the target DNA sequence followed by a protospacer-adjacent motif. 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. At least one gRNA may target the introns of a gene. At least one gRNA may target the exons of a gene. At least one gRNA may target the promoter region of a gene selected from the following genes: ASCL1 , BRN2 , MYT1L , NANOG , VEGFA , TERT , IL1B , IL1R2 , IL1RN , HBG1 , HBG2 and MYOD1 The at least one gRNA may comprise at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625.

[0012] The present invention relates to a DNA targeting system comprising Cas9 and at least one guide RNA (gRNA) bound to a dystrophin gene. The at least one gRNA may target an intron of the dystrophin gene. The at least one gRNA may target an exon of the dystrophin gene. The at least one guide RNA may comprise at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625. The DNA targeting system may comprise between 1 and 10 different gRNAs.

[0013] The present invention relates to an isolated polynucleotide encoding the fusion protein or the DNA targeting system.

[0014] The present invention relates to a vector comprising the isolated polynucleotide.

[0015] The present invention relates to a cell comprising the isolated polynucleotide or the vector.

[0016] The present invention relates to a method for regulating mammalian gene expression in a cell. The method comprises contacting the cell with the fusion protein, the DNA targeting system, the isolated polynucleotide or the vector. Gene expression can be induced.

[0017] The present invention relates to a method for transdifferentiating or inducing cell differentiation. The method comprises contacting a cell with the fusion protein, the DNA targeting system, the isolated polynucleotide or the vector. The cell may be a fibroblast or an induced pluripotent stem cell. The fibroblast may be transdifferentiated into a neuronal cell or a myogenic cell. The DNA targeting system may be contacted with the cell, and at least one gRNA targets the promoter region of at least one gene selected from the following: ASCL1 , BRN2 , MYOD1 and MYT1L DNA targeting systems can include targeting ASCL1 At least one gRNA and targeting the promoter region of a gene BRN2 At least one gRNA targeting the promoter region of a gene. The DNA targeting system may include between 1 and 20 different gRNAs. The DNA targeting system may include 8 or 16 different gRNAs. The DNA targeting system may include dCas9-VP64. The DNA targeting system may be delivered to cells by viral or non-viral methods.

[0018] The present invention relates to a method for correcting a mutant gene in a cell. The method comprises administering the DNA targeting system, the isolated polynucleotide or the vector to a cell. Correction of the mutant gene may include homology-guided repair. The method may further comprise administering donor DNA to a cell. The mutant gene may comprise a frameshift mutation that causes premature stop codons and truncated gene products. Correction of the mutant gene may comprise nuclease-mediated non-homologous end joining. Correction of the mutant gene may comprise deletion of premature stop codons, destruction of splice acceptor sites, deletion of one or more exons or destruction of splice donor sequences. Deletion of one or more exons may result in correction of the reading frame.

[0019] The present invention relates to a method for treating a subject in need thereof having a mutant dystrophin gene. The method comprises administering the DNA targeting system, the isolated polynucleotide or the vector to the subject. The subject may suffer from Duchenne muscular dystrophy.

[0020] The present invention relates to a method for correcting a mutant dystrophin gene of a cell. The method comprises administering the DNA targeting system, the isolated polynucleotide, the vector or the cell to a cell containing a mutant dystrophin gene. The mutant dystrophin gene may comprise a premature stop codon, a damaged reading frame by gene deletion, an abnormal splice acceptor site or an abnormal splice donor site, and wherein the target region is upstream or downstream of the premature stop codon, the damaged reading frame, the abnormal splice acceptor site or the abnormal splice donor site. Correction of the mutant dystrophin gene may comprise homology-directed repair. The method further comprises administering donor DNA to the cell. The mutant dystrophin gene may comprise a frameshift mutation causing a premature stop codon and a truncated gene product. Correction of the mutant dystrophin gene may comprise nuclease-mediated non-homologous end joining. Correction of the mutant dystrophin gene may comprise deletion of a premature stop codon, correction of a damaged reading frame or splicing regulation by disrupting a splice acceptor site or disrupting a splice donor sequence. Correction of the mutant dystrophin gene may include deletion of exons 45-55 or exon 51.

[0021] The present invention relates to a kit comprising the fusion protein, the DNA targeting system, the isolated polynucleotide, the vector or the cell.

[0022] The present invention relates to a method for regulating mammalian gene expression in a cell. The method comprises contacting a cell 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 comprise between 1 and 10 different gRNAs. Different gRNAs may bind to different target 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-about 700 base pairs. Different gRNAs may each bind to at least one different target gene. Different target genes may be located on the same chromosome. Different target genes may be located on different chromosomes. At least one target region may be in a non-open chromatin region, an open chromatin region, a promoter region of a target gene, an enhancer region of a target gene, a transcription region of a target gene, or an upstream region of a transcription start site of a target gene. At least one target region may be located between about 1-about 1000 base pairs upstream of the transcription start site of a target gene. At least one target region may be located between about 1-about 600 base pairs upstream of the transcription start site of a target gene. Gene expression may be induced. The DNA targeting system can include 2 different gRNAs, 3 different gRNAs, 4 different gRNAs, 5 different gRNAs, 6 different gRNAs, 7 different gRNAs, 8 different gRNAs, 9 different gRNAs, or 10 different gRNAs. At least one guide RNA can target a promoter region of a gene selected from: ASCL1 , BRN2 , MYT1L , NANOG , VEGFA , TERT , IL1B , IL1R2 , IL1RN , HBG1 , HBG2 and MYOD1 The at least one guide RNA may comprise at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625. The at least one target region may be within an intron or exon of the target gene.

[0023] The present invention relates to a composition for inducing mammalian gene expression in a cell. The composition comprises the fusion protein and at least one guide RNA (gRNA).

[0024] The present invention relates to a composition for inducing mammalian gene expression in a cell. The composition comprises an isolated polynucleotide sequence encoding the fusion protein and at least one guide RNA (gRNA). At least one guide RNA can target the promoter region of a gene selected from the following: ASCL1 , BRN2 , MYT1L , NANOG , VEGFA , TERT , IL1B , IL1R2 , IL1RN , HBG1 , HBG2 and MYOD1 The at least one guide RNA may comprise at least one of SEQ ID NOs 5-40, 65-144, 492-515, 540-563, and 585-625.

[0025] The present invention relates to a cell comprising the composition for inducing expression of a mammalian gene in a cell.

[0026] The present invention relates to a kit comprising the composition for inducing mammalian gene expression in a cell or the cell comprising the composition for inducing mammalian gene expression in a cell.

[0027] The present invention relates to a kit for inducing mammalian gene expression in cells. The kit comprises the composition for inducing mammalian gene expression in cells or the cell comprising the composition for inducing mammalian gene expression in cells.

[0028] The present invention relates to a composition for genome editing in a subject's muscle. The composition comprises a modified adeno-associated virus (AAV) vector and a nucleotide sequence encoding a site-specific nuclease. The muscle is skeletal muscle or cardiac muscle. The modified AAV vector may have improved cardiac muscle and skeletal muscle tissue tropism. The site-specific nuclease may include a zinc finger nuclease, a TAL effector nuclease, or a CRISPR / Cas9 system. The site-specific nuclease may bind to a gene or locus in a muscle cell. The gene or locus may be a dystrophin gene. The composition may also include donor DNA or a transgene.

[0029] The present invention relates to a kit comprising the composition for genome editing in muscle of a subject.

[0030] The present invention relates to a method for genome editing in a subject's muscle. The method comprises administering the composition for genome editing in a subject's muscle to the muscle, wherein the muscle is skeletal muscle or cardiac muscle. Genome editing may include correcting a mutant gene or inserting a transgene. Correcting a mutant gene may include deletion, rearrangement, or replacement of a mutant gene. Correcting a mutant gene may include nuclease-mediated non-homologous end joining or homology-guided repair.

[0031] The present invention relates to a method for treating a subject. The method comprises administering the composition for genome editing in a subject's muscle to a subject's muscle, wherein the muscle is skeletal muscle or cardiac muscle. The subject may suffer from a skeletal muscle condition or a genetic disease. The subject may suffer from Duchenne muscular dystrophy.

[0032] The present invention relates to a method for correcting a mutant gene in a subject, the method comprising administering the composition for genome editing in a subject's muscle. The muscle is skeletal muscle or cardiac muscle. The composition can be injected into a subject's skeletal muscle. The composition can be injected into a subject systemically. The skeletal muscle can be the tibialis anterior muscle.

[0033] The present invention relates to a modified lentiviral vector for genome editing in a subject, the vector comprising a first polynucleotide sequence encoding the fusion protein and a second polynucleotide sequence encoding at least one sgRNA. The first polynucleotide sequence may be operably connected to a first promoter. The first promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter. The second polynucleotide sequence may encode between 1 and 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 polynucleotide sequence encoding different sgRNAs may be operably connected to a promoter. Each promoter operably connected to different sgRNAs may be the same promoter. Each promoter operably connected to different sgRNAs may be a different promoter. The promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter. The sgRNA can bind to a target gene. Each sgRNA can bind to a different target region within a target locus. Each sgRNA can bind to a different target region within a different locus. The fusion protein can include a Cas9 protein or an iCas9-VP64 protein. The fusion protein can include a VP64 domain, a p300 domain, or a KRAB domain. Two or more endogenous genes can be transcriptionally activated. Two or more endogenous genes can be repressed.

[0034] The present invention relates to a method for activating an endogenous gene in a cell. The method comprises contacting a 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 repressed. The endogenous gene can be stably repressed. The fusion protein can be expressed at a level similar to that of the sgRNA. The fusion protein can be expressed at a different level than that of the sgRNA. The cell can be a primary human cell.

[0035] The present invention relates to a method for performing multiple gene editing in a cell. The method comprises contacting a cell with the modified lentiviral vector. Multiple gene editing may include correcting at least one mutant gene or inserting a transgene. Correcting a mutant gene may include deleting, rearranging or replacing at least one mutant gene. Correcting at least one mutant gene may include nuclease-mediated non-homologous end joining or homology-guided repair. Multiple gene editing may include deleting at least one gene, wherein the gene is an endogenous normal gene or a mutant gene. Multiple gene editing may include deleting at least two genes. Multiple gene editing may include deleting between 2 and 10 genes.

[0036] The present invention relates to a method for regulating gene expression of at least one target gene in a cell. The method comprises contacting a cell with the modified lentiviral vector. Gene expression of at least two genes can be regulated. Gene expression of between 2 genes and 10 genes can be regulated. Gene expression of at least one target gene can be regulated when the gene expression level of at least one target gene is increased or decreased compared to the normal gene expression level of at least one target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 RNA-guided activation of the human IL1RN gene by iCas9-VP64 is shown. (a, b) RNA-guided transcriptional activators were generated by fusing inactive Cas9 (iCas9, D10A / H840A) to the VP64 transactivation domain. iCas9-VP64 recognizes genomic target sites by hybridizing with a 20 bp target sequence via guide RNA (gRNA). (c) IL1RN The expression plasmids of four gRNAs or crRNA / tracrRNAs with sequences in the promoter were co-transfected into HEK293T cells together with the iCas9-VP64 expression plasmid. Evaluation by qRT-PCR IL1RN (d) Four gRNA expression plasmids were co-transfected with iCas9-VP64 separately or in combination. Robust gene activation was observed by qRT-PCR only in response to the combination of gRNAs. (e) Secretion of IL-1ra gene product in the culture medium was evaluated by ELISA to confirm IL1RN Activation of expression. IL-1ra was detected in only 3 of 6 samples treated with a combination of gRNAs. For (ce), data are presented as mean ± sem (n = 3 independent experiments). By Tukey's test, treatment with a combination of gRNAs was significantly different from all other treatments (*P ≤ 0.02). (f) Treatment with an empty expression vector (n = 2) or with an expression plasmid and targeting vector of iCas9-VP64 IL1RN RNA-seq was performed on samples co-transfected with the four gRNAs (n = 2). The only genes with statistically significant changes in expression between these treatments were four IL1RN Increase in isoforms (false discovery rate ≤ 3 x 10-4) and IL32 ed (false discovery rate = 0.03).

[0038] Figure 2RNA-guided human gene activation relevant to cell and gene therapy, genetic reprogramming, and regenerative medicine is shown. HEK293T cells were transfected with iCas9-VP64 expression plasmid and 4 gRNAs alone or in combination. Target gene expression was measured by qRT-PCR and normalized to GAPDH mRNA levels. Data are presented as mean ± sem (n = 3 independent experiments). By Tukey's test, treatment with gRNA combination was statistically significantly different from all other treatments ( *P < 0.05).

[0039] Figure 3 Expression of iCas9-VP64 is shown. Expression of iCas9-VP64 in transfected HEK293 cells was confirmed by western blotting against the N-terminal Flag epitope tag. The wt Cas9 expression plasmid does not contain an epitope tag.

[0040] Figure 4 The gRNA target sites and DNA enzyme hypersensitivity locations of human target genes are shown. The four gRNA target sites for each locus are marked as custom tracks above each gene, and DNase-seq data indicating DNA enzyme hypersensitive open chromatin regions are displayed below each gene. DNase-seq was performed in HEK293T cells to identify DNA enzyme hypersensitive regions as described previously (Song et al., 2011). Cold Spring Harbor protocols 2010, pdb prot5384 (2010); Song et al. Genome Res 21, 1757-1767 (2011). The results suggest that open chromatin is not a necessary condition for gene activation by the combination of gRNA and iCas9-VP64.

[0041] Figure 5 The wild-type Cas9 or inactivated (D10A, H840A) iCas9-VP64 expression plasmids were combined with the targeting IL1RN The expression plasmids of four different guide RNAs of the promoter were co-transfected together. The nuclease activity was determined by the Surveyor assay (Guschin et al., Methods Mol Biol 649, 247-256(2010)). The lower molecular weight bands indicating nuclease activity and DNA repair by non-homologous end joining were present only after treatment with wild-type Cas9, supporting that nuclease activity was abolished by iCas9-VP64.

[0042] Figure 6 Targeting for display HBG1 and HBG2RNA-seq of samples treated with gRNA of control empty expression vector (n = 3) or with expression plasmid and targeting vector of iCas9-VP64 HBG1 RNA-seq was performed on samples co-transfected with 4 gRNAs (n = 2). Three of these gRNAs also targeted HBG2 Observed HBG1 and HBG2 Both showed increases relative to the control, but were not statistically significant due to low expression levels. The only statistically significant changes in gene expression between these treatments were IL32 (false discovery rate = 0.0007) and TNFRS9 (false discovery rate = 0.002) decreased.

[0043] Figure 7 HEK293T cells were treated with iCas9-VP64 and targeted ASCL1 or HBG1 The levels of Ascl1 and γ-globin protein production were assessed by Western blot. In two independent experiments, low levels of these proteins were detected in HEK293T cells, whereas increased expression was detected after iCas9-VP64 treatment.

[0044] Figure 8 Figure 2 shows activation of Ascl1 downstream targets in iCas9-VP64 treated mouse embryonic fibroblasts. Mouse embryonic fibroblasts (MEFs) were transfected with a control GFP expression plasmid or a combination of iCas9-VP64 expression plasmid and four gRNA expression plasmids targeting ASCL1 at a ratio of 50:50 or 75:25. (a) Human ASCL1 The gRNA target site in the promoter (SEQ ID NO: 3) in mouse ASCL1 The target site is indicated by a solid line, and the transcription region is indicated by a dashed line. (b) qRT-PCR assay showed that 2 days after iCas9-VP64 / gRNA treatment, the expression of VP64 in MEFs was significantly increased. ASCL1Expression increased. (ch) After 10 days in neural induction medium, cells were stained for early neuronal differentiation markers Ascl1 and Tuj1 (cd) or more mature neuronal differentiation markers Tuj1 and MAP2 (df). Some Tuj1-positive cells exhibited neuronal morphology (fg) and single cells were found to be positive for Tuj1 and MAP2 (g). (h) Tuj1-positive cells were easily identified in iCas9-VP64 / gRNA treated cultures (approximately 0.05%) but not in controls. n = 3 independent samples and data are expressed as mean ± standard error of the mean. gRNA 75 / 25 was significantly different from gRNA 50 / 50 and control ( *P < 0.01, Tukey test).

[0045] FIG. 9 shows (a) the iCas9-VP64 protein sequence (SEQ ID NO: 1) and (b) the sequence of the gRNA expression cassette with a U6 promoter (SEQ ID NO: 2).

[0046] Fig.10 The standard curve of qRT-PCR is shown. For each gene, the experimental sample with the highest expression level was diluted to draw a standard curve determined by qRT-PCR to ensure effective amplification within the appropriate dynamic range. The efficiency of all amplification reactions was within 90-115%.

[0047] Fig.11 (a)-11(b) show validation of RNA-guided repair. Fig.11 (a) Shows the results of Surveyor assay of genomic DNA harvested from HEK 293T cells 2 days after Cas9 was co-transfected into cells with an empty vector (negative control) or gRNA. Fig.11 (b) Shows the location of the gRNA target. Fig.11 (c) Shows the expected cleavage site of each gRNA.

[0048] Fig.12 Shown is RNA-directed repair in DMD 8036 (del48-50) cells as shown by Surveyor assay.

[0049] Fig.13 RNA-directed repair in DMD 8036 (del48-50) cells shown by PCR across the entire locus. PCR of the wild-type dystrophin gene produced a fragment of 1447 bp in size, while PCR of the mutant gene in the DMD 8036 cell line showed a deletion of approximately 817 bp. The deletion band after introduction of the CRISPR / Cas9-based system was approximately 630 bp.

[0050] Fig.14 Shown is RNA-directed repair in DMD 8036 (del48-50) cells as revealed by western blotting with MANDYS8 (anti-dystrophin antibody) and GAPDH antibody (positive control).

[0051] Fig.15 Shown are ChIP sequencing data illustrating specific binding of iCas9-VP64 targeting the IL1RN promoter. HEK 293T cells were transfected with iCas9-VP64 targeting the IL1RN promoter.

[0052] Fig.16 CRISPR / Cas9 targeting of the dystrophin gene is shown. (A) sgRNA sequences are designed to bind to sequences in the exon 45-55 mutation hotspot of the dystrophin gene, allowing gene editing to restore dystrophin expression for multiple patient-specific mutations. Arrows for introns indicate sgRNA targets designed to delete entire exons from the genome. Arrows for exons indicate sgRNA targets designed to produce targeted frameshifts in the dystrophin gene. (B) An example of frame correction after the introduction of a small insertion or deletion by NHEJ DNA repair of exon 51 using CR3 sgRNA. (C) Schematic diagram of multiple sgRNA targets designed to delete exon 51 and restore the dystrophin reading frame in patient mutations with exon 48-50 deletions. (D) Schematic diagram of multiple sgRNA targets designed to delete the entire exon 45-55 region to overcome multiple DMD patient mutations.

[0053] Fig.17 An image of a TBE-PAGE gel used to quantify the Surveyor assay results to measure the day 3 gene modifications in Table 7 is shown. Asterisks indicate the expected band size indicating nuclease activity.

[0054] Fig.18 An image of a TBE-PAGE gel used to quantify the Surveyor assay results to measure the day 10 gene modifications in Table 7 is shown. Asterisks indicate the expected band size indicating nuclease activity.

[0055] Fig.19Fluorescence-activated flow sorting showing enrichment of genetically modified DMD myoblasts. (A) Plasmids expressing human codon-optimized SpCas9 protein linked to a GFP marker were co-electroporated into human DMD myoblasts with one or both plasmids carrying sgRNA expression cassettes using a T2A ribosomal skipping peptide sequence. (B) The sgRNA expression cassettes shown were independently co-transfected into HEK293T with each plasmid expressing SpCas9 by a T2A ribosomal skipping peptide sequence with (bottom) or without (top) a GFP marker linked to SpCas9. Gene modification frequency was evaluated by the Surveyor assay 3 days after transfection. (C) DMD myoblasts with exons 48-50 deleted in the dystrophin gene were treated with sgRNAs that corrected the dystrophin reading frame in these patient cells. Gene modification was evaluated in unsorted (bulk) or GFP+ sorted cells 20 days after electroporation. (D) GFP expression in DMD myoblasts 3 days after electroporation with the indicated expression plasmids. Transfection efficiency and sorted cell populations are indicated by the gated regions.

[0056] Fig. 20 Shown is a targeted frameshift that restores the dystrophin reading frame using CRISPR / Cas9. (A) The 5' region of exon 51 was targeted using the sgRNA, CR3, that 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 the CR3 expression cassette. The sequence of each clone was determined by Sanger sequencing. The top sequence (bold, exons in red) is the native unmodified sequence. The number of clones for each sequence is indicated in parentheses. (C) Overview of the overall gene editing efficiency and frame conversion resulting from the gene modifications shown in (B). (D) Expression of sgRNAs using SpCas9 and the CR3 sgRNA expression cassette ( Fig.19 C) Western blot of dystrophin expression in human DMD myoblasts treated to produce a targeted frameshift that restores the dystrophin reading frame. Dystrophin expression was probed after 6 days of differentiation using an antibody directed against the rod domain of dystrophin.

[0057] Fig.21Deletion of exon 51 from the human genome using multiplex CRISPR / Cas9 gene editing is shown. (A) End-point genomic PCR spanning the exon 51 locus in human DMD myoblasts with exon 48-50 deletion. The top arrow indicates the expected position of the full-length PCR amplicon, and the two lower arrows indicate the expected position of the PCR amplicon with deletions caused by the specified sgRNA combination. (B) The PCR products of (A) were cloned and each clone was sequenced to determine the insertions and deletions present at the targeted locus. The top row shows the wild-type unmodified sequence, and the triangles indicate the SpCas9 cleavage site. On the right is a representative chromatogram showing the sequence at the expected deletion junction. (C) End-point RT-PCR analysis of dystrophin mRNA transcripts in CRISPR / Cas9-modified human Δ48-50 DMD myoblasts treated with the specified sgRNAs. A representative chromatogram of the expected deletion PCR product is shown on the right. Asterisk: band resulting from hybridization of the deletion product strand to the unmodified strand. (D) Rescue of dystrophin expression by CRISPR / Cas9 genome editing was assessed by Western blotting against dystrophin with GAPDH as a loading control. Arrows indicate the expected restored dystrophin band.

[0058] Fig. 22 Deletion of the entire exon 45-55 region in human DMD myoblasts by multiplex CRISPR / Cas9 gene editing is shown. (A) End-point genomic PCR of genomic DNA detecting the deleted region between intron 44 and intron 55 after treatment of HEK293T or DMD myoblasts with the indicated sgRNAs. (B) Individual clones of the expected size PCR product of the deletion from DMD myoblasts in (A) were analyzed by Sanger sequencing to determine the sequence of the genomic deletion present at the targeted locus. The lower figure is a representative chromatogram showing the sequence at the expected deletion junction. (C) End-point RT-PCR analysis of dystrophin mRNA transcripts in CRISPR / Cas9-modified human Δ48-50 DMD myoblasts treated with the indicated sgRNAs. A representative chromatogram of the expected deletion PCR product is shown on the right. (D) Dystrophin expression restored by Western blot analysis after electroporation of DMD myoblasts with sgRNAs targeting intron 44 and / or intron 55.

[0059] Fig.23Demonstration of flow cytometry-based enrichment of genetically modified DMD myoblasts for in vivo cell transplantation experiments is shown. DMD myoblasts were treated with Cas9 with or without sgRNA expression vectors for CR1 and CR5, and GFP+ cells were sorted by flow cytometry. Deletions at the exon 51 locus were detected by end-point PCR using primers flanking the locus. Negative control: DMD myoblasts treated with Cas9 alone and sorted for GFP+ cells.

[0060] Fig.24 Shows restored in vivo expression of human dystrophin after CRISPR / Cas9-treated human DMD myoblasts were transplanted into immunodeficient mice. Fig.19 Human Δ48-50 DMD myoblasts were treated with SpCas9, CR1 and CR5 to delete exon 51 and sorted for GFP expression as shown. 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 evaluated 4 weeks after transplantation. Cryosections were stained with anti-human spectrin (which is expressed by both uncorrected and corrected myoblasts fused into mouse muscle fibers) or anti-human dystrophin antibodies as specified. White arrows indicate that muscle fibers are positive for human dystrophin.

[0061] Fig.25 Additional immunofluorescence images probing human dystrophin expression are shown. The top left inset shows serial sections of an area stained with anti-human spectrin. (AC) Sections of muscle injected with untreated human DMD myoblasts. (DF) Sections of muscle injected with CR1 / 5 treated human DMD myoblasts enriched by flow cytometry. White arrows indicate dystrophin positive fibers.

[0062] Fig.26 Figure 2 shows the evaluation of CRISPR / Cas9 toxicity in human cells and the off-target effect of CR1 / CR5-mediated deletion of exon 51. (A) Results of cytotoxicity assays in HEK293T cells treated with human optimized SpCas9 and specified sgRNA constructs. Cytotoxicity is based on the survival of GFP-positive cells co-transfected with the specified nucleases. I-SceI is a well-characterized non-toxic large-range nuclease and GZF3 is a known toxic zinc finger nuclease. (B) Surveyor analysis of off-target sites in sorted hDMD cells treated with expression cassettes encoding Cas9 and specified sgRNAs. The three off-target sites measured in hDMD cells were identified from a set of 50 predicted sites tested in HEK293T cells ( Fig. 27and Table 4). TGT: on-target locus of the specified sgRNA. OT: off-target locus. (C, D) End-point nested PCR for detection of chromosomal translocations in (C) HEK293T cells treated with Cas9 and CR1 or (D) in sorted hDMD cells treated with Cas9, CR1, and CR5. Schematic diagram showing 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 position of the predicted sgRNA cleavage site at each locus. Asterisks indicate bands detected at the expected size. The identity of the band in (C) was confirmed by Sanger sequencing from each end ( Fig.30 ). Representative chromatograms of P2 / P5 translocation in HEK293T cells are shown.

[0063] Fig. 27 An image of a TBE-PAGE gel used to quantify the Surveyor assay results to measure on-target and off-target gene modifications in Table 4 is shown. Asterisks indicate the expected band size indicating nuclease activity.

[0064] Fig.28 Endpoint nested PCR for chromosome translocation caused by CRISPR / Cas9 off-target activity for CR3 and CR6 / CR36 in human cells is shown. In (A) HEK293T or sorting hDMD cells treated with Cas9 and CR3 as shown, (B) HEK293T cells treated with Cas9 and CR36 only, or (C) sorting hDMD cells treated with Cas9, CR6 and CR36 expression cassettes, nested endpoint PCR analysis is used to detect translocation. The second nested PCR reaction of translocation is amplified using custom primers for each predicted translocation locus to maximize specificity (see Table 4). Schematic diagram represents the relative position of the nested primer pairs used to detect the presence of translocation. First, each possible translocation event is amplified from genomic DNA isolated from cells treated with or without specifying sgRNA. The second nested PCR reaction is performed using primers in the predicted PCR amplicon that may be generated in the translocation. The expected size is estimated according to the specified primer binding site and the predicted sgRNA cleavage site on each locus. * represents the band detected with the expected size, which is confirmed by Sanger sequencing from each end. # indicates an amplicon where Sanger sequencing showed sequences outside the predicted translocation, likely the result of mispriming during nested PCR.

[0065] Fig.29 show Fig.28Sanger sequencing chromatogram of the bands detected in, which arise from the translocation between CR3 and CR3-OT1 on chromosomes X and 1, respectively, in HEK293T cells treated with Cas9 and CR3 gene cassettes. Arrows show regions homologous to the designated chromosomes near the expected breakpoints caused by the appropriate sgRNA. Note that the sequencing reads become out of phase near the breakpoints due to the error-prone nature of DNA repair by non-homologous end joining.

[0066] Fig.30 show Fig.26 Sanger sequencing chromatogram of the bands detected in C, which result from translocations between CR1 and CR1-OT1 on chromosomes X and 16, respectively, in HEK293T cells treated with Cas9 and the CR1 gene cassette. Arrows show regions homologous to the designated chromosomes near the expected breakpoints caused by the appropriate sgRNA. Note that sequencing reads become out of phase near the breakpoints due to the error-prone nature of DNA repair by non-homologous end joining.

[0067] Fig.31 An overview of in vivo AAV injection and tissue harvesting is shown.

[0068] Fig.32 Surveyor analysis of Rosa26 ZFN activity in skeletal muscle in vitro and in vivo following delivery of AAV-SASTG-ROSA is shown. Arrows indicate expected bands resulting from Surveyor cleavage. nd: not detected. (a) Proliferating C2C12 were transduced with the indicated amounts of virus and harvested 4 days post infection. Arrows indicate expected band sizes resulting from Surveyor cleavage. (b) C2C12 were incubated in differentiation medium for 5 days and then transduced with the indicated amounts of AAV-SASTG-ROSA virus in 24-well plates. Samples were collected 10 days after transduction. (c) The indicated amounts of AAV-SASTG-ROSA were injected directly into the tibialis anterior muscle of C57BL / 6J mice and the muscles were harvested 4 weeks post infection. The harvested TA muscle was divided into 8 separate pieces for genomic DNA analysis, each of which is shown in a separate lane.

[0069] Fig.33 The Rosa T2A opt DNA sequence (SEQ ID NO: 434) and the Rosa T2A opt protein sequence (SEQ ID NO: 435) are shown.

[0070] Fig.34 The SASTG capsid DNA sequence (SEQ ID NO:436) and the SASTG capsid peptide sequence (SEQ ID NO:437) are shown.

[0071] Fig.35 The DZF16 ZFN target site sequence (SEQ ID NO: 442), the entire amino acid sequence on the left side of DZF16-L6 (SEQ ID NO: 443), and the entire amino acid sequence on the right side of DZF16-R6 (SEQ ID NO: 444) are shown.

[0072] Fig.36 The E51C3 ZFN target site sequence (SEQ ID NO: 445), the entire amino acid sequence to the left of E51C3-3L (SEQ ID NO: 446), and the entire amino acid sequence to the right of E51C3-3R (SEQ ID NO: 447) are shown.

[0073] Fig.37 Shown are the DZF15 ZFN target site sequence (SEQ ID NO: 448), the entire amino acid sequence on the left side of DZF15-L6 (SEQ ID NO: 449), the entire amino acid sequence on the right side of DZF15-R6 (SEQ ID NO: 450), the entire amino acid sequence on the left side of DZF15-L5 (SEQ ID NO: 451), and the entire amino acid sequence on the right side of DZF15-R5 (SEQ ID NO: 452).

[0074] Fig.38 The E51C4 ZFN target site sequence (SEQ ID NO: 453), the entire amino acid sequence to the left of E51C4-4L (SEQ ID NO: 454), and the entire amino acid sequence to the right of E51C4-4R (SEQ ID NO: 455) are shown.

[0075] Fig.39 Schematic diagram showing "single vector, multiplex CRISPR system", "binary vector, multiplex CRISPR system" and "single vector, single gRNA system".

[0076] Fig.40 The nucleotide sequences of SaCas9-NLS (wherein NLS is underlined) (SEQ ID NO: 64) and SaCas9 gRNA (SEQ ID NO: 116) are shown.

[0077] Fig.41The nucleotide sequences of NmCas9 (wherein NLS 1 is underlined, NLS 2 is underlined and bold, and the HA tag is in bold), NmCas9 short hairpin from Thomson PNAS 2013 (SEQ ID NO: 118), and NmCas9 long hairpin from ChurchNature Biotech 2013 (SEQ ID NO: 119) are shown.

[0078] Fig.42 Validation of sgRNA and lentiviral Cas9 expression constructs is shown. (a) Constructs encoding a unique Pol III promoter expressing sgRNA targeting the AAVS1 locus or a construct containing the hU6 promoter followed by polythymidine to terminate expression ("PolyT") were transfected into HEK293T cells. Endpoint RT-PCR was used 2 days after transfection to probe the expression of each indicated promoter / sgRNA construct. -RT: no reverse transcriptase control. (b) HEK293T were transfected with expression vectors encoding the AAVS1 zinc finger nuclease or Cas9-T2A-GFP and the indicated promoter / sgRNA expression cassettes, and the Surveyor assay was used to evaluate the level of gene modification 3 days after transfection. (c) HEK293T cells were transduced with lentiviral constructs encoding the indicated Cas9-T2A-GFP constructs without sgRNA, and 7 days after transduction, Cas9 expression was evaluated by Western blot by probing for the FLAG epitope tag on the N-terminus of the Cas9 protein.

[0079] Fig.43 Golden Gate assembly showing a single lentiviral CRISPR / Cas9 expression cassette.

[0080] Fig.44 Single lentiviral delivery showing multiplexed CRISPR / Cas9 system. (a) Four sgRNAs targeting different genomic loci were cloned into a lentiviral vector expressing an active Cas9 nuclease. (b) HEK293T and primary human dermal fibroblasts were transduced with lentivirus expressing the indicated sgRNAs and cleavage events were determined using the Surveyor assay. HEK293T were assayed 7 days after transduction. Human fibroblasts were assayed 10 days after transduction.

[0081] Fig.45Figure 2 shows transient gene activation in HEK293T cells stably expressing dCas9-VP64. HEK293T cells were transduced with lentivirus to stably express dCas9-VP64 and subsequently transfected with plasmids expressing the specified sgRNA combination. By varying the number of sgRNAs delivered, regulatable endogenous gene activation of endogenous IL1RN (a) and HBG1 (b) loci was achieved 3 days after transfection. Peak levels of endogenous IL1RN (c) and HBG1 (d) were observed 3-6 days after transfection, with activation levels returning to background levels between days 15-20. Importantly, the cell line was able to respond after the second transfection on day 20, although at lower levels than previously observed.

[0082] Fig.46 Figure 2 shows stable gene activation in HEK293T using multiple dCas9-VP64 vectors from a single lentivirus. HEK293T were transduced with lentivirus to stably express the specified combination of dCas9-VP64 and gRNA. By varying the number of sgRNAs delivered, regulatable endogenous gene activation of the endogenous IL1RN (a) and HBG1 (b) loci was achieved 7 days after transduction. Peak levels of endogenous IL1RN (c) and HBG1 (d) were observed 6 days after transduction and activation levels were maintained until day 21.

[0083] Fig.47 IL1RN mRNA expression levels are shown.

[0084] Fig.48 Shown is a schematic diagram representing the direct conversion of fibroblasts into neurons by ectopic expression of BAM neuronal transcription factors.

[0085] Fig.49 (A) Schematic diagram showing the dCas9-VP64 construct. dCas9-VP64 is a catalytically inactive form of the Cas9 protein fused to a tetramer of the VP16 transcriptional activation domain. (B) Schematic diagram showing the mechanism of RNA-guided recruitment of dCas9-VP64 to genomic targets. (C) Schematic diagram of the experimental protocol for generating iNs with CRISPR / Cas9 transcription factors.

[0086] Fig.50 Shown in the transduction with dCas9-VP64 and the targeting ASCL1 Promoter, ASCL1 In MEFs transfected with either cDNA or luciferase gRNA, endogenous ASCL1 Total expression or detection by (B) immunofluorescence ASCL1 Protein. Asterisks (*) indicate that the expression of 8 gRNAs co-delivered compared with 4 gRNAs was significantly higher than that of 4 gRNAs. ASCL1The expression was significantly increased (p<0.05). Compared with that induced by dCas9-VP64 and 8 gRNAs targeting the Ascl1 promoter, ASCL1 Ectopic expression of produced more protein but did not activate the endogenous locus in culture by day 3.

[0087] Fig.51 (A) Expression of ectopic BAM factors or dCas9-VP64 and targeting BRN2 and ASCL1 1. Promoter-gRNA Generation TUJ1 and MAP2 Positive cells, (B) cells with neuronal morphology expressing hSyn-RFP reporter at day 11 in N3 medium.

[0088] Fig.52 Shown (A) are cells with neuronal morphology that are positive for the GCaMP5 calcium indicator in culture in the presence (bottom) or absence (top) of KCl. (B) Traces showing normalized fluorescence intensity over time as cells depolarize in response to KCl addition.

[0089] Fig.52 The activation of downstream targets of Ascl1 and Brn2 (i.e., master regulatory genes) using dCas9-VP64 transcription factors to convert fibroblasts into neurons in mouse embryonic fibroblasts treated with iCas9-VP64 was shown. Mouse embryonic fibroblasts (MEF) were transfected with a combination of 8 gRNA expression plasmids targeting ASCL1 and BRN2 with a control GFP expression plasmid or an iCas9-VP64 expression plasmid. The dCas9 transcription factor was delivered by virus. After 10 days in neural induction medium, cells were stained for Tuj1 (early markers of neuronal differentiation) and MAP2 (markers of more mature neuronal differentiation). The conversion to neurons is effective.

[0090] Fig.53 CRISPR / Cas9 platform for controlling mammalian gene regulation is shown. A. Cas9-based effectors bind genomic sequences in the presence of a chimeric gRNA molecule consisting of a constant region that forms a complex with Cas9 preceded by an exchangeable 20 bp protospacer that confers target site specificity. B. Cas9-based synthetic transcription factors repress transcription of target genes by interfering with RNA polymerase activity or by binding and blocking binding sites for endogenous transcription factors within the promoter. C. Targeting regulatory elements such as enhancers can also potentially block expression of multiple distal genes.

[0091] Fig.54Targeting of the HS2 enhancer using CRISPR / dCas9-KRAB is shown. The HS2 region is a strong enhancer that distally regulates the expression of globin genes >10 kb downstream. A set of single gRNAs were designed to target sites along the enhancer region.

[0092] Fig.55 A single gRNA targeting the HS2 enhancer is shown to achieve efficient transcriptional repression of globin genes. A. Delivery of dCas9 and dCas9-KRAB repressor proteins on lentiviral vectors. Single gRNAs were transiently transfected for screening. K562 expressing dCas9-KRAB achieved up to 80% repression of B. γ-globin, C. ε-globin, and D. β-globin genes when measured by quantitative RT-PCR 3 days after transfection compared to control cells not treated with gRNA. E. Protein expression in cells expressing dCas9 or dCas9-KRAB and treated with Cr4 or Cr8 at day 3 shows modest repression of γ-globin expression compared to β-actin control.

[0093] Figure 56 shows the expression of globin locus genes in cells treated with different doses of gRNA plasmid delivered to A. without lentivirus, B. dCas9 lentivirus or C. dCas9-KRAB lentivirus. Increasing the dose of delivered Cr4 gRNA plasmid increased repression in dCas9-KRAB treated cells, indicating the role of both dCas9-KRAB effector and targeting gRNA in achieving repression.

[0094] Fig.57 Stable delivery of a single gRNA with dCas9-KRAB is shown to silence the expression of globin genes. A. dCas9 and dCas9-KRAB repressor proteins were co-expressed with a single gRNA on a lentiviral vector. When measured by quantitative RT-PCR 7 days after transduction, K562 expressing dCas9-KRAB achieved up to 95% repression of B. γ-globin, C. ε-globin, and D. β-globin genes compared to control cells that did not receive lentiviral treatment.

[0095] Fig.58 Isolation of the p300 HAT "core" was shown for targeted epigenetic modification of histones solely by dCas9 fusions.

[0096] Fig.59 Streptococcus pyogenes ( S. pyogenes ) Schematic diagram of dCas9-VP64 fusion (top) and dCas9-p300 core fusion (bottom). The protospacer adjacent motif (PAM) is shown at the target locus by arrows and the synthetic guide RNA (gRNA) is shown by a hatched arrow.

[0097] Figures 60A-60C Representative data for three human loci showing the activation efficacy using dCas9-p300 relative to dCas9-VP64 and dCas9 in the absence of any fused effector domain in a human 293T cell culture line.

[0098] Figures 61A-61C The amino acid sequences of the dCas9 constructs are shown. Fig.61A All are shown in Figures 61A-61C Legend for .

[0099] Fig.62 showed that the HAT-dCase9-p300 fusion protein failed to activate gene expression.

[0100] Fig.63 The gRNA was shown to also function synergistically with the dCas9-p300 core.

[0101] Fig.64 TALENs are shown to mediate integration of minidystrophin at the 5'UTR of the Dp427m skeletal muscle isoform of dystrophin in skeletal myoblast cell lines derived from human DMD patients carrying different deletions in the dystrophin gene. DMD patient cells were electroporated with constructs encoding TALEN pairs active at the 5'UTR locus and a donor template carrying the minidystrophin gene. (a) Schematic diagram showing how minidystrophin integrates into the 5'UTR. (b) Hygromycin-resistant clonal cell lines were isolated and screened by PCR for successful site-specific integration at the 5'UTR using the primers shown in (a). Asterisks indicate clones selected for further analysis in (c). (c) DMD myoblasts isolated from clones with detected integration events were differentiated for 6 days and the expression of an HA tag fused to the C-terminus of minidystrophin was evaluated. DETAILED DESCRIPTION OF THE INVENTION

[0102] As described herein, it has been found that certain methods and system compositions based on engineered CRISPR / CRISPR-related (Cas) 9 can be used to alter the expression of genes, genome engineering, and correct or reduce the effects of gene mutations involved in genetic diseases. The system based on CRISPR / Cas9 includes a Cas9 protein and at least one guide RNA, which provides the DNA targeting specificity of the system. Specifically, the present disclosure describes Cas9 fusion proteins that combine the DNA sequence targeting function of the CRISPR / Cas9-based system with other activities, thereby allowing changes in gene expression and / or epigenetic states. The system can also be used for genome engineering and correcting or reducing the effects of gene mutations.

[0103] The present disclosure also provides for delivering systems and multiple gRNAs for targeting one or more endogenous genes based on CRISPR / CRISPR related (Cas) 9. Certain compositions and methods. Co-transfection of multiple sgRNAs targeting a single promoter allows for synergistic activation, however, due to differences in copy number, co-transfection of multiple plasmids results in variable expression levels in each cell. In addition, gene activation after transfection is transient due to dilution of plasmid DNA over time. In addition, many cell types are not easily transfected, and transient gene expression may not be sufficient to cause therapeutic effects. In order to overcome these limitations, a single lentiviral system has been developed to express Cas9 and up to 4 sgRNAs from independent promoters. A platform for expressing Cas9 or dCas9 fusion proteins and up to 4 gRNAs from a single lentiviral vector is disclosed. In addition to 1, 2, 3 or 4 gRNAs expressed from independent promoters, lentiviral vectors also express constitutive or inducible Cas9 or dCas9-VP64. The system enables control of both the amplitude and timing of gene regulation based on CRISPR / Cas9. In addition, the lentiviral platform provides efficient and sustained levels of gene expression, which can facilitate the therapeutic application of the CRISPR / Cas9 system in primary cells. Finally, the system can be used to edit multiple genes at the same time, such as the simultaneous knockout of several oncogenes.

[0104] The present disclosure also provides certain compositions and methods for delivering site-specific nucleases to skeletal muscle and myocardium using modified adeno-associated virus (AAV) vectors. Site-specific nucleases that can be engineered can be used to change gene expression, genome engineering, correct or reduce the effects of gene mutations involved in genetic diseases or operate genes involved in other conditions that affect skeletal muscle or myocardium or muscle regeneration. Engineered site-specific nucleases may include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and / or CRISPR / Cas9 systems for genome editing. As described herein, using this unique delivery system, genes in skeletal muscle tissue are successfully edited in vivo. The disclosed invention provides methods for rewriting the human genome for therapeutic applications and target model classes for basic science applications.

[0105] Gene editing is highly dependent on cell cycling and complex DNA repair pathways that vary between tissues. Skeletal muscle is an extremely complex environment, consisting of a large number of muscle fibers with more than 100 nuclei per cell. For decades, gene therapy and biologics have generally been limited by in vivo delivery barriers. These challenges include the stability of in vivo vectors, targeting the correct tissues, obtaining sufficient gene expression and active gene products, and avoiding toxicity that may overwhelm activity, which is common when using gene editing tools. In other cases, other delivery vectors (such as direct injection of plasmid DNA) work in skeletal and cardiac muscle to express genes, but these site-specific nucleases do not work well for obtaining detectable levels of genome editing.

[0106] Although many gene sequences are unstable in AAV vectors and therefore cannot be delivered, these site-specific nucleases are surprisingly stable in AAV vectors. When these site-specific nucleases are delivered and expressed, they remain active in skeletal muscle tissue. The protein stability and activity of site-specific nucleases are highly tissue-type dependent and cell-type dependent. These active and stable nucleases are able to modify gene sequences in the complex environment of skeletal muscle. The present invention describes methods for delivering active forms of such therapeutic agents that are effective and promote successful genome modification to skeletal muscle or cardiac muscle.

[0107] The present disclosure also provides certain fusion epigenetic effector molecules, a dCas9-p300 fusion protein, which provides a more widely applicable tool for synthetic transcriptional regulation than the dCas9-VP64 fusion. Compared with the dCas9-VP64 fusion protein at all loci tested, the target genes activated to a much greater extent. In addition, p300 has inherent endogenous activity on enhancers within the human genome. The dCas9-p300 fusion protein is able to activate endogenous target gene promoters and enhancer regions.

[0108] The dCas9-p300 fusion protein can be used in human tissue culture cell lines to activate gene expression. The fusion protein can be used to accurately and predictably direct the epigenetic state of target loci in human cells to control differentiation, modulate cell regulation, and apply new and effective therapies. Current technologies are limited in the intensity of activation and the extent and sustainability of epigenetic regulation, obstacles that can be eliminated by using this new fusion protein.

[0109] The section headings used in this section and throughout the disclosure disclosed herein are for organizational purposes only and are not intended to be limiting.

[0110] 1. Definition

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art are generally understood. In the event of a conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, but methods and materials similar to those described herein can be used to implement or test the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0112] As used herein, the terms "comprises," "including," "having," "possessing," "may," "containing," and variations thereof are intended to refer to open transitional phrases, terms, or words that do not exclude the possibility of other actions or configurations. The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Whether explicitly stated or not, the present disclosure also includes other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements provided herein.

[0113] For the citation of numerical ranges herein, each intervening number of the same precision is explicitly included. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also included, and for the range of 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 explicitly included.

[0114] "Adeno-associated virus" or "AAV", used interchangeably herein, refers to a small virus belonging to the genus Dependovirus of the family Parvoviridae that infects humans and some other primate species. AAV is not currently known to cause disease, but the virus causes a very mild immune response.

[0115] As used herein, "binding region" refers to the region within the nuclease target region that is recognized and bound by the nuclease.

[0116] "Myocardium" or "heart muscle" used interchangeably herein refers to the type of involuntary striated muscle present in the wall of the heart and the base of the cardiac tissue, the myocardium. The myocardium is composed of cardiomyocytes (cardiomyocytes / myocardiocytes). Cardiomyocytes display striations similar to those in skeletal muscle cells, but contain only a single nucleus, unlike multinucleated skeletal cells.

[0117] As used herein, "cardiomyopathy" refers to diseases related to the myocardium, such as cardiomyopathy, heart failure, arrhythmias, and inflammatory heart diseases.

[0118] As used herein, "coding sequence" or "encoding nucleic acid" means a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may also include start and stop signals operably linked to regulatory elements (including a promoter and a polyadenylation signal) that are capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized.

[0119] As used herein, "complementary sequence" or "complementarity" means a nucleic acid which may imply Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule. "Complementarity" refers to a property shared between two nucleic acid sequences such that when aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0120] As used herein, "correction", "genome editing" and "restoration" refer to changing a mutant gene that encodes a truncated protein or does not encode a protein at all, so that full-length functional or partially full-length functional protein expression is obtained. Correction or restoration of mutant genes may include replacing a region of a gene with a mutation with a repair mechanism (e.g., homology-guided repair (HDR)) or replacing the entire mutant gene with a gene copy that does not have a mutation. Correction or restoration of mutant genes may also include repairing frameshift mutations that cause premature termination codons, abnormal splicing acceptor sites, or abnormal splicing donor sites by generating double-strand breaks in the gene and then repairing them using non-homologous end joining (NHEJ). During the repair period that can restore the appropriate reading frame and exclude premature termination codons, NHEJ may add or delete at least one base pair. Correction or restoration of mutant genes may also include destroying abnormal splicing acceptor sites or splicing donor sequences. Correction or restoration of mutant genes may also include deleting non-essential gene segments by the simultaneous action of two nucleases on the same DNA chain to restore the appropriate reading frame by removing the DNA between the two nuclease target sites and repairing the DNA broken by NHEJ.

[0121] "Donor DNA," "donor template," and "repair template" used interchangeably herein refer to a double-stranded DNA fragment or molecule that includes at least a portion of a target gene. The donor DNA may encode a fully functional protein or a partially functional protein.

[0122] "Duchenne muscular dystrophy" or "DMD," used interchangeably herein, refers to a recessive fatal X-linked disorder that causes muscle degeneration and eventual death. DMD is a common inherited single gene disease that occurs in 1 in 3500 males. DMD is the result of inherited or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. Most dystrophin mutations that cause DMD are exon deletions that disrupt the reading frame and cause premature translation termination of the dystrophin gene. DMD patients typically lose the ability to physically support themselves in childhood, become increasingly frail in their teens, and die in their twenties.

[0123] As used herein, "dystrophin" refers to a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of the muscle fiber to the surrounding extracellular matrix throughout the cell membrane. Dystrophin provides structural stability to the dystrophin complex of the cell membrane, which is responsible for regulating muscle cell integrity and function. The dystrophin gene or "DMD gene," used interchangeably herein, is 2.2 megabases located at locus Xp21. The primary transcript measures approximately 2,400 kb, and its mature mRNA is approximately 14 kb. 79 exons encode a protein of more than 3500 amino acids.

[0124] As used herein, "exon 51" refers to the 51st exon of the dystrophin gene. Exon 51 is often close to frame-destructive deletions in DMD patients and is being targeted for oligonucleotide-based exon skipping in clinical trials. Clinical trials for the exon 51 skipping compound eteplirsen recently reported significant functional benefits at 48 weeks, with an average of 47% dystrophin-positive fibers compared to baseline. Mutations in exon 51 are ideally suited for permanent correction via NHEJ-based genome editing.

[0125] "Frameshift" or "frameshift mutation" used interchangeably herein refers to a type of genetic mutation in which the addition or deletion of one or more nucleotides causes a shift in the reading frame of a codon in mRNA. The shift in the reading frame can result in changes in the amino acid sequence of a protein translation, such as missense mutations or premature stop codons.

[0126] As used herein, "functional" and "fully functional" describe a protein that has biological activity. "Functional gene" refers to a gene that is transcribed into mRNA, which is translated into a functional protein.

[0127] As used herein, "fusion protein" refers to a chimeric protein produced by joining two or more genes that originally encoded separate proteins. Translation of the fusion gene results in a single polypeptide having functional properties derived from each of the original proteins.

[0128] As used herein, "gene construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence includes start and stop signals operably linked to regulatory elements (including a promoter and a polyadenylation signal) that are capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct containing the necessary regulatory elements operably linked to a coding sequence encoding a protein so that the coding sequence can be expressed when present in the cells of an individual.

[0129] As used herein, "genetic disease" refers to a disease caused partially or completely, directly or indirectly by one or more abnormal regions in the genome, especially a condition that exists since birth. Abnormality can be a mutation, insertion or deletion. Abnormality can affect the coding sequence of a gene or its regulatory sequence. Genetic diseases can be, but are not limited to, DMD, hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, hepatolenticular degeneration (Wilson'sdisease), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome (retinoblastoma), retinoblastoma and Tay-Sachs disease.

[0130] "Homology-guided repair" or "HDR", used interchangeably herein, refers to a mechanism for repairing double-stranded DNA damage in cells, mostly during the G2 and S phases of the cell cycle, when homologous segments of DNA are present in the nucleus. HDR uses a donor DNA template to guide repair and can be used to produce specific sequence changes in the genome, including targeted additions of entire genes. If the donor template is provided with a site-specific nuclease (e.g., a CRISPR / Cas9-based system), the cellular machinery will repair the break by homologous recombination, which is increased by several orders of magnitude in the presence of DNA cutting. When homologous DNA segments are present, non-homologous end joining can occur.

[0131] As used herein, "genome editing" refers to changing a gene. Genome editing can include correcting or restoring a mutant gene. Genome editing can include knocking out a gene, such as a mutant gene or a normal gene. Genome editing can be used to treat disease or enhance muscle repair by changing a target gene.

[0132] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a specific percentage of identical residues within a specified region. The percentage can be calculated as follows: by optimally aligning the two sequences, comparing the two sequences within a specified region, determining the number of positions where identical residues occur in the two 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. In the case where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity calculations can be performed manually or by applying a computer sequence algorithm such as BLAST or BLAST 2.0.

[0133] "Mutated gene" or "mutated gene" as used interchangeably herein refers to a gene that has undergone a detectable mutation. A mutant gene has undergone an alteration, such as loss, gain or exchange of genetic material, which affects the normal transmission and expression of the gene. "Disrupted gene" as used herein refers to a mutant gene having a mutation that causes a premature stop codon. A disrupted gene product is truncated relative to the full-length non-disrupted gene product.

[0134] As used herein, "non-homologous end joining (NHEJ) pathway" refers to a pathway for repairing double-strand breaks in DNA by directly joining the broken ends without the need for a homologous template. Template-independent DNA rejoining by NHEJ is a random error-prone repair process that randomly introduces microinsertions and microdeletions (indels) at DNA breakpoints. The method can be used intentionally to interrupt, delete or change the reading frame of a targeted gene sequence. NHEJ typically uses short homologous DNA sequences called microhomologies to guide repair. These microhomologies often exist in single-stranded overhangs at the ends of double-strand breaks. When the overhangs are fully compatible, NHEJ usually accurately repairs the break, however, inaccurate repairs that result in nucleotide loss may also occur, but inaccurate repairs are much more common when the overhangs are incompatible.

[0135] As used herein, "normal gene" refers to a gene that has not undergone changes such as loss, gain or exchange of genetic material. A normal gene undergoes normal gene transmission and gene expression.

[0136] As used herein, "nuclease-mediated NHEJ" refers to NHEJ that begins after a nuclease (eg, Cas9) cuts double-stranded DNA.

[0137] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also includes the complementary strand of the single strand. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Therefore, nucleic acids also include substantially identical nucleic acids and their complementary sequences. The single strand provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Therefore, nucleic acids also include probes that hybridize under stringent hybridization conditions.

[0138] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic and cDNA), RNA, or hybrids, wherein nucleic acids may contain a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or recombinant methods.

[0139] As used herein, "operably linked" means that the expression of a gene is under the control of a promoter that is spatially linked thereto. A promoter may be located 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene may be about the same as the distance between the promoter and the gene it controls (from which the promoter is derived). As is known in the art, variations in this distance may be accommodated without loss of promoter function.

[0140] As used herein, "partially functional" describes a protein encoded by a mutant gene and having biological activity that is lower than that of a functional protein but higher than that of a non-functional protein.

[0141] "Premature stop codon" or "out-of-frame stop codon", used interchangeably herein, refers to a nonsense mutation in a DNA sequence that results in a stop codon at a position that is not normally present in the wild-type gene. A premature stop codon may cause a protein to be truncated or shorter than the full-length form of the protein.

[0142] As used herein, "promoter" means a molecule of synthetic or natural origin that can confer, activate or increase the expression of nucleic acids in cells. A promoter may comprise one or more specific transcriptional regulatory sequences to further provide expression and / or alter the spatial expression and / or temporal expression of the sequence. A promoter may also comprise a distal enhancer or repressor protein element, which may be located up to several thousand base pairs from the transcription start site. A promoter may be derived from the following sources, including viruses, bacteria, fungi, plants, insects and animals. In terms of the cells, tissues or organs in which expression occurs, or in terms of the developmental stage when expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions or inducers, a promoter may constitutively or differentially regulate the expression of a gene component. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator 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.

[0143] "Repeating variable di-residues" or "RVDs", used interchangeably herein, refer to pairs of adjacent amino acid residues within a DNA recognition motif (also referred to as "RVD modules") that include 33-35 amino acids of the TALE DNA binding domain. The RVDs determine the nucleotide specificity of the RVD modules. RVD modules can be combined to produce RVD arrays. "RVD array length" as used herein refers to the number of RVD modules that is equivalent to the length of the nucleotide sequence within the TALEN target region (i.e., the binding region) recognized by the TALEN.

[0144] As used herein, "site-specific nuclease" refers to an enzyme that can specifically recognize and cleave a DNA sequence. Site-specific nucleases can be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR / Cas9-based systems.

[0145] As used herein, "skeletal muscle" refers to a type of striated muscle that is under the control of the somatic nervous system and is connected to bone by bundles of collagen fibers called tendons. Skeletal muscle is composed of components called myocytes or "muscle cells", sometimes referred to as "myofibers". Myocytes are formed by the fusion of developing myoblasts (a type of embryonic progenitor cell that produces muscle cells) in a process called myogenesis. These long, cylindrical, multinucleated cells are also called myofibers.

[0146] As used herein, "skeletal muscle condition" refers to conditions associated with skeletal muscle, such as muscular dystrophy, aging, muscle degeneration, wound healing, and muscle weakness or atrophy.

[0147] "Spacer" and "spacer region" are used interchangeably herein to refer to the region within the target region of a TALEN or ZFN that is between the binding regions of two TALENs or ZFNs but is not a part of them.

[0148] "Subject" and "patient" used interchangeably herein refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys, such as cynomolgous or macaques, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human. Other forms of treatment may be performed on the subject or patient.

[0149] As used herein, "target gene" refers to any nucleotide sequence encoding a known or putative gene product. The target gene may be a mutant gene involved in a genetic disease.

[0150] As used herein, "target region" refers to the region of a target gene to which a site-specific nuclease is designed to bind and cleave.

[0151] As used herein, "transcription activator-like effector" or "TALE" refers to a protein structure that recognizes and binds to a specific DNA sequence. "TALE DNA binding domain" refers to a DNA binding domain comprising a series of 33-35 amino acid repeats in series, also known as RVD modules, each of which specifically recognizes a single base pair of DNA. The RVD modules can be arranged in any order to assemble an array that recognizes a specified sequence.

[0152] The binding specificity of the TALE DNA binding domain is determined by a single truncated repeat sequence of 20 amino acids followed by an RVD array. The TALE DNA binding domain may have 12-27 RVD modules, each of which contains an RVD and recognizes a single base pair of DNA. Specific RVDs that recognize each of the four possible DNA nucleotides (A, T, C, and G) have been identified. Because the TALE DNA binding domain is modular, repeat sequences that recognize four different DNA nucleotides can be linked together to recognize any specific DNA sequence. These targeted DNA binding domains can then be combined with catalytic domains to produce functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.

[0153] "Transcription activator-like effector nuclease" or "TALEN" as used interchangeably herein refers to the catalytic domain of a nuclease (e.g., an endonuclease FokI) and an engineered fusion protein of a designed TALE DNA binding domain that can be targeted by 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 TALEN target region.

[0154] As used herein, "transgenic" refers to genes or genetic material containing gene sequences isolated from one organism and introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or may alter the normal function of the genetic code of the transgenic organism. The introduction of a transgene has the potential to change the phenotype of an organism.

[0155] As used herein, "variant" with respect to a nucleic acid means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto under stringent conditions.

[0156] A "variant" of a peptide or polypeptide refers to a variant that differs in amino acid sequence by insertion, deletion or conservative substitution of amino acids but retains at least one biological activity. A variant may also refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is considered in the art to generally involve minor changes. As is understood in the art, these minor changes can be identified, in part, by considering the hydropathic index of the amino acids. Kyte et al., J. Mol. Biol . 157:105-132 (1982). The hydropathic index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. On the one hand, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that can produce proteins that retain biological function. Considering the hydrophilicity of amino acids in the case of peptides allows calculation of the maximum local average hydrophilicity of the peptide. Substitutions can be made with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chains of the amino acids. It is understood that, consistent with this observation, amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly the side chains of those amino acids, as shown by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0157] As used herein, "vector" means a nucleic acid sequence containing a replication origin. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RN vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. For example, the vector may encode an iCas9-VP64 fusion protein comprising the amino acid sequence of SEQ ID NO: 1 or at least one gRNA nucleotide sequence of any one of SEQ ID NO: 5-40, 65-144, 492-515, 540-563, and 585-625. Alternatively, the vector may encode Cas9 and SEQ ID NO: 5-40, 65-144, 492-515, 540-563, and 585-625. At least one gRNA nucleotide sequence of any one of 5-40, 65-144, 492-515, 540-563, and 585-625.

[0158] 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 about 30 amino acids, and the domain usually acts by binding to 3 consecutive base pairs of DNA through the interaction of a single amino acid side chain per base pair.

[0159] "Zinc finger nuclease" or "ZFN," used interchangeably herein, refers to a chimeric protein molecule comprising at least one zinc finger DNA binding domain operably linked to at least one nuclease or a portion of a nuclease that is capable of cleaving DNA when fully assembled.

[0160] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure have the meanings commonly understood by those of ordinary skill in the art. For example, any term used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization and techniques thereof described herein are well-known and commonly used terms in the art. The meaning and scope of the terms should be clear; however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. In addition, unless otherwise required herein, terms in the singular should include the plural form, and plural terms should include the singular.

[0161] 2. Compositions for genome editing

[0162] The present invention relates to a composition for genome editing, genome alteration or altering the gene expression of a target gene. The composition may include a viral vector and, for example, a site-specific nuclease or a fusion protein of a CRISPR / Cas9 system with at least one gRNA.

[0163] a. Compositions for genome editing in muscle

[0164] The present invention relates to a composition for a target gene in skeletal muscle or myocardium of a genome editing subject. The composition includes a modified AAV vector and a nucleotide sequence encoding a site-specific nuclease. The composition delivers the site-specific nuclease in an active form to skeletal muscle or myocardium. The composition may also include donor DNA or a transgenic. These compositions can be used for genome editing, genome engineering, and correction or reduction of mutations in genes involved in genetic diseases and / or other skeletal or myocardial conditions.

[0165] The target gene may participate in the differentiation of cells or other processes in which activation, suppression or interruption of genes may be required, or may have mutations such as deletions, frameshift mutations or nonsense mutations. If the target gene has a mutation that causes premature termination codons, abnormal splicing acceptor sites or abnormal splicing donor sites, site-specific nucleases can be designed to recognize and bind to the nucleotide sequences upstream or downstream of premature termination codons, abnormal splicing acceptor sites or abnormal splicing donor sites. Site-specific nucleases can also be used to interrupt normal gene splicing by targeting splicing acceptors and donors to induce premature termination codon jumps or restore interrupted reading frames. Site-specific nucleases may or may not mediate off-target changes to become protein coding regions of genomes.

[0166] 3. CRISPR system

[0167] "Clustered regularly interspaced short palindromic repeats" and "CRISPR" used interchangeably herein refer to loci containing multiple short direct repeats present in the genomes of about 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system that participates in the defense of invading phages and plasmids that provide acquired immunity. The CRISPR locus in the microbial host contains a combination of CRISPR-associated (Cas) genes and non-coding RNA elements that can program CRISPR-mediated nucleic acid cleavage specificity. A short foreign DNA segment (called a spacer) is integrated into the genome between CRISPR repeats and used as a "memory" after exposure. Cas9 forms a complex with the 3' end of the sgRNA, and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5' end of the sgRNA sequence and a predetermined 20 bp DNA sequence (called the protospacer). The complex is directed to the homologous locus of the pathogen DNA through the region encoded in the crRNA (i.e., the protospacer) and the protospacer sequence adjacent motif (PAM) in the pathogen genome. The non-coding CRISPR array within the direct repeat sequence is transcribed and cleaved into short crRNAs containing each spacer sequence, which guide the Cas nuclease to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed sgRNA, the Cas9 nuclease can be directed to a new genomic target. The CRISPR spacer is used to recognize and silence exogenous genetic elements in a manner similar to RNAi in eukaryotes.

[0168] 3 categories (I, II and III type effector systems) of known CRISPR systems. The II type effector system uses a single effector enzyme Cas9 to target DNA double-strand breaks in 4 successive steps to cut dsDNA. Compared with the I type and III type effector systems that require multiple different effectors to act as complexes, the II type effector system can work in non-traditional situations (such as eukaryotic cells). The II type effector system is composed of long pre-crRNA (pre-crRNA) (which is transcribed from the CRISPR locus containing the spacer), Cas9 protein and tracrRNA involved in pre-crRNA processing. TracrRNA hybridizes with the repeat region of the spacer separating the pre-crRNA, and therefore starts dsRNA cutting by endogenous RNase III. The cutting is then followed by the second cutting event of Cas9 in each spacer, producing a mature crRNA that retains the association with tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNA complex.

[0169] Cas9:crRNA-tracrRNA complex unwinds DNA duplex and searches for matching crRNA sequence for cutting. Target recognition occurs when the complementarity between the "protospacer" sequence in the target DNA and the remaining spacer sequence in the crRNA is detected. If the corrected protospacer sequence adjacent motif (PAM) is also present at the 3' end of the protospacer, Cas9 mediates the cutting of the target DNA. For protospacer targeting, the sequence must be immediately adjacent to the protospacer sequence adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease necessary for DNA cutting. Different type II systems have different PAM requirements. The Streptococcus pyogenes CRISPR system may have a PAM sequence of this Cas9 (SpCas9) as 5'-NRG-3', where R is A or G, and is characterized by the specificity of the system in human cells. The unique ability of the CRISPR / Cas9 system is the direct ability to simultaneously target multiple different genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, the Streptococcus pyogenes type II system naturally prefers the "NGG" sequence, where "N" can be any nucleotide, but other PAM sequences, such as "NAG" in engineered systems (Hsu et al., Nature Biotechnology (2013) doi:10.1038 / nbt.2647). Similarly, from Neisseria meningitidis ( Neisseria meningitidis ) Cas9 (NmCas9) typically has a natural PAM of NNNNGATT, but has activity across a variety of PAMs, including the highly degenerate NNNNGNNN PAM (Esvelt et al., Nature Methods (2013) doi:10.1038 / nmeth.2681).

[0170] 4. CRISPR / Cas9-based systems

[0171] An engineered version of the type II effector system of Streptococcus pyogenes for genome engineering that functions in human cells is shown. In this system, the Cas9 protein is directed to a genomic target site by a synthetically reconstructed "guide RNA" ("gRNA", also interchangeably referred to herein as a chimeric single guide RNA ("sgRNA")), which is a crRNA-tracrRNA fusion that generally avoids the need for RNase III and crRNA processing (see Fig.53A). Provided herein are CRISPR / Cas9-based engineered systems for genome editing and treatment of genetic diseases. CRISPR / Cas9-based engineered systems can be designed to target any gene, including genes involved in genetic diseases, aging, tissue regeneration, or wound healing. A CRISPR / Cas9-based system may include a Cas9 protein or a Cas9 fusion protein and at least one gRNA. The Cas9 fusion protein may include, for example, domains with different activities that are endogenous to Cas9, such as a transactivation domain.

[0172] The target gene may participate in the differentiation of cells or may need to activate genes or may have other processes such as frameshift mutations or nonsense mutations. If the target gene has a mutation that causes premature termination codons, abnormal splicing acceptor sites or abnormal splicing donor sites, the system based on CRISPR / Cas9 can be designed to identify and combine the nucleotide sequence upstream or downstream of premature termination codons, abnormal splicing acceptor sites or abnormal splicing donor sites. The system based on CRISPR-Cas9 can also be used to interrupt normal gene splicing by targeting splicing acceptors and donors to induce premature termination codon jumps or restore interrupted reading frames. The system based on CRISPR / Cas9 may mediate or not mediate off-target changes to become the protein coding region of the genome.

[0173] a. Cas9

[0174] The system based on CRISPR / Cas9 may include Cas9 protein or Cas9 fusion protein.Cas9 protein is an endonuclease that cuts nucleic acid, encoded by CRISPR locus, and participates in type II CRISPR system.Cas9 protein may be from any bacterial or archaeal species, such as Streptococcus pyogenes.Cas9 protein may be mutated to inactivate nuclease activity.Inactivated Cas9 protein (iCas9, also known as "dCas9") from Streptococcus pyogenes without endonuclease activity has recently been targeted by gRNA to genes in bacteria, yeast and human cells to silence gene expression by steric hindrance.Both "iCas9" and "dCas9" used herein refer to Cas9 proteins with amino acid substitutions D10A and H840A and inactivated nuclease activity.For example, a system based on CRISPR / Cas9 may include SEQ ID NO: Cas9 of 459 or 461.

[0175] b. Cas9 fusion protein

[0176] The CRISPR / Cas9-based system may include a fusion protein. The fusion protein may include two heterologous polypeptide domains, wherein the first polypeptide domain includes a Cas protein, and the second polypeptide domain has an activity, such as transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, or demethylase activity. The fusion protein may include the above-mentioned Cas9 protein or a mutant Cas9 protein fused to a second polypeptide domain having the following activities: for example, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, or demethylase activity.

[0177] (1) Transcriptional activation activity

[0178] The second polypeptide domain may have transcriptional activation activity, i.e., a transactivation domain. For example, a fusion protein of iCas9 and a transactivation domain may be targeted to a mammalian promoter by a combination of gRNAs to achieve gene expression of endogenous mammalian genes such as human genes. The transactivation domain may include VP16 protein, a variety of VP16 proteins, such as a VP48 domain or a VP64 domain or a p65 domain of NFκB activating factor activity. For example, the fusion protein may be iCas9-VP64.

[0179] (2) Transcription repression activity

[0180] The second polypeptide domain may have transcriptional repression activity. The second polypeptide domain may have Kruppel-related box activity, such as KRAB domain, ERF repressor protein domain activity, Mxi1 repressor protein domain activity, SID4X repressor protein domain activity, Mad-SID repressor protein domain activity or TATA box binding protein activity. For example, the fusion protein may be dCas9-KRAB.

[0181] (3) Transcriptional release factor activity

[0182] The second polypeptide domain may have transcriptional release factor activity. The second polypeptide domain may have eukaryotic release factor 1 (ERF1) activity or eukaryotic release factor 3 (ERF3) activity.

[0183] (4) Histone modification activity

[0184] 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. Histone acetyltransferase may be p300 or CREB binding protein (CBP) protein or a fragment thereof. For example, the fusion protein may be dCas9-p300.

[0185] (5) Nuclease activity

[0186] The second polypeptide domain may have a nuclease activity different from the nuclease activity of the Cas9 protein. Nucleases or proteins having nuclease activity are enzymes that can cut the phosphodiester bonds between the nucleotide subunits of a nucleic acid. Nucleases are generally further divided into endonucleases and exonucleases, although some enzymes may fall into both categories. Well-known nucleases are deoxyribonucleases and ribonucleases.

[0187] (6) Nucleic acid binding activity

[0188] The second polypeptide domain may have nucleic acid associating activity, or a nucleic acid binding protein-DNA-binding domain (DBD) is an independently folded protein domain containing 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 associating region is selected from the group consisting of a helix-turn-helix region, a leucine zipper region, a winged helix region, a winged helix-turn-helix region, a helix-loop-helix region, an immunoglobulin fold, a B3 domain, a zinc finger, an HMG box, a Wor3 domain, a TAL effector DNA binding domain.

[0189] (7) Methylase activity

[0190] The second polypeptide domain may have methylase activity, which includes transferring a methyl group to DNA, RNA, protein, small molecule, cytosine or adenine. The second polypeptide domain may include a DNA methyltransferase.

[0191] (8) Demethylase activity

[0192] The second polypeptide domain may have demethylase activity. The second polypeptide domain may include an enzyme that removes methyl groups (CH3-) from nucleic acids, proteins (particularly histones) and other molecules. Alternatively, the second polypeptide may transfer a methyl group to a hydroxymethylcytosine in a mechanism that demethylates DNA. The second polypeptide may catalyze the reaction. For example, the second polypeptide that catalyzes the reaction may be Tet1.

[0193] c. gRNA

[0194] gRNA provides targeting based on CRISPR / Cas9 system. gRNA is a fusion of two non-coding RNAs, namely crRNA and tracrRNA. sgRNA can target any desired DNA sequence by exchanging the sequence of the 20 bp pre-spacer (which confers targeting specificity by complementary base pairing with the desired DNA target). gRNA simulates the naturally occurring crRNA:tracrRNA duplex involved in the type II effector system. Such duplexes, such as 42 nucleotide crRNA and 75 nucleotide tracrRNA, can be included to guide Cas9 to cut the target nucleic acid. "Target region", "target sequence" or "pre-spacer" used interchangeably herein refers to the region of the target gene targeted by the CRISPR / Cas9-based system. The CRISPR / Cas9-based system may include at least one gRNA, wherein the gRNA targets different DNA sequences. The target DNA sequence may be overlapping. The target sequence or pre-spacer is followed by the PAM sequence at the 3' end of the pre-spacer. Different type II systems have different PAM requirements. For example, the type II system of Streptococcus pyogenes uses an "NGG" sequence, wherein "N" can be any nucleotide.

[0195] The number of gRNAs administered to a cell can be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs, at least 7 different gRNAs, at least 8 different gRNAs, at least 9 different gRNAs, at least 10 different gRNAs, at least 11 different gRNAs, at least 12 different gRNAs, at least 13 different gRNAs, at least 14 different gRNAs, at least 15 different gRNAs, at least 16 different gRNAs, at least 17 different gRNAs, at least 18 different gRNAs, at least 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 a cell can be between at least 1 gRNA and at least 50 different gRNAs, at least 1 gRNA and at least 45 different gRNAs, at least 1 gRNA and at least 40 different gRNAs, at least 1 gRNA and at least 35 different gRNAs, at least 1 gRNA and at least 30 different gRNAs, at least 1 gRNA and at least 25 different gRNAs, at least 1 gRNA and at least 20 different gRNAs, at least 1 gRNA and at least 16 different gRNAs, at least 1 gRNA and at least 12 different gRNAs, at least 1 gRNA and at least 8 different gRNAs, at least 1 gRNA and at least 4 different gRNAs, at least 4 gRNAs and at least 50 different gRNAs, at least 4 different gRNAs and at least 45 different gRNAs, at least 4 different gRNAs and at least 40 different gRNAs, at least 4 different gRNAs and at least 35 different gRNAs, at least 4 different gRNAs and at least 50 different gRNAs, at least 4 different gRNAs and at least 45 different gRNAs, at least 4 different gRNAs and at least 40 different gRNAs, at least 4 different gRNAs and at least 35 different gRNAs, at least 4 different gRNAs and at least 45 different gRNAs. NA and at least 30 different gRNAs, at least 4 different gRNAs and at least 25 different gRNAs, at least 4 different gRNAs and at least 20 different gRNAs, at least 4 different gRNAs and at least 16 different gRNAs, at least 4 different gRNAs and at least 12 different gRNAs, at least 4 different gRNAs and at least 8 different gRNAs, at least 8 different gRNAs and at least 50 different gRNAs, at least 8 different gRNAs and at least 45 different gRNAs, at least 8 different gRNAs and at least 40 different gRNAs, at least 8 different gRNAs and at least 35 different gRNAs, 8 different gRNAs and at least 30 different gRNAs, at least 8 different gRNAs and at least 25 different gRNAs, 8 different gRNAs and at least 20 different gRNAs, at least 8 different gRNAs and at least 16 different gRNAs, or 8 different gRNAs and at least 12 different gRNAs.

[0196] The gRNA may comprise a complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence. The gRNA may comprise a "G" at the 5' end of the complementary polynucleotide sequence. The gRNA may comprise at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, at least 16 base pairs, at least 17 base pairs, at least 18 base pairs, at least 19 base pairs, at least 20 base pairs, at least 21 base pairs, at least 22 base pairs, at least 23 base pairs, at least 24 base pairs, at least 25 base pairs, at least 30 base pairs, or at least 35 base pairs of a complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence. The PAM sequence may be "NGG", wherein "N" may be any nucleotide. The gRNA may target at least one of the promoter region, enhancer region, or transcription region of the target gene. gRNA may include SEQ ID NO: 5-40, 65-144, 492-515, 540-563, 585-625, 462 ( Fig.40 )、464 ( Fig.41 ) and 465 ( Fig.41 ) of at least one nucleic acid sequence.

[0197] The gRNA can target any nucleic acid sequence. The nucleic acid sequence target can be DNA. The DNA can be any gene. For example, the gRNA can target a gene such as BRN2 , MYT1L , ASCL1 , NANOG , VEGFA , TERT , IL1B , IL1R2 , IL1RN , HBG1 , HBG2 , MYOD1 , OCT4 and DMD .

[0198] (1) Dystrophin

[0199] Dystrophin is a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of the muscle fiber to the surrounding extracellular matrix throughout the cell membrane. Dystrophin provides structural stability to the dystrophin complex of the cell membrane. The dystrophin gene is 2.2 megabases located at locus Xp21. The primary transcript measures approximately 2,400 kb and its mature mRNA is approximately 14 kb. 79 exons encode a protein of over 3500 amino acids. Normal skeletal muscle tissue contains only small amounts of dystrophin, but abnormal expression of what is present results in the development of severe and incurable symptoms. Some mutations in the dystrophin gene result in defective dystrophin and a severe dystrophic phenotype in affected patients. Some mutations in the dystrophin gene result in partially functional dystrophin and a much milder dystrophic phenotype in affected patients.

[0200] DMD is the result of inherited or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. For DMD, naturally occurring mutations and their consequences are relatively well understood. It is known that deletions that conform to the reading frame that occur in the exon 45-55 region contained within the rod domain can produce highly functional dystrophin, and many carriers are asymptomatic or show mild symptoms. In addition, in theory, more than 60% of patients can be treated by targeting the exons of this region of the dystrophin gene. Attempts have been made to restore the interrupted dystrophin reading frame in DMD patients by skipping non-essential exons during mRNA splicing to produce internally deleted but functional dystrophin. The deletion of internal dystrophin exons retains the appropriate reading frame, but causes less severe Becker muscular dystrophy.

[0201] (2) CRISPR / Cas9-based system targeting dystrophin

[0202] Disclosed herein is a CRISPR / Cas9-based system that is specific for the dystrophin gene. The CRISPR / Cas9-based system may include Cas9 and at least one gRNA to target the dystrophin gene. The CRISPR / Cas9-based system may bind to and recognize a target region. A target region may be selected that is immediately upstream of a possible out-of-frame stop codon so that insertions or deletions during the repair process restore the dystrophin reading frame by frame switching. The target region may also be a splice acceptor site or a splice donor site so that insertions or deletions during the repair process interrupt splicing and restore the dystrophin reading frame by splice site interruption and exon exclusion. The target region may also be an abnormal stop codon so that insertions or deletions during the repair process restore the dystrophin reading frame by eliminating or interrupting the stop codon.

[0203] Single or multiple sgRNAs can be designed to restore the dystrophin reading frame by targeting the mutation hotspot at exons 45-55 and introducing small insertions and deletions within exons or large deletions of one or more exons. Dystrophin expression can be restored in Duchenne muscular dystrophy patient muscle cells in vitro after treatment with Cas9 and one or more sgRNAs. Human dystrophin was detected after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplexed gene editing capabilities of the CRISPR / Cas9 system enable efficient generation of large deletions in this mutation hotspot by either universal editing approaches or patient-specific gene editing approaches, which can correct up to 62% of patient mutations.

[0204] CRISPR / Cas9-based systems can use gRNAs of different sequences and lengths. Examples of gRNAs can be found in Table 6. CRISPR / Cas9-based systems can target nucleic acid sequences of SEQ ID NOs: 65-144 or their complements. The gRNA can include nucleotide sequences of SEQ ID NOs: 65-144 or their complements. For example, the disclosed CRISPR / Cas9-based systems are engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. These CRISPR / Cas9-based systems restore dystrophin expression in cells from DMD patients.

[0205] (a) Exons 51 and 45-55

[0206] Exon 51 is often adjacent to frame-destroying deletions in DMD. Exclusion of exon 51 of the dystrophin transcript by exon skipping can be used to treat approximately 15% of all DMD patients. Such DMD mutations are theoretically amenable to permanent correction by NHEJ-based genome editing and HDR. The CRISPR / Cas9-based systems described herein were developed for targeted modification of exon 51 in the human dystrophin gene. These CRISPR / Cas9-based systems were transfected into human DMD cells and mediated efficient gene modification and switching to correct the reading frame. Protein repair was accompanied by frame repair and was detected in a large population of CRISPR / Cas9-based system-treated cells. Similarly, exclusion of exons 45-55 of the dystrophin transcript can be used to treat approximately 62% of all DMD patients.

[0207] (3) AAV / CRISPR constructs

[0208] AAV can be used to deliver CRISPR using different construct configurations (see Fig.39 ). For example, AAV can deliver the Cas9 and gRNA expression cassettes in separate vectors. Alternatively, if using a vector derived from, for example, Staphylococcus aureus ( Staphylococcus aureus) or small Cas9 proteins from species such as Neisseria meningitidis, a single AAV vector within the 4.7 kb packaging limit can combine both Cas9 and up to two gRNA expression cassettes (see Fig.39 ).

[0209] 5. Multiple CRISPR / Cas9-based systems

[0210] The present disclosure relates to multiple CRISPR / Cas9-based systems (including systems based on CRISPR / CRISPR-related (Cas) 9, such as Cas9 or dCas9) and multiple gRNAs to target one or more endogenous genes. This platform utilizes a convenient Golden Gate cloning method to quickly incorporate up to 4 independent sgRNA expression cassettes into a single lentiviral vector. Each sgRNA is effectively expressed and can mediate multiple gene editing on different loci in immortalized and primary human cell lines. It has been shown that transient transcriptional activation in cell lines stably expressing dCas9-VP64 is adjustable by the coordinated activation of 1-4 sgRNAs. In addition, a single lentiviral vector can induce persistent long-term endogenous gene expression in immortalized and primary human cells. The system allows rapid assembly of a single lentiviral vector, which makes multiple gene editing or activation in models and primary cell lines possible.

[0211] The system based on multiple CRISPR / Cas9 provides the efficacy of adjustable induction of transcriptional activation and transcriptional activation. In addition to the expression of 1, 2, 3 or 4 sgRNAs from independent promoters, the final vector easily produced by Golden Gate assembly expresses constitutive Cas9 or dCas9-VP64. Each promoter can effectively express sgRNAs that instruct similar levels of Cas9 nuclease activity. In addition, the lentiviral delivery of a single vector expressing Cas9 and 4 sgRNAs targeting independent loci leads to simultaneous multiple gene editing of all 4 loci. Using lentiviral delivery of Cas9 with or without sgRNA, adjustable transcriptional activation of 2 endogenous genes in both transient and unstable conditions is achieved. Very effective long-term gene activation in primary human cells is achieved. This system is therefore an attractive and effective method for producing multiple gene editing and long-term transcriptional activation in human cells.

[0212] Multiple CRISPR / Cas9-based systems allow for efficient multiplexed gene editing to inactivate multiple genes simultaneously. The CRISPR / Cas9 system can simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs, making the system particularly suitable for multiplexed gene editing or synergistic activation applications. The CRISPR / Cas9 system greatly accelerates the process of molecular targeting to new sites by simply modifying the expressed sgRNA molecules. A single lentiviral vector can be combined with various methods to achieve inducible control of these components through chemical or optogenetic regulation to facilitate the study of the dynamics of gene regulation in both time and space.

[0213] A system based on multiple CRISPR / Cas9 can activate two or more endogenous genes in transcription. A system based on multiple CRISPR / Cas9 can suppress two or more endogenous genes in transcription. For example, at least two endogenous genes, at least 3 endogenous genes, at least 4 endogenous genes, at least 5 endogenous genes or at least 10 endogenous genes can be activated or suppressed by a system based on multiple CRISPR / Cas9. Genes between 2 and 15, genes between 2 and 10, genes between 2 and 5, genes between 5 and 15 or genes between 5 and 10 can be activated or suppressed by a system based on multiple CRISPR / Cas9.

[0214] (1) Modified lentiviral vector

[0215] The system based on multiple CRISPR / Cas9 includes a modified lentiviral vector. The modified lentiviral vector includes a first polynucleotide sequence encoding a fusion protein and a second polynucleotide sequence encoding at least one sgRNA. The fusion protein can be a fusion protein of the above-mentioned CRISPR / Cas9-based system. The first polynucleotide sequence can be operably connected to a promoter. The promoter can be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter.

[0216] 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 can encode between 1 sgRNA and 50 sgRNAs, 1 sgRNA and 45 sgRNAs, 1 sgRNA and 40 sgRNAs, 1 sgRNA and 35 sgRNAs, 1 sgRNA and 30 sgRNAs, 1 sgRNA and 25 different sgRNAs, 1 sgRNA and 20 sgRNAs, 1 sgRNA and 16 sgRNAs, 1 sgRNA and 8 different sgRNAs, 4 different sgRNA and 50 different sgRNAs, 4 different sgRNAs and 45 different sgRNAs, 4 different sgRNAs and 40 different sgRNAs, 4 different sgRNAs and 35 different sgRNAs, 4 different sgRNAs and 30 different sgRNAs, 4 different sgRNAs and 25 different sgRNAs, 4 different sgRNAs and 20 different sgRNAs, 4 different sgRNAs and 16 different sgRNAs, 4 different sgRNA and 8 different sgRNAs, 8 different sgRNAs and 50 different sgRNAs, 8 different sgRNAs and 45 different sgRNAs, 8 different sgRNAs and 40 different sgRNAs, 8 different sgRNAs and 35 different sgRNAs, 8 different sgRNAs and 30 different sgRNAs, 8 different sgRNAs and 25 different sgRNAs, 8 different sgRNAs and 20 different sgRNAs, 8 different sgRNAs and 16 different sgRNAs, 16 different sgRNAs and 50 different sgRNAs, 16 different sgRNAs and 45 different sgRNAs, 16 different sgRNAs and 40 different sgRNAs, 16 different sgRNAs and 35 different sgRNAs, 16 different sgRNAs and 30 different sgRNAs, 16 different sgRNAs and 25 different sgRNAs, or 16 different sgRNAs and 20 different sgRNAs. Each polynucleotide sequence encoding different sgRNAs can be operably linked to a promoter. The promoters operably linked to different sgRNAs can be the same promoter. The promoters operably linked to different sgRNAs can be different promoters. The promoter can be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter.

[0217] At least one sgRNA can bind to a target gene or locus. If more than one sgRNA is included, each sgRNA binds to a different target region within one target locus or each sgRNA binds to a different target region within different loci. The fusion protein can include a Cas9 protein or an iCas9-VP64 protein. The fusion protein can include a VP64 domain, a p300 domain, or a KRAB domain.

[0218] 6. Site-specific nucleases

[0219] The above-mentioned composition includes a nucleotide sequence encoding a site-specific nuclease that binds and cuts the target area. The site-specific nuclease can be engineered. For example, the engineered site-specific nuclease can be a CRISPR / Cas9-based system, ZFN or TALEN. The site-specific nuclease can bind to and cut a gene or locus of the genome of a cell in skeletal muscle or cardiac muscle. For example, the gene or locus can be a Rosa26 locus or a dystrophin gene.

[0220] a. CRISPR / Cas9-based systems

[0221] The CRISPR / Cas9-based system described above can be used to introduce site-specific double-strand breaks at targeted genomic loci.

[0222] b. Zinc Finger Nuclease (ZFN)

[0223] Site-specific nucleases can be ZFNs. A single zinc finger contains about 30 amino acids, and the domain acts by binding to three consecutive base pairs of DNA through the interaction of a single amino acid side chain per base pair. The modular structure of the zinc finger motif allows several domains to be connected in series, allowing recognition and targeting of multiple extended sequences of 3 nucleotides. These targeted DNA binding domains can be combined with nuclease domains (e.g. FokI ) to produce site-specific nucleases that can introduce site-specific double-strand breaks at targeted genomic loci, called "zinc finger nucleases" (ZFNs). This DNA cleavage stimulates the natural DNA repair machinery, leading to 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 Tables 1 and Figure 35-38 In Table 1, the DNA recognition helix is ​​underlined, and "Fok ELD-S" and "Fok KKR-S" refer to the FokI nuclease domain fused to the zinc finger protein DNA binding domain. Figure 35-38In the figure, the target DNA sequences in the target site (i.e., in SEQ ID NOs: 442, 445, 448, and 453) and the DNA recognition helices in the ZFN amino acid sequences (i.e., in SEQ ID NOs: 443, 444, 446, 447, 449-452, 454, and 455) are underlined, respectively.

[0224] Table 1 Complete amino acid sequences of identified ZFNs.

[0225]

[0226] c. TAL Effector Nuclease (TALEN)

[0227] TALEN can be used to introduce site-specific double-strand breaks into targeted genomic loci. When 2 independent TALENs bind to nearby DNA sequences, allowing FokI to dimerize and cut the target DNA, site-specific double-strand breaks are produced. Due to the high rate of its successful and effective genetic modification, TALEN has advanced genome editing. This DNA cutting can stimulate the natural DNA repair machinery, leading to one of two possible repair pathways: homology-guided repair (HDR) or non-homologous end joining (NHEJ) pathways. TALEN can be designed to target any gene involved in genetic diseases.

[0228] TALEN may include a nuclease and a TALE DNA binding domain that binds to a target gene in a TALEN target region. The target gene may have a mutation such as a frameshift mutation or a nonsense mutation. If the target gene has a mutation that causes an early stop codon, TALEN can be designed to recognize and bind to a nucleotide sequence upstream or downstream of the early stop codon. A "TALEN target region" includes a binding region of 2 TALENs and a spacer region that appears between the binding regions. 2 TALENs bind to different binding regions within the TALEN target region, after which the TALEN target region is cut. Examples of TALEN are described in International Patent Application No. PCT / US2013 / 038536, which is incorporated herein by reference in its entirety.

[0229] 7. Transcription Activators

[0230] The above composition includes a nucleotide sequence encoding a transcription activator that activates a gene. The transcription activator can be engineered. For example, the engineered transcription activator can be a CRISPR / Cas9-based system, a zinc finger fusion protein, or a TALE fusion protein.

[0231] a. CRISPR / Cas9-based systems

[0232] The above-mentioned CRISPR / Cas9-based system can be used to activate the transcription of the target gene together with RNA. The CRISPR / Cas9-based system can include the above-mentioned fusion protein, wherein the second polypeptide domain has transcriptional activation activity or histone modification activity. For example, the second polypeptide domain can include VP64 or p300.

[0233] b. Zinc finger fusion protein

[0234] The transcription activator can be a zinc finger fusion protein. The zinc finger targeting the above DNA binding domain can be combined with a domain having transcription activation activity or histone modification activity. For example, the domain can include VP64 or p300.

[0235] c. TALE fusion protein

[0236] TALE fusion proteins can be used to activate the transcription of target genes. TALE fusion proteins can include a TALE DNA binding domain and a domain with transcriptional activation activity or histone modification activity. For example, the domain can include VP64 or p300.

[0237] 8. Composition

[0238] The present invention relates to compositions for altering gene expression and engineering or altering genomic DNA in cells or subjects. The compositions may also include a viral delivery system.

[0239] a. Compositions for genome editing in muscle

[0240] The present invention relates to a composition for genome editing of a target gene in a subject's skeletal muscle or myocardium. The composition includes a modified AAV vector and a nucleotide sequence encoding a site-specific nuclease. The composition delivers an active form of the site-specific nuclease to skeletal muscle or myocardium. The composition may also include donor DNA or a transgenic. These compositions can be used for genome editing, genome engineering, and correction or reduction of mutations in genes involved in genetic diseases and / or other skeletal or myocardial conditions.

[0241] The target gene may participate in the differentiation of cells or other processes in which activation, suppression or interruption of genes may be required, or may have mutations such as deletions, frameshift mutations or nonsense mutations. If the target gene has a mutation that causes premature termination codons, abnormal splicing acceptor sites or abnormal splicing donor sites, site-specific nucleases can be designed to recognize and bind to the nucleotide sequences upstream or downstream of premature termination codons, abnormal splicing acceptor sites or abnormal splicing donor sites. Site-specific nucleases can also be used to interrupt normal gene splicing by targeting splicing acceptors and donors to induce premature termination codon jumps or restore interrupted reading frames. Site-specific nucleases may or may not mediate off-target changes to become protein coding regions of genomes.

[0242] b. Adeno-associated virus vector

[0243] The above composition includes a modified adeno-associated virus (AAV) vector. The modified AAV vector may have improved myocardial and skeletal muscle tissue tropism. The modified AAV vector will be able to deliver and express site-specific nucleases in 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 may deliver nucleases to bones and myocardium in vivo. The modified AAV vector may be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9. Modified AAV vectors can be based on AAV2 pseudotypes with alternative myotropic AAV capsids that effectively transduce skeletal or cardiac muscle by systemic and local delivery, such as AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, and AAV / SASTG vectors (Seto et al., Current Gene Therapy (2012) 12:139-151).

[0244] c. CRISPR / Cas9-based systems

[0245] The present disclosure also provides a DNA targeting system or at least one composition of the above-mentioned CRISPR / Cas9-based system. These compositions can be used for genome editing, genome engineering, and correcting or reducing the effects of gene mutations involved in genetic diseases. The composition includes a CRISPR / Cas9-based system, which includes the above-mentioned Cas9 protein or Cas9 fusion protein. The CRISPR / Cas9-based system may also include at least one of the above-mentioned gRNAs.

[0246] d. Multiple CRISPR / Cas9-based systems

[0247] The present disclosure also provides the above-mentioned multiple CRISPR / Cas9-based systems. These compositions can be used for genome editing, genome engineering and correction or reduction of the effects of gene mutations involved in genetic diseases. These compositions can be used to target more than one gene. The composition includes a modified lentiviral vector comprising a CRISPR / Cas9-based system including the above-mentioned Cas9 protein or Cas9 fusion protein and more than one of the above-mentioned gRNAs.

[0248] 9. How to use

[0249] The potential applications of the composition are diverse in multiple fields of science and bioengineering. The disclosed composition can be used to repair genetic mutations that cause diseases. The disclosed composition can be used to interrupt genes so that gene interruption leads to muscle regeneration or increased muscle strength, or muscle aging weakens. The disclosed composition can be used to introduce therapeutic genes for systemic expression from skeletal muscle or cardiac muscle, such as coagulation factors or monoclonal antibodies. The disclosed composition can be used to regulate mammalian gene expression. The disclosed composition can be used to transdifferentiate or induce cell differentiation or correct mutant genes in cells. Examples of activating genes related to cell and gene therapy, genetic reprogramming and regenerative medicine are provided. RNA-guided transcriptional activators can be used to reprogram cell lineage specifications. For genetic reprogramming, transdifferentiation and / or induced differentiation, activating endogenous genes encoding key regulators of cell fate, rather than forcing these factors to overexpress, may result in faster, more effective, stable and specific methods.

[0250] 10. Methods for genome editing in muscle

[0251] The present disclosure relates to methods for genome editing in skeletal muscle or myocardium of a subject. The method comprises administering a composition as described above to skeletal muscle or myocardium of a subject for genome editing in skeletal muscle or myocardium. Genome editing may include correcting a mutant gene or inserting a transgene. Correcting a mutant gene may include deletion, rearrangement or replacement of a mutant gene. Correcting a mutant gene may include nuclease-mediated NHEJ or HDR.

[0252] 11. Methods using CRISPR / Cas9-based systems

[0253] The potential applications of the system based on CRISPR / Cas9 are diverse in multiple fields of science and bioengineering. The disclosed system based on CRISPR / Cas9 can be used to regulate mammalian gene expression. The disclosed system based on CRISPR / Cas9 can be used to transdifferentiate or induce cell differentiation or correct mutant genes in cells. Examples of activating genes related to cell and gene therapy, genetic reprogramming and regenerative medicine are provided. RNA-guided transcriptional activators can be used to reprogram cell lineage specifications. Although reprogramming is incomplete and inefficient in these experiments, there are still many ways to improve the method, including repeated transfection of iCas9-VP64 / gRNA combinations, stable expression of these factors and targeting multiple genes, such as Brn2 and Myt1l, except Ascl1 for transdifferentiation into neuronal phenotypes. For genetic reprogramming and transdifferentiation or induced differentiation of cells, activating endogenous genes encoding key regulatory factors of cell fate, rather than forcing these factors to overexpress, may lead to faster, more effective and stable specific methods. Finally, Cas9 fusions to other domains, including repression and epigenetic modification domains, could provide a greater diversity of RNA-guided transcriptional regulators to complement other RNA-based tools for mammalian cell engineering.

[0254] a. Methods of activating gene expression

[0255] The present disclosure provides a mechanism for activating the expression of endogenous genes (e.g., mammalian genes) by targeting the transcriptional activator of the promoter by RNA using the above-mentioned CRISPR / Cas9-based system. This is fundamentally different from the aforementioned method based on engineered sequence-specific DNA binding proteins, and can provide opportunities for targeted gene regulation. Because the production of gRNA expression plasmids only includes the synthesis of 2 short custom oligonucleotides and 1 cloning step, it is possible to quickly and economically produce many new gene activators. gRNA can also be directly transfected into cells after in vitro transcription. A variety of gRNAs targeting a single promoter are shown, but it will also be possible to target multiple promoters at the same time. Using RNA rather than protein to identify genomic target sites can also overcome the limitations of sites (e.g., methylated DNA) targeted for epigenetic modification.

[0256] In contrast to current approaches based on engineered DNA binding proteins, Cas9 fused to a transcriptional activation domain can be targeted by a combination of guide RNA molecules to induce expression of endogenous human genes. This simple and versatile approach for targeted gene activation overcomes the need for engineering new proteins and allows for widespread synthetic gene regulation.

[0257] The method may include administering the above-mentioned CRISPR / Cas9-based system, a polynucleotide or vector encoding the CRISPR / Cas9-based system, or a DNA targeting system or a composition of at least one CRISPR / Cas9-based system to a cell or subject. The method may include administering a CRISPR / Cas9-based system, such as administering 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 such as a VP16 protein or a transcriptional coactivator such as a p300 protein.

[0258] (1) dCas9-VP16

[0259] The Cas9 fusion protein may include a transcriptional activation domain such as a VP16 protein. The transcriptional activation domain may contain at least 1 VP16 protein, at least 2 VP16 proteins, at least 3 VP16 proteins, at least 4 VP16 proteins (i.e., VP64 activation domain), at least 5 VP16 proteins, at least 6 VP16 proteins, at least 6 VP16 proteins, or at least 10 VP16 proteins. The Cas9 protein may be a Cas9 protein in which the nuclease activity is inactivated. For example, the Cas9 protein in the fusion protein may be iCas9 (SEQ ID NO: 1 amino acids 36-1403), which includes amino acid substitutions of D10A and H840A. The Cas9 fusion protein may be iCas9-VP64.

[0260] (2) dCas9-p300

[0261] The Cas9 fusion protein may include a transcriptional coactivator domain such as p300 protein. p300 protein (also known as EP300 or E1A binding protein p300) is composed of EP300Gene encoding, regulating the activity of many genes in the whole body tissue. p300 protein plays a role in regulating cell growth and division, promoting cell maturation and specialized functions (differentiation) and preventing the growth of cancerous tumors. p300 protein activates transcription by connecting transcription factors to protein complexes that perform transcription in the nucleus. p300 interacts with transcription factors under the control of one or more of the following p300 domains: nuclear receptor interaction domain (RID), CREB and MYB interaction domain (KIX), cysteine / histidine region (TAZ1 / CH1 and TAZ2 / CH3) and interferon response binding domain (IBiD). The last 4 domains of p300, KIX, TAZ1, TAZ2 and IBiD, each tightly bind to the sequence of the two transactivation domains 9aaTAD spanning the transcription factor p53. The protein plays the role of histone acetyltransferase that regulates transcription by chromatin reconstruction, and is important in cell proliferation and differentiation. It mediates cAMP-gene regulation by specifically binding to phosphorylated CREB protein.

[0262] p300 protein can activate mothers against decapentaplegichomolog 7, MAF, TSG101, peroxisome proliferator-activated receptor α, NPAS2, PAX6, DDX5, MYBL2, mothers against decapentaplegichomolog 1, mothers against decapentaplegichomolog 2, lymphoid enhancer-binding factor 1, factor), SNIP1, TRERF1, STAT3, EID1, RAR-related orphan receptor alpha, ELK1, HIF1A, ING5, peroxisome proliferator-activated receptor gamma, SS18, TCF3, Zif268, estrogen receptor alpha, GPS2, MyoD, YY1, ING4, PROX1, CITED1, HNF1A, MEF2C, MEF2D, MAML1, Twist transcription factor, PTMA, IRF2, DTX1, Flap structure-specific endonuclease 1, myocyte-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.

[0263] The transcriptional coactivation domain may include a human p300 protein or a fragment thereof. The transcriptional coactivation domain may include a wild-type human p300 protein or a mutant human p300 protein or a fragment thereof. The transcriptional coactivation domain may include a core lysine-acetyltransferase domain of a human p300 protein, i.e., a p300 HAT core (also referred to as a "p300 WT core"; see Fig.58 ). The Cas9 protein can be a Cas9 protein in which the nuclease activity is inactivated. For example, the Cas9 protein in the fusion protein can be iCas9 (amino acids 36-1403 of SEQ ID NO: 1), which includes amino acid substitutions of D10A and H840A. The Cas9 fusion protein can be iCas9-p300 WT core.

[0264] (3) gRNA

[0265] The method may also include administering a CRISPR / Cas9-based system and at least one gRNA to a cell or subject, wherein the gRNA targets different DNA sequences. The target DNA sequences may be overlapping. The number of gRNAs administered to a cell 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 a cell can be between at least 1 gRNA and at least 50 different gRNAs, at least 1 gRNA and at least 45 different gRNAs, at least 1 gRNA and at least 40 different gRNAs, at least 1 gRNA and at least 35 different gRNAs, at least 1 gRNA and at least 30 different gRNAs, at least 1 gRNA and at least 25 different gRNAs, at least 1 gRNA and at least 20 different gRNAs, at least 1 gRNA and at least 16 different gRNAs, at least 1 gRNA and at least 12 different gRNAs, at least 1 gRNA and at least 8 different gRNAs, at least 1 gRNA and at least 4 different gRNAs, at least 4 gRNAs and at least 50 different gRNAs, at least 4 different gRNAs and at least 45 different gRNAs, at least 4 different gRNAs and at least 40 different gRNAs, at least 4 different gRNAs and at least 35 different gRNAs, at least 4 different gRNAs and at least 30 different gRNAs, at least 4 different gRNAs and at least 25 different gRNAs, at least 4 different gRNAs and at least 20 different gRNAs, at least 4 different gRNAs and at least 16 different gRNAs, at least 4 different gRNAs and at least 12 different gRNAs, at least 4 different gRNAs and at least 8 different gRNAs, at least 8 different gRNAs and at least 50 different gRNAs, at least 8 different gRNAs and at least 45 different gRNAs, at least 8 different gRNAs and at least 40 different gRNAs, at least 8 different gRNAs and at least 35 different gRNAs, 8 different gRNAs and at least 30 different gRNAs, at least 8 different gRNAs and at least 25 different gRNAs, 8 different gRNAs and at least 20 different gRNAs, at least 8 different gRNAs and at least 16 different gRNAs, 8 different gRNAs and at least 12 different gRNAs, at least 8 different gRNAs and at least 8 different gRNAs.

[0266] The gRNA may comprise a complementary polynucleotide sequence of the target DNA sequence followed by NGG. The gRNA may comprise a "G" at the 5' end of the complementary polynucleotide sequence. The gRNA may comprise at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, at least 16 base pairs, at least 17 base pairs, at least 18 base pairs, at least 19 base pairs, at least 20 base pairs, at least 21 base pairs, at least 22 base pairs, at least 23 base pairs, at least 24 base pairs, at least 25 base pairs, at least 30 base pairs, or at least 35 base pairs of a complementary polynucleotide sequence of the target DNA sequence followed by NGG. The gRNA may target at least one of a promoter region, an enhancer region, or a transcription region of a target gene. The gRNA may include a nucleic acid sequence of at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625.

[0267] b. Methods of inhibiting gene expression

[0268] The present disclosure provides a mechanism for suppressing the expression of endogenous genes (e.g., mammalian genes) by targeting genomic regulatory elements (e.g., distal enhancers) using the above-mentioned CRISPR / Cas9-based system through RNA. Cas9 fusion proteins may include transcriptional repressors, such as KRAB repressors. The Cas9 fusion protein may be dCas9-KRAB. dCas9-KRAB can also affect epigenetic gene regulation by recruiting heterochromatin formation factors to targeted loci. The CRISPR / dCas9-KRAB system can be used to suppress the transcription of genes, but can also be used to target genomic regulatory elements (e.g., that were previously unreachable by traditional repression methods (e.g., RNA interference) Fig.53 B). Delivery of dCas9-KRAB using gRNA targeting a distal enhancer can disrupt the expression of multiple genes regulated by the targeted enhancer (see Fig.53 C) The targeted enhancer can be any enhancer of a gene, such as the HS2 enhancer.

[0269] a. Transdifferentiation or differentiation induction methods

[0270] The present disclosure provides a mechanism for transdifferentiating cells or inducing cell differentiation by activating endogenous genes through RNA using the above-mentioned CRISPR / Cas9-based system.

[0271] (1) Transdifferentiation

[0272] CRISPR / Cas9-based systems can be used to transdifferentiate cells. Transdifferentiation, also known as lineage reprogramming or direct conversion, is a process in which a cell is converted from one differentiated cell type to another without going through an intermediate pluripotent state or progenitor cell type. It is a type of metaplasia that includes all cell fate changes, including the interconversion of stem cells. Transdifferentiation of cells has potential uses in disease modeling, drug development, gene therapy, and regenerative medicine. For example BRN2 , MYT1L , ASCL1 , NANOG and / or MYOD1 Activation of endogenous genes such as mitochondria, myocytes, fibroblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, or smooth muscle cells can lead to the transdifferentiation of several cell types such as fibroblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, or smooth muscle cells into neuronal and myogenic phenotypes, respectively.

[0273] (2) Inducing differentiation

[0274] The CRISPR / Cas9-based system can be used to induce differentiation of cells such as stem cells, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells or smooth muscle cells. For example, stem cells, such as embryonic stem cells or pluripotent stem cells, can be induced to differentiate into myocytes or vascular endothelial cells, i.e., induce neuronal or myogenic differentiation.

[0275] 12. Application of multiple CRISPR / Cas9-based systems

[0276] Multiple CRISPR / Cas9-based systems take advantage of the ease and low cost of sgRNA design and can employ CRISPR / Cas9 technology to facilitate advances in high-throughput genomic research. For example, a single lentiviral vector described herein can be used to express Cas9 and multiple sgRNAs in different cell lines (e.g., primary fibroblasts described herein). Fig.47 ). Multiple CRISPR / Cas9-based systems can be used in the same manner as the CRISPR / Cas9-based systems described above.

[0277] In addition to the transcriptional activation and nuclease functionality, the system can also be used to express other new Cas9-based effectors that control epigenetic modifications for a variety of purposes, including the study of genome architecture and endogenous gene regulatory pathways. Due to the delicate balance of endogenous gene regulation between multiple enzymes, multiple Cas9 systems with different functionalities will allow the examination of complex interactions between different regulatory signals. The vectors described herein should be compatible with aptamer-modified sgRNAs and orthologous Cas9s to enable independent genetic manipulation using a set of sgRNAs.

[0278] A system based on multiple CRISPR / Cas9 can be used to activate at least one endogenous gene in a cell. The method includes contacting the cell with a modified lentiviral vector. The endogenous gene can be transiently activated or stably activated. The endogenous gene can be transiently repressed or stably repressed. The fusion protein can be expressed at a level similar to that of the sgRNA. The fusion protein can be expressed at a different level than that of the sgRNA. The cell can be a primary human cell.

[0279] A system based on multiple CRISPR / Cas9 can be used for a method of performing multiple gene editing in a cell. The method includes contacting the cell with a modified lentiviral vector. Multiple gene editing may include correcting a mutant gene or inserting a transgene. Correcting a mutant gene may include deleting, rearranging, or replacing a mutant gene. Correcting a mutant gene may include nuclease-mediated non-homologous end joining or homology-guided repair. Multiple gene editing may include deleting or correcting at least one gene, wherein the gene is an endogenous normal gene or a mutant gene. Multiple gene editing may include deleting or correcting at least two genes. For example, at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, or at least 10 genes may be deleted or corrected.

[0280] A system based on multiple CRISPR / Cas9 can be used for a method of multiplex regulation of gene expression in a cell. The method includes contacting a cell with a modified lentiviral vector. The method may include regulating the gene expression level of at least one gene. When the gene expression level of at least one target gene is increased or decreased compared to the normal gene expression level of at least one target gene, the gene expression of at least one target gene is regulated. The gene expression level can be RNA or protein level.

[0281] 13. Methods for correcting mutated genes and treating subjects

[0282] The present disclosure also relates to a method for correcting a mutant gene of a subject. The method includes administering a composition to a subject's skeletal muscle or cardiac muscle for genome editing in the above-mentioned skeletal muscle or cardiac muscle. The composition delivers site-specific nucleases to skeletal muscle or cardiac muscle, and can restore the expression of fully functional or partially functional proteins with a repair template or donor DNA, which can replace the entire gene or a region containing a mutation. Site-specific nucleases can be used to introduce site-specific double-strand breaks into targeted genomic loci. When the site-specific nuclease binds to the target DNA sequence, it allows the target DNA to be cut, resulting in site-specific double-strand breaks. This DNA cutting can stimulate natural DNA repair machinery, resulting in one of two possible repair pathways: homology-guided repair (HDR) or non-homologous end joining (NHEJ) pathways.

[0283] The present disclosure relates to genome editing with site-specific nucleases in the absence of a repair template, which can effectively correct the reading frame and restore the expression of functional proteins involved in genetic diseases. The disclosed site-specific nucleases may include correction methods using homology-guided repair or nuclease-mediated non-homologous end joining (NHEJ), which makes it possible to effectively correct in primary cell lines with restricted proliferation that may not be suitable for homologous recombination or selection-based gene correction. This strategy integrates the rapid and robust assembly of active site-specific nucleases with effective gene editing methods for the treatment of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts, premature stop codons, abnormal splicing donor sites, or abnormal splicing acceptor sites.

[0284] a. Nuclease-mediated non-homologous end joining

[0285] Restoration of protein expression from endogenous mutant genes can be achieved through template-free NHEJ-mediated DNA repair. In contrast to transient approaches that target target gene RNA, correction of the target gene reading frame in the genome by transient expression of site-specific nucleases can result in permanent restoration of target gene expression in individual modified cells and all their progeny.

[0286] Nuclease-mediated NHEJ gene correction can correct mutated target genes and provide several potential advantages over HDR approaches. For example, NHEJ does not require a donor template that may cause nonspecific insertion mutagenesis. In contrast to HDR, NHEJ operates effectively at all stages of the cell cycle, so it can be effectively used in both cells in the cycle and post-mitotic cells (such as muscle fibers). In addition to the pharmacological forced read-through of oligonucleotide-based exon skipping or stop codons, this provides an alternative robust permanent gene repair, and in theory will require as little as one drug treatment. In addition to the plasmid electroporation method described herein, the gene correction based on NHEJ using a system based on CRISPR / Cas9, and other engineered nucleases including meganucleases and zinc finger nucleases can be combined with other current in vitro and in vivo platforms for cell-based and gene-based therapies. For example, the delivery of a CRISPR / Cas9-based gene transfer system or as a purified cell-permeable protein can make a genome editing method without DNA possible, which can overcome any possibility of insertion mutagenesis.

[0287] b. Homology-guided repair

[0288] Repair of protein expression from endogenous mutant genes may include homology-directed repair. The above method also includes administering a donor template to the cell. The donor template may include a nucleotide sequence encoding a fully functional protein or a partially functional protein. For example, the donor template may include a miniaturized dystrophin construct, referred to as a minidystrophin ("minidys"), a fully functional dystrophin construct for restoring a mutant dystrophin gene or a fragment of a dystrophin gene, which results in repair of the mutant dystrophin gene after homology-directed repair.

[0289] c. Methods of using CRISPR / Cas9 to correct mutant genes and treat subjects

[0290] The present disclosure also relates to genome editing with a repair template or donor DNA to restore the expression of fully functional or partially functional proteins with a CRISPR / Cas9-based system, which can replace the entire gene or a region containing a mutation. A system based on CRISPR / Cas9 can be used to introduce site-specific double-strand breaks into a targeted genomic locus. When a system based on CRISPR / Cas9 utilizes gRNA to bind to a target DNA sequence, thereby allowing the target DNA to be cut, site-specific double-strand breaks are caused. The system based on CRISPR / Cas9 has the advantage of advanced genome editing due to its high rate of successful and effective genetic modification. This DNA cutting can stimulate the natural DNA repair machinery, resulting in one of two possible repair pathways: homology-guided repair (HDR) or non-homologous end joining (NHEJ) pathways. For example, a system based on CRISPR / Cas9 involving a dystrophin gene may include a gRNA having a nucleic acid sequence of any one of SEQ ID NO: 65-115.

[0291] The present disclosure relates to genome editing based on CRISPR / Cas9 systems without the need for repair templates, which can effectively correct reading frames and restore the expression of functional proteins involved in genetic diseases. The disclosed CRISPR / Cas9-based systems and methods may include correction methods using homology-guided repair or nuclease-mediated non-homologous end joining (NHEJ), which makes it possible to make effective corrections in primary cell lines with restricted proliferation that may not be suitable for homologous recombination or selection-based gene correction. This strategy integrates the rapid and robust assembly of active CRISPR / Cas9-based systems with effective gene editing methods for the treatment of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts, premature stop codons, abnormal splicing donor sites, or abnormal splicing acceptor sites.

[0292] The present disclosure provides methods for correcting mutant genes in cells and treating subjects with genetic diseases (e.g., DMD). The method may include administering the above-mentioned CRISPR / Cas9-based system, a polynucleotide or vector encoding the CRISPR / Cas9-based system, or a composition of the CRISPR / Cas9-based system to a cell or subject. The method may include administering a CRISPR / Cas9-based system, such as administering a Cas9 protein or a Cas9 fusion protein containing a second domain with nuclease activity, a nucleotide sequence encoding the Cas9 protein or Cas9 fusion protein, and / or at least one gRNA, wherein the gRNA targets different DNA sequences. The target DNA sequence may be overlapping. The number of gRNAs administered to the cell 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 15 different gRNAs, at least 20 different gRNAs, at least 30 different gRNAs, or at least 50 different gRNAs as described above. The gRNA may include at least one nucleic acid sequence of SEQ ID NOs: 65-115. The method may include homology-guided repair or non-homologous end joining.

[0293] 14. Methods of treating diseases

[0294] The present disclosure relates to methods for treating subjects in need thereof. The methods include administering the composition to a subject's tissue as described above for changing gene expression and engineering or altering genomic DNA in cells or subject genome editing. The methods may include administering the composition to a subject's skeletal muscle or myocardium for genome editing in the above-mentioned skeletal muscle or myocardium. The subject may suffer from a skeletal muscle or myocardial condition that causes degeneration or weakness or a genetic disease. For example, the subject may suffer from Duchenne muscular dystrophy as described above.

[0295] a. Duchenne muscular dystrophy

[0296] The above methods can be used to correct the dystrophin gene and restore the fully functional or partially functional protein expression of the mutated dystrophin gene. In some aspects and embodiments, the present disclosure provides methods for reducing the effects (e.g., clinical symptoms / indications) of DMD in a patient. In some aspects and embodiments, the present disclosure provides methods for treating DMD in a patient. In some aspects and embodiments, the present disclosure provides methods for preventing DMD in a patient. In some aspects and embodiments, the present disclosure provides methods for preventing further development of DMD in a patient.

[0297] 15. Constructs and Plasmids

[0298] The above-mentioned composition may include a gene construct encoding a system based on CRISPR / Cas9 disclosed herein. A gene construct (e.g., a plasmid) may include a nucleic acid encoding at least one of a system based on CRISPR / Cas9 such as a Cas9 protein and a Cas9 fusion protein and / or a gRNA. The above-mentioned composition may include a gene construct encoding a modified AAV vector and a nucleic acid sequence encoding a site-specific nuclease disclosed herein. A gene construct (e.g., a plasmid) may include a nucleic acid encoding a site-specific nuclease. The above-mentioned composition may include a gene construct encoding a modified lentiviral vector disclosed herein. A gene construct (e.g., a plasmid) may include a nucleic acid encoding a Cas9-fusion protein and at least one sgRNA. A gene construct may be present in a cell as an extrachromosomal molecule that functions. A gene construct may be a linear minichromosome, including a centromere, a telomere, a plasmid, or a cosmid.

[0299] The gene construct can also be part of the genome of a recombinant viral vector including a recombinant lentivirus, a recombinant adenovirus, and a recombinant adenovirus-associated virus. The gene construct can be part of the genetic material of a live attenuated microorganism or a recombinant microbial vector living in a cell. The gene construct can include regulatory elements for gene expression of a nucleic acid coding sequence. The regulatory element can be a promoter, an enhancer, a start codon, a stop codon, or a polyadenylation signal.

[0300] The nucleic acid sequence may constitute a gene construct that may be a vector. The vector will be able to express a fusion protein, such as a Cas9-fusion protein or a site-specific nuclease, in a mammalian cell. The vector may be a recombinant. The vector may include a heterologous nucleic acid encoding a fusion protein (e.g., a Cas9-fusion protein or a site-specific nuclease). The vector may be a plasmid. The vector may be used to transfect cells with nucleic acids encoding Cas9-fusion proteins or site-specific nucleases, and culture and maintain transformed host cells under conditions where expression of the Cas9-fusion protein or site-specific nuclease system occurs.

[0301] The coding sequence can be optimized for stability and high levels of expression. In some cases, codons are selected to reduce secondary structure formation of the RNA, such as secondary structure formed by intramolecular bonding.

[0302] The vector may include a heterologous nucleic acid encoding a system or site-specific nuclease based on CRISPR / Cas9, and may additionally include a start codon (which may be an upstream of a system or site-specific nuclease encoding sequence based on CRISPR / Cas9) and a stop codon (which may be a downstream of a system or site-specific nuclease encoding sequence based on CRISPR / Cas9). The start and stop codons may be in frame with a system or site-specific nuclease encoding sequence based on CRISPR / Cas9. The vector may also include a promoter operably connected to a system or site-specific nuclease encoding sequence based on CRISPR / Cas9. The promoter operably linked to the CRISPR / Cas9-based system or site-specific nuclease coding sequence can be a promoter from Simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter such as bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter, Epstein-Barr virus (EBV) promoter or Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from human genes such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter can also be a natural or synthetic tissue-specific promoter, such as a muscle or skin-specific promoter. Examples of the promoter are described in U.S. Patent Application Publication No. US20040175727, the contents of which are incorporated herein by reference in their entirety.

[0303] The vector may also include a polyadenylation signal, which may be a system based on CRISPR / Cas9 or a downstream of a site-specific nuclease. 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 from a pCEP4 vector (Invitrogen, SanDiego, CA).

[0304] The vector may also include a CRISPR / Cas9-based system (i.e., Cas9 protein or Cas9 fusion protein encoding sequence or sgRNA) or an enhancer upstream of a site-specific nuclease. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as those from CMV, HA, RSV, or EBV. Polynucleotide functional enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94 / 016737, the contents of which are fully incorporated by reference. The vector may also include a mammalian origin of replication to maintain the vector outside the chromosome, and to produce multiple copies of the vector in the cell. The vector may also include a regulatory sequence, which is very suitable for gene expression in mammals or human cells to which the vector is given. The vector may also include a reporter gene, such as green fluorescent protein ("GFP") and / or a selection marker, such as hygromycin ("Hygro").

[0305] The vector can be an expression vector or a system for producing proteins by conventional techniques and readily available starting materials, including Sambrook et al., Molecular Cloning and Laboratory Manual, 2nd Edition, Cold Spring Harbor (1989), which is fully incorporated by reference. In some embodiments, the vector may include a nucleic acid sequence encoding a CRISPR / Cas9-based 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, the gRNA comprising a nucleic acid sequence of at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, and 585-625.

[0306] 16. Pharmaceutical Compositions

[0307] The composition may be a pharmaceutical composition. The pharmaceutical composition may include about 1 ng to about 10 mg of DNA encoding a CRISPR / Cas9-based system or a CRISPR / Cas9-based system protein component (i.e., Cas9 protein or Cas9 fusion protein). The pharmaceutical composition may include about 1 ng to about 10 mg of DNA of a modified AAV vector and a nucleotide sequence encoding a site-specific nuclease. The pharmaceutical composition may include about 1 ng to about 10 mg of DNA of a modified lentiviral vector. The pharmaceutical composition of the present invention is formulated according to the mode of administration to be adopted. In the case where the pharmaceutical composition is an injectable pharmaceutical composition, they are sterilized, pyrogen-free and particle-free. It is preferred to use isotonic preparations. In general, additives for isotonicity may include sodium chloride, glucose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, vasoconstrictors are added to the preparation.

[0308] Compositions can also include pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients can be functional molecules as carriers, adjuvants, carriers or diluents. Pharmaceutically acceptable excipients can be transfection facilitators, which can include surfactants, such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known transfection facilitators.

[0309] Transfection facilitator is polyanion, polycation, including poly L-glutamic acid (LGS) or lipid. Transfection facilitator is poly L-glutamic acid, more preferably poly L-glutamic acid is present in the composition at a concentration of less than 6 mg / ml for genome editing in skeletal muscle or myocardium. Transfection facilitator may also include surfactants, such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used to be administered in combination with gene constructs. In some embodiments, the DNA vector encoding the composition may also include transfection facilitator such as lipid, liposome, including lecithin liposome or other liposomes known in the art, such as DNA-liposome mixture (see, for example, W09324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known transfection facilitators. Preferably, transfection facilitator is polyanion, polycation, including poly L-glutamic acid (LGS) or lipid.

[0310] 17. Delivery Method

[0311] Provided herein is a method for delivering a pharmaceutical preparation (preferably the above-mentioned composition) for providing a gene construct. Delivery of the composition can be transfection or electroporation of the composition as a nucleic acid molecule expressed in a cell and delivered to the cell surface. The nucleic acid molecule can be electroporated using a BioRad Gene Pulser Xcell or Amaxa Nucleofector IIb device. Several different buffers can be used, including BioRad electroporation solution, Sigma phosphate buffered saline product #D8537 (PBS), Invitrogen OptiMEM I (OM) or Amaxa Nucleofector solution V (NV). Transfection can include a transfection reagent, such as Lipofectamine 2000.

[0312] When the composition is delivered to the tissue, the vector thus enters the mammalian cells, and the transfected cells will express the fusion protein, such as a CRISPR / Cas9-based system and / or a site-specific nuclease. The composition can be administered to a mammal to alter gene expression or to remodel or alter the genome. For example, the composition can be administered to a mammal to correct the dystrophin gene of the mammal. The mammal can be a human, a non-human primate, a cow, a pig, a sheep, a goat, an antelope, a bison, a buffalo, a bovine, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, or a chicken, preferably a human, a cow, a pig, or a chicken.

[0313] a. CRISPR / Cas9-based systems

[0314] Vectors encoding protein components of a CRISPR / Cas9-based system (i.e., Cas9 protein or Cas9 fusion protein) can be delivered to mammals by DNA injection (also known as DNA vaccination) with or without in vivo electroporation, liposome mediation, nanoparticle promotion, and / or recombinant vectors. The recombinant vector can be delivered by any viral means. The viral means can be a recombinant lentivirus, a recombinant adenovirus, and / or a recombinant adeno-associated virus.

[0315] Nucleotides encoding system protein components based on CRISPR / Cas9 (i.e., Cas9 protein or Cas9 fusion protein) can be introduced into cells to genetically correct the target gene or change the gene expression of the gene, such as activating or suppressing endogenous genes. For example, nucleotides encoding system protein components based on CRISPR / Cas9 (i.e., Cas9 protein or Cas9 fusion protein) directed to mutant dystrophin genes by gRNA can be introduced into myoblasts of DMD patients. Alternatively, they can be introduced into fibroblasts of DMD patients, and the genetically corrected fibroblasts are treated with MyoD to induce differentiation into myoblasts, which can be implanted into subjects (e.g., damaged muscles of subjects) to confirm that the corrected dystrophin is functional and / or treats the subject. The modified cells can also be stem cells, such as induced pluripotent stem cells, bone marrow-derived progenitor cells, skeletal muscle progenitor cells, human skeletal myoblasts of DMD patients, CD133+ cells, mesoangioblasts, and MyoD- or Pax7-transduced cells or other myogenic progenitor cells. For example, the CRISPR / Cas9-based system can induce neuronal or myogenic differentiation of induced pluripotent stem cells.

[0316] 18. Route of administration

[0317] The composition can be administered to the subject by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, by inhalation, by oral administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal and intraarticular or a combination thereof. For veterinary use, the composition is administered as a suitable acceptable formulation in accordance with normal veterinary practice. Veterinarians can easily determine the dosage regimen and route of administration that is most suitable for a specific animal. The composition can be administered by conventional syringes, needleless injection devices, "microprojectile bombardment gene guns" or other physical methods such as electroporation ("EP"), "fluid power methods" or ultrasound.

[0318] The composition can be delivered to a mammal by several techniques including DNA injection (also known as DNA vaccination), with or without in vivo electroporation, liposome mediation, nanoparticle promotion, recombinant vectors such as recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus-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.

[0319] 19. Cell Type

[0320] Any of these delivery methods and / or routes of administration can be used with a variety of cell types, such as those currently under investigation for cell-based therapies. The cell types can be fibroblasts, pluripotent stem cells, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, or the K562 human erythroleukemia cell line.

[0321] a. DMD

[0322] The cell types currently under investigation for cell-based therapies for DMD include immortalized myoblasts, such as wild-type and DMD patient-derived lines, such as Δ48-50 DMD, DMD 8036 (del48-50), C25C14 and DMD-7796 cell lines, original DMD dermal fibroblasts, induced pluripotent stem cells, bone marrow-derived progenitor cells, skeletal muscle progenitor cells, human skeletal myoblasts of 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. Immortalization of human myoblasts can be used for clonal derivation of genetically corrected myoblasts. Cells can be modified in vitro to isolate and amplify clonal populations of immortalized DMD myoblasts containing genetically corrected dystrophin genes and free of other nuclease-directed mutations in genomic protein coding regions. Alternatively, transient in vivo delivery of nucleases via nonviral or nonintegrating viral gene transfer, or via direct delivery of purified proteins and gRNA containing cell-permeable motifs, may enable highly specific in situ correction with minimal or no risk of foreign DNA integration.

[0323] 20. Medicine Box

[0324] Provided herein is a medicine kit that can be used to edit the genome in skeletal muscle or myocardium, such as to correct a mutant gene. The medicine kit comprises a composition for genome editing in the above-mentioned skeletal muscle or myocardium and instructions for using the composition. The instructions included in the medicine kit may be attached to the packaging material or may be included as a drug instruction sheet. Although instructions are generally written or printed materials, they are not limited thereto. The present disclosure contemplates any medium capable of storing the instructions and communicating them to an end user. The medium includes, but is not limited to, electronic storage media (e.g., disks, tapes, cassettes, chips), optical media (e.g., CD ROMs), etc. The term "instructions" as used herein may include an Internet website address providing instructions.

[0325] The composition for genome editing in skeletal muscle or cardiac muscle may include a modified AAV vector and a nucleotide sequence encoding the above-mentioned site-specific nuclease. The site-specific nuclease may include a ZFN, TALEN, or the above-mentioned CRISPR / Cas9-based system that specifically binds to and cuts the mutant gene. The above-mentioned site-specific nuclease may be included in the kit to specifically bind to and target a specific region of the mutant gene. The site-specific nuclease may be specific to the above-mentioned mutant dystrophin gene. The kit may also include the above-mentioned donor DNA, gRNA, or transgene.

[0326] a. CRISPR / Cas9-based systems

[0327] Provided herein is a medicine box that can be used to correct mutant genes. The medicine box includes instructions for correcting at least one component of the mutant gene and using a system based on CRISPR / Cas9. The instructions included in the medicine box may be attached to the packaging material or may be included as a drug instruction sheet. Although instructions are generally written or printed materials, they are not limited thereto. The present disclosure contemplates any medium capable of storing the instructions and communicating them to an end user. The medium includes, but is not limited to, electronic storage media (e.g., disks, tapes, cassettes, chips), optical media (e.g., CD ROMs), etc. The term "instructions" as used herein may include an Internet website address that provides instructions.

[0328] At least one component may include at least one of the above-mentioned CRISPR / Cas9-based systems for specific gene targeting. The kit may include a Cas9 protein or a Cas9 fusion protein, a nucleotide sequence encoding the Cas9 protein or the Cas9 fusion protein, and / or at least one gRNA. The kit may include the above-mentioned CRISPR / Cas9-based system to specifically bind to and target a specific target region upstream, inside, or downstream of the target gene coding region. For example, a CRISPR / Cas9-based system may be specific to the promoter region of the target gene or a CRISPR / Cas9-based system may be specific to a mutant gene (e.g., the above-mentioned mutant dystrophin gene). The kit may include the above-mentioned donor DNA.

[0329] 21. Example

[0330] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the disclosure described herein are very applicable and worthy of attention, and may be implemented using suitable equivalent methods without departing from the scope of the disclosure or aspects of the embodiments disclosed herein. Having now described the disclosure in detail, the disclosure will be more clearly understood by reference to the following examples, which are not intended only to illustrate certain aspects and embodiments of the disclosure and should not be construed as limiting the disclosure. The disclosures of all journal references, U.S. patents, and publications cited herein are incorporated herein by reference in their entirety.

[0331] The present invention has several aspects, which are illustrated by the following non-limiting examples.

[0332] Example 1

[0333] Materials and methods

[0334] Cell culture and transfection. HEK293T cells were obtained from the American Tissue Collection Center (ATCC) through the Duke University Cancer Center Facilities and maintained at 37°C, 5% CO2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transfection efficiency was routinely higher than 80%, as determined by fluorescence microscopy after delivery of a control eGFP expression plasmid. The Cas9 expression plasmid was transfected at a mass ratio of 3:1 with individual gRNA expression plasmids or an equal amount of gRNA expression plasmids (consisting of a mixture of equal amounts of 4 gRNAs).

[0335] Primary mouse embryonic fibroblasts (PMEF-HL, Millipore, Billerica, MA) were seeded (75,000 / well) in 24-well TCPS plates (BD, Franklin Lakes, NJ) in a 1% PBS-containing medium supplemented with 10% Premium Select FBS (Atlanta Biologicals, Lawrenceville, GA), 25 μg mL -1 MEFs were maintained in complete MEF medium consisting of high glucose DMEM supplemented with gentamicin (Invitrogen), 1XGlutaMAX, nonessential amino acids, sodium pyruvate, and β-mercaptoethanol (Invitrogen) at 37°C and 5% CO2. MEFs were transfected as described previously with a single 1 µg cm -2The total plasmid DNA was delivered as cationic nanocomplexes after electrostatic condensation with serum-free and antibiotic-free OptiMEM containing poly(CBA-ABOL) (Adler et al., Molecular therapy. Nucleic acids 1, e32 (2012). At 4 h post-transfection, OptiMEM was replaced with complete MEF medium. At 48 h post-transfection, MEFs were processed for qRT-PCR or complete MEF medium was replaced with N3 neural induction medium containing: DMEM / F-12 (Invitrogen), 1X N-2Supplement (Invitrogen), 10 ng mL -1 Human bFGF2 (Stemgent, Cambridge, MA) and 25 µg mL -1 Gentamycin (Invitrogen). A GFP reporter vector (pmax-GFP, 3486 bp, Amaxa, Cologne, Germany) was used to optimize transfection conditions. The Cas9 expression plasmid was transfected in a 3:1 or 1:1 mass ratio with the same mixture of the four gRNA expression plasmids.

[0336] Plasmids Plasmids encoding wild-type and H840A Cas9 were obtained from Addgene (plasmid #39312 and plasmid #39316; Jinek et al., Science 337, 816-821 (2012)). The H840A Cas9 was cloned in frame at the N-terminus into the vector pcDNA3.1 with the FLAG epitope tag and the nuclear localization sequence (NLS) of the primer pair that introduced the D10A mutation. The VP64 domain, NLS and HA epitope tag were cloned in frame with the Cas9 ORF at the C-terminus ( Figure 1 a, Figure 9a). The tracrRNA and crRNA expression cassettes were ordered using gBlocks (Integrated DNA Technologies (IDT)) (Cong et al., Science 339, 819-823 (2013)) and cloned into a pZ donor plasmid (Sigma) with KpnI and SacII sites. Chimeric guide RNA expression cassettes were ordered using gBlocks modified to include a BbsI restriction site to facilitate rapid cloning of new guide RNA spacer sequences (Mali et al., Science 339, 823-826 (2013)) (Figure 9b). Oligonucleotides containing the target sequence were obtained from IDT, hybridized, phosphorylated, and cloned into appropriate plasmids using the BbsI site. The target sequences are shown in Table 2.

[0337]

[0338]

[0339] Western blotting . Cells were lysed in 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, and 0.1% SDS. The lysate was mixed with loading buffer, boiled for 5 minutes, and equal volumes of protein were electrophoresed in NuPAGE® Novex 4-12% or 10% Bis-Tris gel polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific antibody binding was blocked with 50 mM Tris / 150 mM NaCl / 0.1% Tween-20 (TBS-T) containing 5% skim milk for 30 minutes. The membranes were incubated overnight with primary antibodies (HRP-conjugated anti-Flag (Cell Signaling, catalog number 2044) diluted 1:1000 in TBS-T containing 5% BSA; incubated for 30 minutes with anti-GAPDH (Cell Signaling, clone 14C10) diluted 1:5000 in TBS-T containing 5% milk; incubated with anti-ASCL1 (Santa Cruz, clone sc-48449) diluted 1:500 in 5% BSA; or with anti-g-globin (Santa Cruz, clone sc-48449) diluted 1:500 in 5% milk. Cruz, clone 51-7) and the membrane was washed with TBS-T for 30 minutes. The membrane labeled with the primary antibody was incubated with 1:5000 dilution of anti-rabbit HRP conjugated antibody (Sigma-Aldrich), anti-goat (1:3000) or anti-mouse (1:5000) for 30 minutes and washed with TBS-T for 30 minutes. The membrane was observed using Immun-Star WesternC™ chemiluminescent kit (Bio-Rad), and images were taken using ChemiDoc™ XRS+ system and processed using ImageLab software (Bio-Rad).

[0340] ELISA. Serum-free medium (OPTI-MEM) was collected and frozen at -80°C. According to the manufacturer's protocol, human IL-1ra (R&D Systems, catalog number DY280) secreted into the culture medium was quantitatively determined by enzyme-linked immunosorbent assay (ELISA). The recombinant human IL-1ra was diluted in OPTI-MEM to prepare a standard curve, and the IL-1ra in the culture medium was measured without dilution. The sample was concentrated about 8 times (Amicon Ultra, catalog number UFC500396) by centrifugation through a 3 kDa MWCO filter for 20 minutes. The reported value was corrected by the concentration factor of each sample.

[0341] The optical density at 450 nm was measured with wavelength correction at 540 nm. Each standard and sample was determined in duplicate. The duplicate readings were averaged and normalized by subtracting the average zero standard optical density. A standard curve was generated by log-transforming the data and a linear regression of IL-1ra concentration and optical density was performed. The reported values ​​are the means and standard errors of the means of 3 independent experiments performed in technical duplicate on different days with each experiment averaged ( n = 3).

[0342] qRT-PCR Total 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 using PerfeCTa® SYBR® Green FastMix was performed using the CFX96 Real-Time PCR Detection System (Bio-Rad) using the oligonucleotide primers reported in Table 3 designed using Primer3Plus software and purchased from IDT.

[0343]

[0344] Primer specificity was confirmed by agarose gel electrophoresis and melting curve analysis. Reaction efficiencies were calculated within an appropriate dynamic range to ensure linearity of the standard curve ( Fig.10 ). The results are expressed as ΔΔC T Methods Fold increase in target gene mRNA expression normalized to GAPDH expression. Values ​​reported are means and standard errors of the means of three independent experiments performed on different days in technical duplicate with each experiment averaged ( n = 3).

[0345] RNA-Seq. RNA seq library was constructed. Briefly, the first strand cDNA was synthesized from mRNA captured by oligo dT Dynabead® (Invitrogen) using SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). The second strand cDNA was synthesized using DNA polymerase I (New England Biolabs). The cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter), and the sequencing primers were simultaneously fragmented and inserted into the double-stranded cDNA using Nextera transposase (Illumina; 5 minutes at 55°C). The transposition reaction was terminated using QG buffer (Qiagen), and the fragmented cDNA was purified on AMPure XP beads. By 6 PCR cycles, an indexed sequencing library was generated.

[0346] The libraries were sequenced using 50-bp single-end reads on 2 lanes of an Illumina HiSeq 2000 instrument, generating 29 million and 74 million reads per library. Reads were aligned to human RefSeq transcripts using Bowtie (Langmeadet et al., Genome biology 10, R25 (2009)). DESeq was used to calculate the statistical significance of differential expression, including correction for multiple hypothesis testing (Anders et al., Genome biology 11, R106 (2010). The raw RNA-seq reads and the number of reads aligned to each RefSeq transcript are deposited in the Gene Expression Omnibus (GEO) for public access, with an accession number currently pending.

[0347] Immunofluorescence stainingFor the detection of Tuj1 and MAP2 expression, transfected MEFs were fixed with 4% PFA (EMS, Hatfield, PA) at room temperature (RT) for 20 min at day 10 of culture in N3 medium. The cells were then incubated with rabbit anti-Tuj1 (Covance, Princeton, New Jersey, clone TUJ1 1-15-79, 1:500) and mouse anti-MAP2 (BD, clone Ap20, 1:500) in blocking buffer containing 0.2% Triton X-100, 3% w / v BSA, and 10% goat serum (Sigma-Aldrich, Saint Louis, MO) for 2 h at RT or with mouse anti-Ascl1 (BD clone 24B72D11.1, 1:100) for 24 h at 4°C. The cells were then washed three times with PBS, incubated with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen, 1:200) in blocking buffer for 1 hour at room temperature, and washed three times with PBS. The stained MEFs were then scanned with a Nikon Eclipse TE2000-U inverted fluorescence microscope with a ProScanII engine stage (Prior Scientific, Rockland, MA) to generate large mosaic images of each complete culture area. These mosaics were processed with FIJI macro to automatically and uniformly threshold the images according to local contrast, exclude small debris, and perform a quantification of Tuj1 in each well. + The number of cells was counted.

[0348] Statistics. Statistical analyses were performed in JMP 10 Pro using the Tukey test with α equal to 0.05.

[0349] Example 2

[0350] result

[0351] To generate a CRISPR / Cas9-based transcriptional activation system, the catalytic residues of Cas9 (D10A, H840A) were mutated to generate iCas9 and genetically fused to the C-terminal VP64 acidic transactivation domain ( Figure 1 a, b). Robust iCas9-VP64 expression was observed from transfected plasmids in human embryonic kidney (HEK) 293T cells by Western blotting of the N-terminal Flag epitope tag ( Figure 3The CRISPR system recognizes its target through base pairing of a 20 bp sequence in the gRNA with a complementary DNA target followed by an NGG protospacer adjacent motif (PAM) sequence, where N is any base pair. Combinations of synthetic transcription factors targeting endogenous human promoters result in synergistic and robust activation of gene expression. Thus, a 20 bp sequence within 500 bp of the transcription start site is activated. IL1RN Four gRNA target sites followed by NGG PAM sequences were identified in the promoter of the gene ( Figure 4 , Table 2). In order to compare the crRNA-based and gRNA-based targeting strategies, the four target site sequences were introduced into the crRNA and gRNA expression plasmids. 17 , and co-transfected with iCas9-VP64 expression plasmid into HEK293T cells. Although significant induction was observed by qRT-PCR in samples treated with the combination of crRNAs IL1RN expression, but much higher levels were achieved with combinations of gRNAs ( Figure 1 c). No changes in gene expression were observed in cells treated with gRNA and iCas9 expression plasmid without VP64, indicating the key role of the activation domain in regulating gene expression ( Figure 1 c). By performing Surveyor assays in samples treated with iCas9-VP64 and wild-type Cas9 to detect DNA repair events, it was confirmed that nuclease activity at these target sites was abolished in the iCas9-VP64 system ( Figure 5 ). Targeting multiple sites in the promoter with combinations of gRNAs showed robust increases in gene expression by transfecting each of the 4 gRNAs alone or in combination ( Figure 1 d) High levels of mitochondrial expression were observed only when the gRNA combination was co-transfected with iCas9-VP64. IL1RN Express( Figure 1 d), as observed with other classes of engineered transcription factors. Similarly, in 3 different experiments, 3 of 6 samples treated with only the gRNA combination observed IL1RN Gene-encoded IL-1 receptor antagonist (IL-1ra) protein production was not observed in samples treated with single gRNA or control plasmid ( Figure 1 e). To examine the specificity of gene activation caused by iCas9-VP64, global gene expression of HEK293T cells treated with combinations of four gRNAs was evaluated by RNA-seq ( Figure 1 f). Obviously, only genes with significantly increased expression relative to the control (false discovery rate ≤ 3 x 10 -4 ) is from IL1RN The locus expresses four isoforms ( Figure 4), indicating a high level of gene activation specificity.

[0352] To demonstrate the general applicability of this system, four gRNAs were designed to target each of the promoters of eight other genes related to medicine and bioengineering, including ASCL1 , NANOG , HBG1 / 2 , MYOD1 , VEGFA , TERT , IL1B and IL1R2 ( Figure 4 , Table 2). ASCL1 and MYOD1 Forced expression of induced transdifferentiation of several cell types into neuronal and myogenic phenotypes, respectively. NANOG It is a marker of pluripotency and is also used in genetic reprogramming strategies. It encodes a homologue of γ-globin during embryonic development. HBG1 and HBG2 activation of β-globin, which could be used as a therapeutic strategy to compensate for β-globin mutations in sickle cell disease. VEGFA Upregulation of synthetic transcription factors as a strategy to enhance tissue regeneration and wound healing. TERT Forced expression of a gene encoding telomerase can be used to immortalize cell lines. IL1B Encodes the IL-1β cytokine that mediates inflammation and autoimmunity. IL1R2 The expression of the encoded decoy receptor blocked IL-1β signal transduction. By qRT-PCR, the expression of each of these genes was increased by co-transfection of iCas9-VP64 and the expression plasmids of the four gRNAs into HEK293T cells ( Figure 2 In some cases, 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 2 ad). Apparently, chromatin accessibility as measured by DNase-seq is not a predictor of successful gene activation ( Figure 4 ). In the use of iCas9-VP64 and targeting HBG1 4 gRNAs (3 of which also target HBG2 This revealed that RNA-seq could not distinguish HBG1 and HBG2 Specific and reproducible increases in expression of both, although statistical significance was not achieved due to low overall expression levels ( Figure 6 The increase in protein expression of Ascl1 and γ-globin after treatment with iCas9-VP64 and 4 gRNAs was detected by Western blotting ( Figure 7), confirming the higher mRNA levels observed by qRT-PCR ( Figure 2 ). Low baseline levels of Ascl1 and γ-globin protein expression were detectable in the empty vector control. Since preliminary evidence that gene target activation by iCas9-VP64 can lead to secondary changes in gene networks and cellular phenotypes, iCas9-VP64 and targeted ASCL1 The expression plasmids of four gRNAs were co-transfected into mouse embryonic fibroblasts (MEF) ( Figure 8 Forced expression of Ascl1 in MEFs showed partial activation of a neuronal gene network, including the downstream target Tuj1. ASCL1 The promoter is conserved ( Figure 8 a), and thus also observed in MEFs treated with plasmids encoding iCas9-VP64 and four gRNAs ASCL1 Activation of expression ( Figure 8 b). In addition, in the iCas9-VP64 / gRNA treated samples, cells expressing Ascl1 and the neuronal marker Tuj1 were easily detected by immunofluorescence staining 12 days after transfection ( Figure 8 ch). No Tuj1-positive cells were observed in cells treated with the control plasmid.

[0353] To date, there has not been any comprehensive investigation of the specificity of Cas9 / CRISPR activity in mammalian cells. Using RNA-seq, it was shown that there was no strong specificity between targeted gene activation and detectable off-target gene activation ( Figure 1 f, Figure 6 ). For this specific analysis select IL1RN and HBG1 / 2 , as the gene products IL-1ra and γ-globin do not exert secondary effects on gene expression in HEK293T cells. In contrast to the use of a single strong activator, exploiting the synergistic activity of multiple weak transcriptional activators may increase specific gene regulation, as multiple adjacent off-target sites would be unlikely to exist at another locus. Interestingly, compared with control samples treated with an empty expression plasmid alone, off-target sites were significantly reduced in samples treated with iCas9-VP64 and the targeted IL1RN or HBG1 / 2 In the two samples treated with gRNA, IL32 Genes moderately downregulated (false discovery rate < 0.03) ( Figure 1 f. Figure 6 ). Because targeting IL1RN and HBG1 / 2 Both samples were equally affected, so it is unlikely that this is a result of off-target iCas9-VP64 activity related to target sequence identity.

[0354] To evaluate the specificity of iCas9-VP64 binding to the genome, ChIP sequencing was performed on cells treated with iCas9-VP64 and four gRNAs targeting the IL1RN promoter using anti-HA antibodies. The experiment showed that iCas9 targeting the IL1RN promoter ( Fig.15 ). In addition, the experiments showed an extremely high level of specificity. iCas9 had only 10 potential off-target binding sites (FDR < 5%). To further query specificity, RNA sequencing experiments were performed with iCas9 EGEM and found that only the IL1RN gene isoform had increased expression relative to the control (FDR ≤ 3 x 10.4).

[0355] Example 3

[0356] CRISPR targeting the dystrophin gene—methods and materials

[0357] Plasmid constructs . As previously described, an expression cassette of Streptococcus pyogenes sgRNA and human codon-optimized Cas9 (hCas9) nuclease was used (Perez-Pinera et al., Nat Methods 10:973-976 (2013)). In order to generate a fluorescent reporter system to enrich CRISPR / Cas9-modified cells, GeneBlock (IDT) was synthesized. It contains a portion of the 3' end of the Cas9 coding sequence fused to the T2A jumping peptide immediately upstream of multiple cloning sites, which is then cloned into the hCas9 expression vector. The eGFP reporter gene is then cloned into the T2 vector for co-translation of Cas9 and eGFP proteins from the same expression vector (hCas9-T2A-GFP, SEQ ID NO: 116).

[0358] Cell culture and transfection HEK293T cells were obtained from the American Tissue Collection (ATCC) 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 Muscle1, 1-11 (2011)) (one from a wild-type donor and two from the Δ48-50 DMD patient-derived line) were maintained in skeletal muscle medium (PromoCell) supplemented with 20% fetal calf serum (Sigma), 50 μg / ml fetuin, 10 ng / ml human epidermal growth factor (Sigma), 1 ng / ml human basic 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 Stock (GM05162A, Δ46-50) and maintained in DMEM supplemented with 10% fetal calf serum, 1 ng / mL human basic fibroblast growth factor, and 1% penicillin / streptomycin. All cell lines were maintained at 37°C and 5% CO2.

[0359] HEK293T cells were transfected with 400 ng of each expression vector in 24-well plates using Lipofectamine 2000 (Invitrogen) 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 PBS as the electroporation buffer using optimized conditions for each line ( Figure 1 ) (Ousterout et al., Mol Ther 21:1718-1726 (2013)). Transfection efficiency was measured by delivering an eGFP expression plasmid (pmaxGFP, Clontech) and using flow cytometry. These efficiencies were typically ≥95% for HEK293T and ≥70% for primary fibroblasts and immortalized myoblasts. For all experiments, the indicated mass of electroporated plasmid was equivalent to the amount used for each CRISPR / Cas9-based system.

[0360] Cel-I Quantification of Endogenous Gene Modification (Surveyor Assay) . Surveyor nuclease assay was used to quantify the damage induced by the CRISPR / Cas9-based system at endogenous target sites (Guschin, DY et al., Meth Mol Biol649, 247-256 (2010)), which can detect the mutation characteristics of nuclease-mediated NHEJ. After transfection, the cells were incubated at 37°C for 3 or 10 days, and genomic DNA was extracted using a DNeasy Blood and Tissue kit (QIAGEN). The target locus was amplified by 35 PCR cycles using primers specific for each locus (see Table 4), such as 5'-GAGTTTGGCTCAAATTGTTACTCTT-3' (SEQ ID NO: 60) and 5'-GGGAAATGGTCTAGGAGAGTAAAGT-3' (SEQ ID NO: 61) using an AccuPrime High Fidelity PCR kit (Invitrogen).

[0361] Table 4 Summary of the top 10 off-target sites predicted in silico and the activity of each site detected by Surveyor assay in HEK293T cells transfected with Cas9 and the indicated sgRNA expression cassettes. nd: not detected.

[0362]

[0363]

[0364]

[0365] The resulting PCR products were randomly melted and reannealed on a thermocycler with a rate of −0.3°C / s between steps according to the following program: 95°C for 240 s, followed by 85°C for 60 s, 75°C for 60 s, 65°C for 60 s, 55°C for 60 s, 45°C for 60 s, 35°C for 60 s, and 25°C for 60 s. After reannealing, 8 μL of PCR product was mixed with 1 μL Surveyor Nuclease S and 1 μL Enhancer S (Transgenomic) and incubated at 42°C for 1 hour. After incubation, 6 μL of the digestion product was loaded on a 10% TBE polyacrylamide gel and electrophoresed at 200 V for 30 minutes. The gel was stained with ethidium bromide and quantified by densitometry using ImageLab (Bio-Rad) as described previously (Guschin et al., 2011). Meth Mol Biol 649, 247-256 (2010)).

[0366] Fluorescence-activated cell sorting of myoblastsDMD myoblasts were electroporated with 5 μg each of hCas9-T2A-GFP and sgRNA expression vectors and incubated at 37°C and 5% CO2. Three days after electroporation, cells were trypsinized and FACS sorts were collected using a FACSvantage II sorter. GFP-positive cells were collected and grown for analysis.

[0367] PCR-based assay for detecting genomic deletions Exon 51 or exon 45-55 loci were amplified from genomic DNA by PCR (Invitrogen AccuPrime High Fidelity PCR Kit) using primers flanking each locus. For exon 51, the flanking primers were CelI-CR1 / 2-F and CelI-CR5-R, or for exon 45-55 analysis, CelI-CR6-F and CelI-CR36-R (Table 4). PCR products were analyzed in TAE-agarose gels and stained with ethidium bromide for analysis.

[0368] PCR-based detection of translocations . The predicted loci of possible translocations were amplified by two-step nested PCR (Invitrogen AccuPrime High Fidelity PCR kit for each step) from genomic DNA of cells transfected with only Cas9 (control) or Cas9 and sgRNA. In the first step, a combination of Surveyor primers for each locus modified to include restriction sites that facilitate cloning and sequencing analysis was used to amplify the translocations that may occur in each on-target and off-target sgRNA target site by 35 PCR cycles (Table 4). Using a nested primer set custom-designed for each individual predicted translocation, 1 microliter of each PCR reaction was subjected to a second round of 35 rounds of PCR amplification (Table 4). Each second nested PCR primer binds in the same approximate region within the main amplicon; however, Primer3 online bioinformatics software was used to optimize each pair to ensure specific detection of each translocation. Purify the PCR amplicon (QIAGEN Gel Extraction kit) equivalent to the expected length of the predicted translocation and only present in cells treated with sgRNA, and analyze by Sanger sequencing.

[0369] mRNA analysis. Immortalized myoblasts were differentiated into myofibers and collected 5 days before trypsinization of the cells by replacing the growth medium with DMEM supplemented with 1% insulin-transferrin-selenium (Invitrogen #51500056) and 1% penicillin / streptomycin (Invitrogen #15140). Total RNA was isolated from these cells using the RNeasy Plus Mini kit (QIAGEN) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using the VILO cDNA synthesis kit (Life Technologies #11754) and 1.5 μg RNA at 42°C for 2 hours according to the manufacturer's instructions. The target locus was amplified by 35 PCR cycles using an AccuPrime High Fidelity PCR kit (Invitrogen) using primers annealing to exons 44 and 52 to detect exon 51 deletions through CR1 / 5 or CR2 / 5 or primers annealing to exons 44 and 60 to detect exon 45-55 deletions through CR6 / 36. The PCR products were electrophoresed on TAE-agarose gels and stained with ethidium bromide for analysis. The dissolved PCR bands were cloned and analyzed by Sanger sequencing to confirm the expected exon junctions. Table 5 lists the sequences of the primers used in Example 4.

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382] Western blot analysis.To evaluate dystrophin expression, immortalized myoblasts were differentiated into myofibers by replacing the growth medium with DMEM supplemented with 1% insulin-transferrin-selenium (Invitrogen) and 1% antibiotic / antimycotic (Invitrogen) for 4-7 days, such as 6 or 7 days. Fibroblasts were transdifferentiated into myoblasts by inducing MyoD overexpression and incubating the cells in DMEM supplemented with 1% insulin-transferrin-selenium (Invitrogen), 1% antibiotic / antimycotic (Invitrogen) and 3 μg / mL doxycycline for 15 days. Dystrophin expression was evaluated 3 days after transfection of HEK293T cells. Cells were trypsinized, collected, lysed in RIPA buffer (Sigma) supplemented with a protease inhibitor cocktail (Sigma), and the total protein amount was quantified using the bicinchoninic acid assay according to the manufacturer's instructions (Pierce). The samples were then mixed with NuPAGE loading buffer (Invitrogen) and 5% β-mercaptoethanol and heated to 85°C for 10 minutes. 25 micrograms of protein were separated on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) with MES buffer (Invitrogen). The proteins were transferred to a nitrocellulose membrane in a transfer buffer containing 10-20% methanol (e.g., 10% methanol) and 0.01% SDS for 1-2 hours. The blot was then blocked with 5% milk-TBST at room temperature for 1 hour. The blot was probed with the following primary antibodies: MANDYS8 to detect dystrophin (1:100, Sigma D8168) and rabbit anti-GAPDH (1:5000, Cell Signaling 2118S). The blots were then incubated with mouse or rabbit horseradish peroxidase-conjugated secondary antibodies (SantaCruz) and visualized using the ChemiDoc chemiluminescent system (BioRad) and Western-C ECL substrate (BioRad).

[0383] Transplanted into immunodeficient mice.All animal experiments were performed according to protocols approved by the Duke Institutional Animal Care & Use Committee. Cells were trypsinized, harvested, and washed in 1X Hank's Balanced Salt Solution (HBSS, Sigma). Two million cells were pelleted and resuspended in 5 µL 1X HBSS (Sigma) supplemented with cardiotoxin (Sigma #C9759) just before injection. These cells were transplanted into the tibialis anterior (TA) muscle of the hind limb of NOD.SCID.γ (NSG) mice (Duke CCIF Breeding Core) by intramuscular injection. Four weeks after injection, mice were euthanized and TA muscles were harvested.

[0384] Immunofluorescence staining . After incubation in 30% glycerol overnight at 4°C, harvested muscles were mounted in Optimal Cutting Temperature compound and frozen. Serial 10-micron sections were obtained by cryosectioning the embedded muscle tissue at -20°C. The frozen sections were then washed in PBS to remove the OCT compound and subsequently blocked in PBS containing 10% heat-inactivated fetal bovine serum (for spectrin detection) or 5% heat-inactivated fetal bovine serum (for dystrophin detection) for 30-60 minutes at room temperature. The frozen sections were incubated overnight at 4°C with the following primary antibodies specific for human epitopes only: 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-based immunofluorescence signal amplification detection kit (Life Technologies, TSA Kit #22, catalog number T-20932). Briefly, cryosections were incubated with 1:200 secondary goat anti-mouse biotin-XX (Life Technologies #B2763) in blocking buffer for 1 hour at room temperature. Signals were then amplified using streptavidin-HRP conjugate (1:100, from TSA Kit) in blocking buffer for 1 hour at room temperature. Finally, cryosections were incubated with tyramide-AlexaFluor488 conjugate (1:100, TSA Kit) in amplification buffer provided by the manufacturer for 10 minutes at room temperature. Stained cryosections were then mounted in ProLongAntiFade (Life Technologies #P36934) and observed using conventional fluorescence microscopy.

[0385] Cytotoxicity assay. To quantitatively evaluate potential sgRNA or SpCas9 nuclease-related cytotoxicity, HEK293T cells were transfected with 10 ng GFP reporter and 100 ng SpCas9 expression vector and 100 ng sgRNA expression vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The percentage of GFP-positive cells was evaluated at 2 and 5 days by flow cytometry. The survival rate was calculated as the decrease in GFP-positive cells from day 2 to day 5, normalized to cells transfected with an empty nuclease expression vector as described (Cornu et al., Meth Mol Biol 649:237-245 (2010)).

[0386] Example 4

[0387] CRISPR targeting the dystrophin gene—results

[0388] A CRISPR / Cas9-based system was designed to target the dystrophin gene. Different gRNAs were selected to target different regions of the human and mouse dystrophin genes based on NNNNN NNNNN NNNNN NNNNN NGG and GNNNN NNNNN NNNNN NNNNN NGG (see Tables 6, 7, and 8).

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396] Table 7

[0397] name Notes % Mod DCR1 Deletion of exon 51 6.6 DCR2 Deletion of exon 51 10.3 DCR3 Frameshift 13 DCR4 Deletion of exon 51 11.9 DCR5 Deletion of exon 51 12.4 DCR6 In intron 44 as close to exon 44 as possible (in case of patient deletion) 16.1 DCR7 Within intron 55 and as close to exon 56 as possible (in case of patient deletion) 6.8 DCR8 Only exon 42-43 deletion (-1 / +2) can be corrected, (-2 / +1) cannot be corrected by this 17.3 DCR9 Skipped exon 44 (5') 14.4 DCR10 Frameshift 14.9 DCR11 Correction downstream of exon 45 <1 DCR12 5' splice acceptor / frameshift <1 DCR13 Correction downstream of exon 46 16.9 DCR14 Frameshift 17.2 DCR15 Correction downstream of exon 47 15.4 DCR16 Frameshift 11.5 DCR17 Correction downstream of exon 48 <1 DCR18 5' splice acceptor / frameshift 1.8 DCR19 Correction downstream of exon 49 33.7 DCR20 5' splice acceptor 14.9 DCR21 Correction downstream of exon 50 24.1 DCR22 Frameshift 25.9 DCR23 Correction downstream of exon 52 25.2 DCR24 Frameshift (can only correct +1 reading frame) 24.8 DCR25 Correction downstream of exon 53 2.6 DCR26 Frameshift 24.5 DCR27 Correction downstream of exon 54 13.4 DCR28 5' splice acceptor 21.6 DCR29 Correction downstream of exon 55 19.2 DCR30 Integration of micro-dystrophin in exon 1 Not detected DCR31 Correction downstream of exon 51 18.9 DCR32 Missing stop codon Not detected DCR33 Alternatives to CR6 1.3 DCR34 Alternatives to CR6 13.2 DCR35 CR7's alternative 22.5 DCR36 CR7's alternative 26.4 DCR40 Disruption of exon 23 5' splice donor (corrects mdx mutation) DCR41 Disruption of exon 23 5' splice donor (corrects mdx mutation) DCR42 Deletion of exon 53 MDX4CV mutation DCR43 Disruption of myostatin receptors

[0398]

[0399]

[0400]

[0401] Specifically, 400 ng of Cas9 was co-transfected into HEK 293T cells with 400 ng of empty vector or gRNAs targeting the region including exon 51, namely CR1, CR2, CR3, CR4, and CR5 (see Fig.11 (b)). Genomic DNA was harvested 2 days after transfection and analyzed using the Surveyor assay (see Fig.11 (a) and 11(c)).

[0402] The CRISPR / Cas9-based system was used in DMD 8036 (del48-50) cells to determine whether the system could repair the mutant dystrophin gene. 5 µg of Cas9 was co-transfected into DMD 8036 (del48-50) cells along with 7.5 µg of 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 harvested 3 days after transfection and analyzed using the Surveyor assay ( Fig.12 ) or PCR analysis ( Fig.13 ) were analyzed. The locus was amplified by PCR using primers flanking the region containing the genomic targets for CR1 and CR5 (forward primer: 5'-gagaggttatgtggctttacca (SEQ ID NO:457), reverse primer: 5'-ctgcgtagtgccaaaacaaa (SEQ ID NO:458)) to give a 1447 bp band for the wild-type locus or the expected size of approximately 630 bp for the deletion locus. After 7 days of differentiation, Western blot of treated cells showed expression of dystrophin (see Fig.14 ).

[0403] Example 5

[0404] Targeting CRISPR / Cas9 to hotspot mutations in the human dystrophin gene

[0405] To correct widespread dystrophin mutations using the CRISPR / Cas9 gene editing platform, 12 sgRNAs were generated targeting the hotspot mutation region between exons 45-55 ( Fig.16pyogenes system using human codon-optimized SpCas9 nuclease and chimeric single guide RNA (sgRNA) expression vectors to guide efficient site-specific gene editing. Similar to Example 4, TALEN was used to target exon 51, and the protospacer at the 5' and 3' ends of exons 45-55 was selected to target, which meets the 5'-NRG-3' PAM requirement of SpCas9. Small insertions or deletions generated by NHEJ-based DNA repair within these exons can generate targeted frameshift mutations that overcome various dystrophin mutations around each exon ( Fig.16 A-16B). For example, CR3 is designed to correct a dystrophin mutation or deletion around exon 51 by introducing a small insertion or deletion at the 5' end of exon 51 to restore the downstream dystrophin reading frame ( Fig.16 B). Alternatively, sgRNAs were designed to exploit the multiplex power of the CRISPR / Cas9 system and specifically delete individual exons or series of exons to restore the dystrophin reading frame, similar to oligonucleotide-based exon skipping approaches. For this purpose, sgRNAs were targeted to exon 51 ( Fig.16 C) or exons 45-55 ( Fig.16 These sgRNAs were intentionally targeted to sites either downstream or upstream of the exons to be included in the resulting transcripts to minimize the likelihood that background patient deletions would include the intronic sgRNA target site.

[0406] Example 6

[0407] Screening of sgRNAs targeting the dystrophin gene in human cells

[0408] The frequency of gene editing in the human HEK293T cell line was evaluated to quickly determine the targeting efficiency of different sgRNAs. HEK293T was transfected with constructs encoding human codon-optimized SpCas9 and specified sgRNAs. As specified, each sgRNA was designed to modify the dystrophin gene. The frequency of gene modification was determined by the Surveyor assay on days 3 or 10 after transfection. The ratio of the Surveyor signals measured on days 3 and 10 was calculated to quantify the stability of the gene editing frequency of each sgRNA in human cells. As determined by the Surveyor assay 3 days after transfection, 29 / 32 (about 90%) of the measured sgRNAs were able to mediate efficient gene modification in the expected locus (Table 9, Fig.17 ). The gene editing frequency was stable for almost all sgRNAs (the signal change from day 3 to day 10 was <25%, Table 9, Fig.18), indicating that gene editing mediated by each individual sgRNA is extremely well tolerated. A notable exception is CR33, which has no detectable activity at day 10, although the activity may be lower than the sensitivity of the Surveyor assay (estimated to be about 1%).

[0409]

[0410]

[0411] Example 7

[0412] Enrichment of gene-edited cells using a fluorescence-based reporter system

[0413] sgRNAs were selected to correct specific mutations in DMD patient myoblast cell lines. After transfection into DMD myoblasts, unexpectedly low or undetectable gene modification activity was observed as measured by the Surveyor assay ( Fig.19 C, whole group). Flow cytometry was used to select transfected cells co-expressing GFP by 2A ribosomal skipping peptide linked to SpCas9 protein ( Fig.19 A). Adding this fluorescent reporter to the SpCas9 expression vector does not appear to significantly affect gene editing activity in HEK293T cells ( Fig.19 B) Three days after electroporation, a low percentage of transfected myoblasts (approximately 0.5-2%) expressed the fluorescent reporter, despite the high transfection efficiency of the control GFP-expressing plasmid (typically >70%, Fig.19 D, pmaxGFP). Given the high levels of CRISPR / Cas9 activity in the easily transfected HEK293T line, inefficient transgene expression after electroporation of SpCas9-T2A-GFP and sgRNA constructs into DMD cells could explain the low measured gene editing efficiency in unsorted cells. After sorting GFP-positive DMD myoblasts, a large increase in detectable activity was observed at most sgRNA target loci ( Fig.19 C) Therefore, all subsequent experiments used cells sorted for SpCas9 expression by expression of this fluorescent reporter.

[0414] Example 8

[0415] Repair of dystrophin expression by targeted frameshift

[0416] Small indels generated by NHEJ DNA repair can be used to generate targeted frameshifts to correct abnormal reading frames. sgRNA CR3 was designed to restore the dystrophin reading frame by introducing small indels within exon 51 ( Fig.16B, 20A). Alleles of genomic DNA from HEK293T cells co-transfected with expression plasmids for SpCas9 and CR3 sgRNA were evaluated by Sanger sequencing for the types of insertions and deletions generated by CRISPR / Cas9 at the locus ( Fig. 20 B). Apparently, insertions and deletions result in conversion to all three reading frames ( Fig. 20 B, 20C). To confirm genetic correction in relevant patient cell lines, expression plasmids for SpCas9 and CR3 sgRNA were electroporated into a DMD myoblast cell line with a deletion of exons 48-50 that is correctable by generating a frameshift in exon 51. Treated cells were sorted and confirmed to have gene modification activity by Surveyor assay (CR3, Fig.19 C sorting population) and differentiated into myotubes to test restored dystrophin expression. Dystrophin expression was observed to be accompanied by detectable nuclease activity ( Fig. 20 D). The S. pyogenes CRISPR / Cas9 system provides a powerful method to rapidly generate targeted frameshifts to overcome a variety of patient mutations and restore human dystrophin gene expression.

[0417] Example 9

[0418] Multiplex CRISPR / Cas9 gene editing mediates genomic deletion of exon 51 and rescues dystrophin expression

[0419] The multiplexing capabilities of the CRISPR / Cas9 system provide a new approach to efficiently generate genomic deletions of specific exons for targeted gene correction. Fig.19 In this study, DMD patient myoblasts with background deletions correctable by exon 51 skipping were treated with two combinations of sgRNAs flanking exon 51 (CR1 / CR5 or CR2 / CR5) and sorted to enrich for gene-edited cells. As detected by end-point PCR of genomic DNA from these treated cells, the expected genomic deletion was only present when both sgRNAs were electroporated into cells with SpCas9 ( Fig.21 A). For both deletions, Sanger sequencing confirmed the expected junction of the distal chromosome segments ( Fig.21 B). After differentiation of sorted myoblasts, deletion of exon 51 from mRNA transcripts was observed only in cells treated with both sgRNAs ( Fig.21 C). Finally, restored dystrophin expression was detected in treated cells accompanied by a loss of exon 51 at both the genomic and mRNA levels ( Fig.21 D).

[0420] Example 10

[0421] Rescue of dystrophin by large multi-exon genomic deletions

[0422] Although overcoming patient-specific mutations is a major use of the CRISPR / Cas9 system, it would be advantageous to develop a single method that can overcome multiple common patient deletions. For example, a promising strategy is to exclude the entire exon 45-55 region as a method to correct up to 62% of known patient deletions. Multiplex CRISPR / Cas9-based gene editing was tested to determine whether it could produce effective deletions of the exon 45-55 locus in human cells. After transfection into HEK293T cells, the expected deletion of approximately 336,000 bp ( Fig. 22 A). Similarly, the deletion was detected by PCR of genomic DNA from SpCas9 / sgRNA-treated DMD patient cells, which carried a background deletion of unknown length of exons 48-50 ( Fig. 22 A). Sanger sequencing of this deletion band of genomic DNA from treated DMD cells showed that the expected junction of intron 44 and intron 55 is directly adjacent to the sgRNA target site ( Fig. 22 B). Upon differentiation of treated DMD cells, the expected deletion of exons 45-55 was detected in the dystrophin mRNA transcript, which was confirmed by Sanger sequencing to be a fusion of exons 44 and 56 ( Fig. 22 C). Restored protein expression was observed by western blotting in sorted cell populations containing CRISPR / Cas9-induced deletions from exons 45-55 of the genome and the resulting mRNA transcripts ( Fig. 22 D) These data demonstrate that multiplex CRISPR / Cas9 editing provides a single universal method to restore the dystrophin reading frame in more than 60% of DMD patient mutations.

[0423] Embodiment 11

[0424] Transplantation of corrected myoblasts into immunodeficient mice

[0425] A promising approach for DMD therapy is to correct a population of autologous patient muscle progenitor cells that can be implanted into patient skeletal muscle tissue to rescue dystrophin expression. To confirm the ability of the corrected cells to express human dystrophin in vivo, a population of DMD myoblasts treated with sgRNAs CR1 and CR5 flanking exon 51 were implanted as described previously and sorted for GFP expression ( Fig.19 , Fig.23 ). After 4 weeks, myofibers positive for human spectrin expressed by both corrected and uncorrected cells were observed in frozen sections of injected muscle tissue ( Fig.24Many of these fibers were also positive for human dystrophin, whose expression was localized to the sarcolemma, indicating functional gene correction in these cells ( Fig.24 , Fig.25 In sections from mice injected with untreated DMD myoblasts, no fibers positive for human dystrophin were observed ( Fig.24 , Fig.25 ), indicating that CRISPR / Cas9-modified cells are the source of human dystrophin expression.

[0426] Example 12

[0427] Off-target and cytotoxicity analysis

[0428] As previously described, the relative cytotoxicity of the CRISPR / Cas9 system in human cells was evaluated for selected sgRNAs by adapting a flow cytometry-based GFP retention assay (Ousterout et al., Mol Ther 21:1718-1726 (2013)). Minimal cytotoxicity was observed for SpCas9 co-expression with or without sgRNA after transfection into human cells ( Fig.26 A). Publicly available tools are available to evaluate and prioritize potential CRISPR / Cas9 activity at off-target loci based on the predicted positional bias of a given mismatch in the sgRNA protospacer sequence and the total number of expected target site mismatches (Hsu et al., Nat Biotechnol 31:827-826 (2013)). This public web server was used to predict the most likely off-target sites for the sgRNAs used to correct the dystrophin gene in this study (Table 4). The top 10 potential off-target sites in HEK293T cells treated with SpCas9 and each sgRNA expression cassette of CR1, CR3, CR5, CR6, or CR36 were evaluated by the Surveyor assay. CR1, CR3, and CR36, each with one of the 10 predicted off-target loci, showed significant levels of gene modification (Tables 4 and Fig. 27 Interestingly, the CR3 off-target sequences had essentially the same and similar modification frequencies as the intended on-target sequences (9.3% on OT-1 vs. 13.3% on the intended site) (Tables 4 and Fig. 27 ). Apparently, CR3-OT1 was the only one of the three off-target sites that showed significant levels of activity in sorted hDMD cells by the Surveyor assay ( Fig.26 B).

[0429] By reconnecting at the far end between the cut target and the off-target locus on different chromosomes, the nuclease activity on the off-target site may cause unexpected chromosome rearrangement.Due to the potential increase of off-target activity caused by using two or more nucleases (such as in multiple CRISPR / Cas9 gene editing), this poses a major concern for the gene correction strategy based on deletion.In both single and multiple CRISPR / Cas9 editing strategies, a highly sensitive nested genome PCR assay is used to detect potential translocations to detect the translocations on the confirmed off-target locus (Table 4).Using this assay, in the model HEK293T cell line that also shows high levels of off-target activity, it is easy to detect translocations between the on-target and off-target sites ( Fig.26 C and Fig.28 A, 28B). Sanger sequencing of PCR amplicons confirmed the identity of the predicted translocation event for each primer pair ( Figure 29-30 ). One type of translocation detected in HEK293T cells was also detected in sorted hDMD myoblasts by nested PCR, although the signal was much weaker and sequence identity was not confirmed due to low product yields ( Fig.26 D and Fig.28 A, 28C). No translocation was detected using this assay in HEK293T or sorted hDMD cells treated with CR6 or CR6 / CR36, respectively ( Fig.28 ), with only low levels of off-target activity at CR6-OT3 in HEK293T cells (Table 4). These results emphasize the importance of selecting highly specific sgRNAs, especially for multiplex editing applications, and suggest that this approach could benefit from ongoing efforts to improve the specificity of the CRISPR / Cas9 system. These data demonstrate that the selected sgRNAs were able to correct the dystrophin gene without significant toxicity and with only a single strongly predicted off-target site with detectable levels of activity.

[0430] Example 13

[0431] Discussion

[0432] Genome editing is a powerful tool for correcting genetic diseases, and the recent development of the CRISPR / Cas9 system has significantly accelerated progress in this field. It has been shown that DMD, the most common genetic disease for which there is currently no approved treatment option, has been corrected. Many gene-based and cell-based therapies for DMD are in preclinical studies and clinical trials, and genome editing methods are compatible with many of these methods. For example, genome editing can be combined with patient-specific cell-based therapies for DMD. As shown, the CRISPR / Cas9 system can work in human pluripotent stem cells and other human cell lines as well as human skeletal myoblasts. Importantly, gene editing with CRISPR / Cas9 does not completely destroy the myogenic capacity of these cells, as demonstrated by effective dystrophin expression in vitro and in vivo in transplanted immunodeficient mice. Therefore, this strategy should be compatible with cell-based therapies for DMD.

[0433] Additionally, enriched pools of gene-corrected cells showed expression of human dystrophin in vivo after implantation into immunodeficient mice. CRISPR / Cas9 gene editing had no apparent toxic 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 of 50 predicted off-target sites among the five sgRNAs, and gene editing activity was detectable between on-target and off-target sites. CRISPR / Cas9 technology is an efficient and versatile method for correcting most dystrophin mutations and can be used as a general platform for the treatment of genetic diseases.

[0434] In addition, in contrast to the plasmid-based delivery methods used herein, direct transfection of sgRNA and Cas9 mRNA can be used to improve specificity and completeness by reducing the duration of Cas9 expression and eliminating the possibility of random plasmid integration. Alternatively, for in vivo genome editing and translation of this method, the CRISPR / Cas9 system can be delivered directly to the skeleton and / or myocardium via a viral, plasmid, or RNA delivery vector. The large size of the Streptococcus pyogenes Cas9 gene (approximately 4.2 kilobases) poses a challenge to its use in size-restricted adeno-associated virus vectors. However, from other species (such as Neisseria meningitidis and Streptococcus thermophilus ( S. thermophilus ) is short enough to efficiently package both Cas9 and sgRNA expression cassettes into a single AAV vector for in vivo gene editing applications.

[0435] The CRISPR / Cas9 system enabled efficient modification of almost 90% of the targets tested, consistent with other reports of robust activity of the system at different loci. The robustness and versatility of this technology is a major advance for creating patient-specific gene edits at will. Low levels of dystrophin, including as little as 4% of wild-type expression, can be sufficient to improve survival, motor function, and cardiac function in mouse models. The level of CRISPR / Cas9 activity may be sufficient for therapeutic benefit.

[0436] The use of deleted exons multiplexed with CRISPR / Cas93 also provides a unique class of opportunities and challenges. Deletion of entire exons from the genome was performed to restore dystrophin expression, in contrast to restoring the reading frame of the dystrophin gene with a small number of gains and losses generated by NHEJ-based DNA repair after the action of a single nuclease. The protein products of the edited genes are predictable and have been characterized in patients with Becker muscular dystrophy with naturally occurring deletions, in contrast to the random gains and losses created by the action of a single nuclease within the exon (which will result in the generation of new epitopes from each DNA repair event). In addition, for each successful gene editing event, the product generated from the exon deletion will result in restored dystrophin, while modifying the gene with random gains and losses within the exon will only restore the reading frame of one-third of the editing events that lead to the corrected reading frame.

[0437] All the sgRNAs tested were not associated with significant cytotoxic effects in human cells. For the five sgRNAs used, three potential off-target sites that restored dystrophin expression were identified from the 50 total tested sites. In addition, in HEK293T cells expressing high levels of Cas9 and sgRNA, chromosomal translocations between the expected on-target sites and these off-target sites were detected by highly sensitive nested PCR assays. Apparently, the off-target activity and translocation identified in HEK293T cells (which are immortalized and aneuploid cell lines expressing extremely high levels of Cas9 and sgRNA) do not occur at high levels, and in some cases cannot be detected in hDMD myoblasts. Importantly, given the severity of DMD, this level of specificity can be tolerated, lacking obvious cytotoxic effects in human cells.

[0438] Embodiment 14

[0439] developed an engineered AAV capsid, termed SASTG ( Fig.34 ; SEQ ID NOs: 436 and 437) for improved cardiac and skeletal muscle tissue tropism (Piacentino et al. (2012) Human Gene Therapy 23:635-646). ZFN targeting the Rosa26 locus ("Rosa26 ZFN"; Fig.33; SEQ ID NO: 434 and 435) were 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 produced and purified by the UNC Viral Vector Core. The Surveyor assay (Guschin et al., Methods Mol Biol 649, 247-256 (2010)) to demonstrate NHEJ mutagenesis at the Rosa26 locus following delivery of an AAV-SASTG Rosa26 ZFN in cultured C2C12 myoblasts that were actively cycling or forced to differentiate by serum deprivation (not shown).

[0440] To confirm that mature postmitotic skeletal muscle was effectively targeted by Rosa26 ZFN after AAV transfer, AAV-SASTG vectors encoding Rosa26 ZFN were directly injected into the tibialis anterior (TA) muscle of 6-week-old C57BL6 / J mice at a titer of 1e10 vector genomes (vg) or 2.5e10 vg / muscle. Mice were killed 4 weeks after injection, and TA muscles were harvested and cut into several segments for genomic DNA extraction and analysis ( Fig.31 ). Genomic DNA was PCR amplified and subjected to Surveyor assay to detect the NHEJ mutational properties of ZFN-induced mutagenesis at the Rosa26 target site ( Fig.32 ). Fig.32 Figure 2 shows Rosa26 ZFN activity in vitro and in vivo in skeletal muscle as analyzed by Surveyor after delivery of AAV-SASTG-ROSA. Proliferating C2C12 cells were transduced with the indicated amounts of virus and harvested 4 days post infection ( Fig.32 (a)). C2C12 cells were incubated in differentiation medium for 5 days and then transduced with the indicated amount of AAV-SASTG-ROSA virus in 24-well plates ( Fig.32 (b)). Samples were collected 10 days after transduction. The indicated amount of AAV-SASTG-ROSA was injected directly into the tibialis anterior muscle of C57BL / 6J mice, and the muscles were harvested 4 weeks after infection. The harvested muscles were divided into 8 separate pieces for genomic DNA analysis, each of which is shown in a separate lane ( Fig.32 (c)). Clearly, at the highest dose (2.5e10 vg), high levels of gene modification were detected in all segments.

[0441] Embodiment 15

[0442] AAV-CRISPR constructs targeting mutant dystrophin genes

[0443] AAV constructs are designed to therapeutically correct mutations in the dystrophin gene that cause Duchenne muscular dystrophy and skeletal and cardiac muscle degeneration. The CRISPR / Cas9 system can be delivered using AAV to restore the dystrophin reading frame by deleting exon 51, deleting exons 45-55, interrupting splice donor or acceptor sites, or creating a frameshift within exon 51 (Ousterout et al., Molecular Therapy 2013) to restore the dystrophin reading frame and protein expression. The CRISPR / Cas9 system can include a Cas9 having a sequence of SEQ ID NO: 64 or 114 (see Fig.40 and 41 ). Provide gRNAs that can be combined with these Cas9s to target their respective PAM sequences (see Fig.40 and 41 ; see also Tables 2 and 3).

[0444] Example 16

[0445] Generation of induced neurons (iN)

[0446] The generation of induced neurons (iNs) from other cell lineages has potential applications in regenerative medicine and research on neurological diseases. Mouse embryonic fibroblasts (MEFs) can be directly converted into functional neuronal cells by delivering three neuronal transcription factors: BRN2 , ASCL1 and MYT1L A mixture of (BAM factors, Fig.48 ) occurs. Other methods may include other factors that induce different neuronal subtypes. These experiments require ectopic delivery of transcription factors and activation of the corresponding endogenous loci to maintain the neuronal phenotype. The CRISPR / Cas9 system is transformed into a versatile transcription factor to activate endogenous genes in mammalian cells with any promoter in the genome targeted by an RNA-guided mechanism ( Fig.49 A, 49B).

[0447] Materials and methods. Activation of coding sequences using CRISPR / Cas9-transcription factors ASCL1 and BRN2 Endogenous genes are used to direct MEF reprogramming into functional induced neurons.

[0448] Cell cultureMEFs were seeded onto 24-well TCPS plates or poly-D-lysine / laminin-coated coverslips. After dCas9-VP64 transduction and gRNA transfection (see Tables 10 and 11 for gRNA sequences), cells were cultured in MEF medium (Adler et al., Mol Ther Nucleic Acids 1:e32 (2012) for 24 hours and then transferred to N3 neural induction medium (Vierbuchen et al., Nature 463:1035-1041 (2010)) Fig.49 B).

[0449]

[0450]

[0451]

[0452] qRT-PCR and IF: In dCas9-VP64 and gRNA, ASCL1 On day 3 after delivery of cDNA or a negative control vector encoding luciferase, endogenous ASCL1 Activation. TUJ1 and MAP2 The generation of iNs was evaluated by co-staining and identification of cells with neuronal morphology and elongated processes.

[0453] Live cell reporter : After 7-8 days in N3 medium, MEFs cultured on poly-D-lysine / laminin-coated coverslips were transduced with viruses carrying hSyn-RFP and MAP2-GCamP5 reporters to identify the majority of mature iNs for functional characterization by calcium imaging and electrophysiology ( Fig.49 B).

[0454] Results. dCas9-VP64 and targeting ASCL1 The co-delivery of 8 gRNAs significantly increased the activation of endogenous genes by 400-fold compared to the 100-fold activation induced by co-delivery of 4 gRNAs (p<0.05). Fig.50 A) Detection of nuclear localization of MEFs by immunofluorescence ASCL1 Compared with the mixture of dCas9-VP64 and gRNA, ectopic Ascl1 Expression produces more ASCL1 protein, but did not activate the endogenous locus by day 3 ( Fig.50 A, 50B). In the delivery of dCas9-VP64 and targeting ASCL1 and BRN2 After 13 days in neurogenesis medium, cells with extended processes were identified by immunofluorescence. TUJ1 and MAP2 Co-positive cells ( Fig.51 A, first row). Similar numbers of TUJ1 and MAP2 Co-positive cells ( Fig.51 A, second row). As early as day 11 in neurogenesis medium, cells expressing the hSyn-RFP reporter with neuronal morphology can be observed in culture ( Fig.51 B). Cells expressing the MAP2-GCaMP5 calcium indicator show KCl-induced depolarization detected by fluorescence microscopy ( Fig.52 A, 52B).

[0455] Mouse embryonic fibroblasts are directly converted into neurons with neuronal morphology TUJ1 and MAP2 Co-positive cells are activated by endogenous transcription factors via CRISPR / Cas9 BRN2 and ASCL1 Although dCas9-VP64 is ASCL1 Ectopic expression of Fig.50 B), but the generation of neuron-like cells is similar. Activation of endogenous loci can induce a reprogramming cascade of events that is mechanistically distinct from that produced by ectopic expression.

[0456] dCas9-VP64 is able to infiltrate heterochromatin and activate stably silenced endogenous genes, a property of the only subclass of "pioneer" transcription factors. Therefore, transformation of cell lineages with CRISPR / Cas9-transcription factors can better overcome epigenetic barriers to reprogramming than ectopic expression of transcription factors, especially difficult to reprogram cell types, such as adult cells. This may have clinical importance in the field of regenerative medicine, as autologous sources are often required in cell replacement therapies.

[0457] Embodiment 17

[0458] Multiplex CRISPR / Cas9-based genome engineering—Materials and methods

[0459] Plasmid constructs. As described above, an expression cassette of Streptococcus pyogenes sgRNA and human codon-optimized Cas9 (hCas9) nuclease was used. Other promoters of 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 promoters were synthesized using GeneBlocks (IDT) and cloned to replace the hU6 sgRNA expression cassette. GeneBlock (IDT) was cloned into the 3' end of the Cas9 coding sequence to fuse the T2A jump peptide and eGFP gene immediately after Cas9 to monitor vector expression. The coding region of hCas9-T2A-GFP (SEQ ID NO: 145) was then transferred into a lentiviral expression vector containing the human ubiquitin C (hUbC) promoter driving the expression of hCas9-T2A-GFP and a restriction site to facilitate Golden Gate cloning of the sgRNA expression cassette immediately upstream of the hUbC promoter ( Fig.42 A).

[0460] Protocol for custom lentiviral vector assembly. Assembly of custom lentiviral vectors expressing up to 4 selected sgRNAs and active Cas9, dCas9, or dCas9-VP64 is completed in less than 5 days. The cloning method uses GoldenGate cloning and a type IIS restriction enzyme that cuts outside its recognition sequence to generate unique overhangs. Golden Gate assembly expedites cloning because all 4 expression cassettes are ligated into the final lentiviral vector in a single step. Lentiviral vectors express active Cas9, cCas9, or dCas9-VP64 in addition to expression of 1, 2, 3, or 4 sgRNAs from independent promoters.

[0461] Step 1: Anneal each single stranded oligonucleotide containing 20 bp protospacer in the manner described to generate sticky ends and ligate into the desired pZ donor-promoter vector. Order 2 single stranded oligonucleotides for each desired genomic target. To anneal complementary oligonucleotides, mix 8 µL sense oligo + 8 µL antisense oligo (both at 10 mM) + 2 µL 10X ligase buffer. Melt the oligonucleotides and re-anneal in a PCR machine with the following program at -0.3°C / sec between steps: 96°C for 300 sec, followed by 85°C for 20 sec, 75°C for 20 sec, 65°C for 20 sec, 55°C for 20 sec, 45°C for 20 sec, 35°C for 20 sec, and 25°C for 20 sec. To phosphorylate the sticky ends, add 1 µL 25mM ATP + 1 microliter T4 polynucleotide kinase (NEB) and incubate at 37°C for 60 minutes followed by 65°C for 20 minutes to heat inactivate the enzyme. Each protospacer was ligated into the desired expression vector using 50 ng of vector and 1 μL of annealed oligonucleotides in a 10 μL reaction volume using T4 DNA ligase (NEB) incubated at 16°C for 60 minutes according to the manufacturer's instructions. 5 microliters of the ligation was transformed into XL1 blue chemically competent bacteria (Agilent) according to the manufacturer's instructions. The plate was changed to LB agar plates containing 50 μg / mL kanamycin (Sigma) and incubated overnight at 37°C. In our experience, >90% of the colonies will contain the desired ligation product. Sequencing using the M13 reverse standard sequencing primer was performed to confirm each final sgRNA construct before proceeding to step 2.

[0462] step 2 : Golden Gate assembly was used to construct the four promoter-gRNA cassettes into the lentiviral destination vector.After completing step 1, there are 4 separate plasmids that each express a different sgRNA from a different promoter. To assemble the 4 different promoter-sgRNA constructs into the desired destination vector, 200 ng of each sgRNA expression plasmid and the desired lentiviral destination vector were mixed with 1 μL T4 DNA ligase (NEB), 1 μL BsmBI FastDigest (FisherScientific), and 2 μL 10X T4 ligase buffer (NEB) in a 20 μL reaction volume. The reaction was incubated as follows: 37°C for 10 minutes, 16°C for 15 minutes, 37°C for 30 minutes, and 80°C for 5 minutes. 5 μL of the ligation reaction was transformed into SURE 2 chemically competent cells (Agilent) according to the manufacturer's instructions. The plate was transferred to an LB agar plate containing 100 μg / mL ampicillin and incubated overnight at 37°C. Optionally, colonies can be screened by lacZ-based blue / white screening using IPTG and X-gal; however, in our experience, >90% of transformants contain the appropriate ligation product. Due to the inverted repeats formed by the reverse sgRNA expression cassette, the final construct may be unstable, so we recommend maintaining these plasmids in the SURE 2 cell line and screening the final plasmids with a pilot PCR using the sense primer 5'-TCGGGTTTATTACAGGGACAGCAG-3' (SEQ ID NO: 464) and the antisense primer 5'-TCTAAGGCCGAGTCTTATGAGCAG-3' (SEQ ID NO: 465). These primers amplify across 4 promoter-gRNA regions. Due to the repetitive nature, different banding patterns should be observed, with the largest product size being approximately 1800 bp.

[0463] Cell culture and transfection. HEK293T cells were obtained from the American Tissue Collection (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, 5% CO2. HEK293T cells were transfected with 200 ng of each sgRNA expression vector (800 ng total pDNA) in 24-well plates using Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol.

[0464] Virus production and transduction. All lentiviral vectors used in this study were second generation and produced using standard virus production methods. Briefly, 3.5 million HEK293T cells were seeded per 10 cm dish. The next day, cells were transfected with 20 μg transfer vector, 6 μg pMD2G, and 10 μg psPAX2 using the calcium phosphate transfection method. The medium was changed 12-14 hours after transfection. The viral supernatant was collected 24 and 48 hours after the medium change, passed through a 0.45 μm filter, and combined. For transduction, the cell culture medium was replaced with viral supernatant supplemented with 4 μg / mL polybrene. After 12-24 hours, the viral supernatant was replaced with fresh culture medium.

[0465] Reverse transcription PCR. RNA was isolated using the miRNeasy Mini RNA Isolation Kit (Qiagen). DNase digestion was performed using a DNA-free kit (Applied Biosystems). cDNA synthesis was performed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNA was PCR amplified using Taq DNA polymerase (NEB) and the resulting products were electrophoresed in TAE agarose gels. Images were captured using the ChemiDoc XRS+ system and processed using ImageLab software (Bio-Rad).

[0466] 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 using PerfeCTa SYBR Green FastMix (Quanta Biosciences) was performed using the CFX96 Real-Time PCR Detection System (Bio-Rad). Primer specificity was confirmed by agarose gel electrophoresis and melting curve analysis. The reaction efficiency was calculated within the appropriate dynamic range to ensure the linearity of the standard curve. The results were expressed as the fold increase in target gene mRNA expression normalized to β-actin expression using the ΔΔCt method. The reported values ​​are the means and SEM of 2 independent experiments (n = 2), where for each experiment, technical duplicates were averaged.

[0467] Western Blot. Cells were lysed in RIPA buffer (Sigma) supplemented with a protease inhibitor cocktail (Sigma). Protein concentration was measured using BCA protein assay reagent (Thermo Scientific) and a BioTek Synergy 2 Multi-Mode microplate reader. Lysates were mixed with loading buffer and boiled for 5 minutes; 25 μg of protein was electrophoresed in a NuPage 10% Bis-Tris gel polyacrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane. Nonspecific antibody binding was blocked with TBST (50 mM Tris, 150 mM NaCl, and 0.1% Tween-20) with 5% skim milk for 1 hour at room temperature. The membrane was incubated with the following primary antibodies under the following conditions: anti-MyoD (1:250, Santa Cruz Sc-32758) in TBST containing 5% BSA at 4°C overnight; anti-Myogenin (1:250, Santa Cruz Sc-12732) in 5% BSA at 4°C overnight; anti-FLAG-HRP (1:1000, Cell Signaling 2044) in TBST containing 5% milk at room temperature for 60 minutes; anti-GAPDH (1:5000, Cell Signaling, clone 14C10) in TBST containing 5% milk at room temperature for 30 minutes. The membrane was then washed 3 times with TBST for a total of 15 minutes. The membrane was incubated with 1:5,000 dilution of anti-rabbit HRP-conjugated antibody (Sigma, 6154) or anti-mouse HRP-conjugated antibody (Santa Cruz, SC-2005) for 30 minutes and washed 3 times with TBST for 15 minutes each. The membrane was observed using ImmunStar WesternC chemiluminescent kit (Bio-Rad), images were taken using ChemiDoc XRS+ system, and processed using ImageLab software (Bio-Rad).

[0468] Cel-I quantification of endogenous gene modification. CRISPR / Cas9 nuclease damage at endogenous target sites was quantified using the Surveyor nuclease assay, which detects mutational properties of nuclease-mediated NHEJ. After transfection or transduction, cells were incubated at 37°C for 3-10 days, and genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen). The target locus was amplified by 35 PCR cycles using the AccuPrime High Fidelity PCR kit (Invitrogen). The resulting PCR product was randomly melted and reannealed at a rate of -0.3°C / second between steps in a PCR machine according to 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. After reannealing, 8 μL of PCR product was mixed with 1 μL Surveyor nuclease S and 1 μL Enhancer S (Transgenomic, Omaha, NE) and incubated at 42°C for 1 hour. After incubation, 6 μL of digestion product was loaded onto a 10% TBE polyacrylamide gel and electrophoresed for 30 minutes at 200 V. The gel was stained with ethidium bromide and quantitatively determined by densitometry using ImageLab (Bio-Rad) (Perez-Pinera et al., Nucleic Acids Res 40:3741-3751 (2012)).

[0469] Statistical analysis. Means and standard errors of the mean were compiled from at least 2 independent experiments. Effects were evaluated using multivariate ANOVA and Dunnett's post hoc test using JMP 10 Pro.

[0470] Embodiment 18

[0471] Development of a single lentiviral vector for multiplex CRISPR / Cas9 applications

[0472] A limitation of current CRISPR / Cas9 gene editing systems, particularly transactivator systems, is the efficient delivery of multiple sgRNAs and Cas9 proteins simultaneously for multiplexed gene editing and coordinated gene activation applications, especially in difficult-to-transfect cell types. 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, the vector expresses four sgRNAs from independent pol III promoters (human U6 promoter, mouse U6 promoter, 7SK, and H1). We confirmed sgRNA expression from each promoter by end-point RT-PCR to detect sgRNAs targeting the AAVS1 locus ( Fig.42 A). To test the activity of each sgRNA expression construct, we co-transfected each promoter construct independently expressing an sgRNA targeting AAVS1 with an active Cas9 expression construct into human HEK293T cells. Clearly, we detected consistent and high levels of gene modification at the target locus by each sgRNA that was comparable to the well-characterized zinc finger nuclease with high activity at the AAVS1 locus ( Fig.42 B). In addition, lentiviral delivery of different Cas9-based constructs, including active Cas9 nuclease, dead Cas9, and dead Cas9 fused to the VP64 transactivator domain, resulted in the expression of full-length Cas9 protein in HEK293T cells, as determined by western blotting ( Fig.42 C).

[0473] Using these components, we developed the Golden Gate cloning method to facilitate the rapid and efficient cloning of multiple sgRNA expression cassettes into a single lentiviral vector expressing the desired Cas9 effector ( Fig.43 ). In the first step, oligonucleotides encoding sgRNA pre-spacer sequences are independently cloned into different expression vectors each with a different promoter driving sgRNA expression. In the second step, each sgRNA expression construct is subcloned into the selected lentiviral Cas9 expression vector by Golden Gate assembly. This strategy allows up to 4 sgRNAs to be quickly and robustly cloned into a single lentiviral vector for gene editing or activation applications. In order to express less than 4 sgRNAs, a PolyT terminator sequence is cloned downstream of an unused promoter to prevent transcription from an unused promoter. Each vector co-expresses the selected Cas9 with eGFP via a 2A skipping peptide to enable fluorescence-activated flow cytometry sorting and enrichment of cells with a high multiplicity of infection. Finally, the entire region containing the sgRNA and Cas9 expression cassettes is flanked by loxP sites to mediate removal by Cre-lox excision.

[0474] Embodiment 19

[0475] Validation of a single lentiviral sgRNA / Cas9 expression vector for multiplex genome engineering

[0476] To confirm the independent activity of each sgRNA, we assembled a single lentiviral vector expressing active Cas9 and four sgRNAs, each targeting an independent locus ( Fig.44 A). As control vectors, we assembled constructs expressing only one sgRNA together with polyT protospacers at the other three positions. We transduced HEK293T and primary fibroblasts with lentiviral vectors expressing the indicated sgRNAs and monitored the gene modification frequency 7 or 10 days after transduction, respectively ( Fig.44 B). A single lentiviral vector mediates efficient multiplex editing at all four loci in both cell types ( Fig.44 B). Interestingly, expression of all four sgRNAs together resulted in higher modification frequencies than a single sgRNA at three of the four loci in fibroblasts ( Fig.44 B). We observed efficient multiplex gene editing in fibroblasts, a cell type that is traditionally difficult to transfect. These data demonstrate that a single lentivirus can effectively express four active sgRNAs and that the lentiviral platform can be used to target four different loci for multiplex CRISPR / Cas9 gene editing.

[0477] Embodiment 20

[0478] Transient RNA-guided gene activation in cell lines stably expressing lentiviral Cas9-based transactivators

[0479] Next, we focused our interest on developing a system that enables transient gene activation by transfecting sgRNA into model cell lines that stably express Cas9. HEK293T were transduced with different Cas9-T2A-GFP, and GFP expression was monitored by flow cytometry. After normal passage every 2-3 days, each cell line showed stable GFP expression until 35 days after transduction. The transduced HEK293T were then transfected with 1-4 separate sgRNA expression constructs targeting the IL1RN or HBG1 promoter. Transient transfection of these sgRNA constructs in cell lines stably expressing dCas9-VP64 resulted in regulatable endogenous gene activation ( Fig.45 A, 45B). After transient transfection of sgRNA constructs in cells expressing dCas9-VP64, gene activation reached maximum activation levels approximately 3-6 days after transfection and decreased to undetectable levels by 20 days after transfection ( Fig.45CD, 45D). In addition, we were able to reactivate each gene by a second transfection with all four sgRNA constructs targeting each promoter, although the level of activation was significantly lower than that observed with the first transfection ( Fig.45 C, 45D). This decrease in activity after the second transfection may be due to reduced vector expression or competitive growth of non-transduced cells. Nevertheless, these data suggest that lentiviral Cas9 combined with transient sgRNA delivery can be used as a versatile system to regulate and transiently activate and reactivate target genes in cell lines stably transduced with Cas9.

[0480] Embodiment 21

[0481] Stable gene activation in HEK293T cells using a single lentiviral sgRNA / Cas9 transactivator expression vector

[0482] Lentiviral delivery may enable long-term stable gene activation via CRISPR / Cas9 transactivation. To test this, HEK293T cells were transduced with a single lentiviral vector encoding dCas9-VP64 and 1-4 sgRNA expression cassettes. Similar to our transient transfection results ( Fig.45 ), we were able to regulatably and robustly activate the expression of endogenous IL1RN and HBG1 genes ( Fig.46 A, 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 after transfection, and gene expression returned to background levels 15-20 days after transfection. In contrast, lentiviral delivery of dCas9-VP64 and the same four sgRNAs targeting IL1RN or HBG1 induced sustained gene activation for more than 20 days after transduction ( Fig.46 C, 46D). Therefore, single lentiviral delivery of multiple dCas9-VP64 transactivators is a useful platform for stable and efficient upregulation of target endogenous genes.

[0483] Embodiment 22

[0484] dCas9-KRAB—Targeting the HS2 enhancer

[0485] The HS2 enhancer is a well-characterized distal regulatory element required for activation of the globin gene locus. dCas9-KRAB was delivered with a gRNA targeting the HS2 enhancer to determine whether this system could repress γ-, ε-, and β-globin expression in the K562 human erythroleukemia cell line ( Fig.54 ). A set of gRNAs targeting different sites along the core region of the HS2 enhancer (SEQ ID NO: 467) was generated. See Table 12.

[0486]

[0487]

[0488]

[0489] Screening for single gRNAs at the globin locus by CRISPR / dCas9. 5-8 days before electroporation with 5 µg of expression plasmid encoding U6-sgRNA, dCas9 and dCas9-KRAB effectors were delivered by lentiviral vectors ( Fig.55 A). Cells without gRNA electroporation (no gRNA) and cells treated with gRNA targeting a different locus (IL1RN) were included as controls. When measured 3 days after transfection, multiple gRNAs achieved effective repression of the ε-, γ-, and β-globin genes, achieving up to 80% knockout ( Fig.55 B, 55C, 55D). Expression of gRNA with dCas9 or dCas9-KRAB inhibited gene expression at the globin locus. Overall, treatment with dCas9-KRAB resulted in stronger repression of a given gRNA compared to dCas9 alone, suggesting an important role for the KRAB domain in recruiting heterochromatin factors that enhance repression. The level of repression achieved depended on the amount of gRNA plasmid delivered by transfection in dCas9-KRAB-treated cells alone ( Fig.56A , 56B , 56C). Increasing the dose of Cr4 gRNA plasmid up to 10 µg increased the level of silencing globin gene in dCas9-KRAB treated cells.

[0490] Stable silencing of the globin gene by dCas9-KRAB. Co-expression of dCas9 / dCas9-KRAB and a single gRNA in K562 cells by lentivirus ( Fig.57 A). Controls included cells not treated with lentivirus (NT), treated with dCas9 / dCas9-KRAB but without gRNA (No gRNA), and treated with dCas9 / dCas9-KRAB and a gRNA targeting a different locus (IL1RN). Cells treated with lentivirus were selected from day 4 to day 7. Multiple gRNAs achieved efficient transcriptional repression of the ε-, γ-, and β-globin genes, achieving up to 95% knockout ( Fig.57 57B, 57C, 57D). ε-globin expression was mostly silenced in response to gRNA targeting the HS2 enhancer. Treatment with dCas9-KRAB and gRNA resulted in significantly more repression than treatment with dCas9 and gRNA.

[0491] These observations demonstrate that dCas9-KRAB targeting the HS2 enhancer by gRNA achieves efficient repression of distal globin genes. This is the first example of targeting distal regulatory elements of epigenetic control by the CRISPR / Cas9 system in mammalian cells. Enhancers regulate development and disease, and the present disclosure provides methods to probe and control enhancer function and can be used to determine the effects of dCas9-KRAB on local chromatin accessibility and genome-wide expression.

[0492] Embodiment 23

[0493] dCas9-p300

[0494] Design of dCas9-p300 fusion protein and comparison with dCas9-VP64 fusion protein (see Fig.59 ). The amino acid constructs constructed by dCas9 are shown in Figures 61A-61C One day before transfection with Lipofectamine 2000 transfection reagent (Life Technologies), human embryonic kidney ( H uman E mbryonic K Cells of the tissue culture line HEK293T (ATCC; CRL-11268) were seeded in 24-well tissue culture dishes at a density of 1.5e5 cells / well. After 24 hours, the cells were transfected with 1 µL Lipofectamine 2000, 375 ng of dCas9 expression constructs (dCas9, dCas9VP64, or dCas9p300, respectively) and 125 ng of combined gRNA expression plasmids (4 types, each in an equimolar ratio). Table 13 shows the gRNA information.

[0495]

[0496]

[0497] Cells were harvested 3 days after transfection and mRNA expression was determined by RT-QPCR. The RT-QPCR primer sequences are listed in Table 14.

[0498] Table 14 RT-QPCR primers

[0499]

[0500] Using ΔΔC t Method, RT-QPCR was normalized to GAPDH Results are expressed as the fold increase in target gene expression relative to cells treated with Lipofectamine alone without DNA transfection ("no DNA") (see Figures 60A-60C ).

[0501] Fig.62 Mutation of residues in the p300 HAT domain was shown to cause loss of its ability to activate gene expression. Fig.63 Multiple gRNAs were shown to synergize with dCas-9-p300, as was shown with dCas-9-VP64.

[0502] Embodiment 24

[0503] Fig.64 TALEN-mediated integration of micro-dystrophin at the 5'UTR of the Dp427m skeletal muscle isoform of dystrophin in skeletal myoblast cell lines derived from human DMD patients carrying different deletions in the dystrophin gene is shown. DMD patient cells were electroporated with a construct encoding a TALEN pair active for the 5'UTR locus and a donor template carrying the micro-dystrophin gene. Fig.64 (a) Schematic showing how micro-dystrophin is integrated into the 5'UTR. Fig.64 (b) shows the isolation of hygromycin-resistant clonal cell lines and the use of Fig.64 (a) The primers shown were used to screen for successful site-specific integration in the 5'UTR by PCR. The asterisks indicate the primers selected for Fig.64 (c) Clones were further analyzed. Fig.64 (c) shows that cloned DMD myoblasts with detected integration events were differentiated for 6 days and evaluated for expression of an HA tag fused to the C-terminus of micro-dystrophin.

[0504] It is to be understood that the above description and the accompanying examples are only illustrative and should not be taken as limiting the scope of the present invention, which is defined solely by the appended claims and their equivalents.

[0505] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention, including but not limited to changes and modifications related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the invention.

[0506] appendix

[0507]

[0508] AAV / Rosa26 construct (SEQ ID NO: 456)

[0509]

[0510] HS2 enhancer target sequence (SEQ ID NO:467)

[0511] Taagcttcagtttttccttagttcctgttacatttctgtgtgtctccattagtgacctcccatagtccaagcatgagcagttctggccaggcccctgtcggggtcagtgccccacccccgccttctggttctgtgtaaccttctaagcaaaccttctggctcaagcacagcaatgctgagtcatgatgagtcatgctgaggcttagggtgtgtgcccagatgttctcagcctagagtgatgactcctatctgggtccccagcaggatgcttacagggcagatggcaaaaaaaaggagaagctgaccacctgactaaaactccacctcaaacggcatcataaagaaaatggatgcctgagacagaatgtgacatattctagaatatatt

[0512] dSpCas9-KRAB sequence (SEQ ID NO:466)

[0513]

Claims

1. A DNA targeting composition comprising: A fusion protein comprising a first polypeptide domain and a second polypeptide domain, wherein the first polypeptide domain comprises a clustered regularly interspaced short palindromic repeats-related 9 (Cas9) protein, wherein the second polypeptide domain comprises a VP64 domain, a KRAB domain or a p300 domain, wherein the second polypeptide domain comprises a p300 domain comprising amino acids 1422-2038 of SEQ ID NO: 676, or wherein the second polypeptide domain comprises a VP64 domain comprising amino acids 1422-1471 of SEQ ID NO: 675, or wherein the second polypeptide domain comprises a KRAB domain encoded by a polynucleotide comprising nucleotides 4222-4564 of SEQ ID NO: 466; and At least one guide RNA (gRNA).

2. The DNA targeting composition of claim 1, wherein the second polypeptide domain comprises a p300 domain comprising amino acids 1422-2038 of SEQ ID NO:

676.

3. The DNA targeting composition of claim 1, wherein the second polypeptide domain comprises a VP64 domain comprising amino acids 1422-1471 of SEQ ID NO:

675.

4. The DNA targeting composition of claim 1, wherein the second polypeptide domain comprises a KRAB domain encoded by a polynucleotide comprising nucleotides 4222-4564 of SEQ ID NO:

466.

5. The DNA targeting composition of claim 1, wherein the Cas9 protein is derived from Streptococcus pyogenes ( Streptococcus pyogenes ), Staphylococcus aureus ( Staphylococcus aureus ) or Neisseria meningitidis ( Neisseria meningitidis ).

6. The DNA targeting composition of claim 1, wherein the Cas9 protein comprises amino acids 36-1403 of SEQ ID NO: 1, or is encoded by a polynucleotide comprising nucleotides 7-4122 of SEQ ID NO: 145, or is encoded by a polynucleotide comprising nucleotides 1-3168 of SEQ ID NO: 459, or is encoded by a polynucleotide comprising nucleotides 28-3270 of SEQ ID NO:

461.

7. The DNA targeting composition of claim 1, wherein the fusion protein comprises SEQ ID NO: 675 or SEQ ID NO: 676, or is encoded by a polynucleotide comprising SEQ ID NO:

466.

8. The DNA targeting composition of claim 1, wherein the at least one guide RNA targets a promoter region of a gene selected from: ASCL1 , BRN2 , MYT1L , NANOG , VEGFA , TERT , IL1B , IL1R2 , IL1RN , HBG1 , HBG2 and MYOD1 ;or wherein the at least one guide RNA comprises a targeting domain encoded by at least one of SEQ ID NOs: 5-40, 65-144, 492-515, 540-563, 585-625, or 679-750, or wherein the at least one guide RNA targets ASCL1 and comprises a targeting domain encoded by at least one of SEQ ID NOs: 5-8, or targets a sequence comprising at least one of SEQ ID NOs: 468-515, or wherein the at least one guide RNA targets NANOG and comprises a targeting domain encoded by at least one of SEQ ID NOs: 9-12, or wherein the at least one guide RNA targets VEGFA and comprises a targeting domain encoded by at least one of SEQ ID NOs: 13-16, or wherein the at least one guide RNA targets TERT and comprises a targeting domain encoded by at least one of SEQ ID NOs: 17-20, or wherein the at least one guide RNA targets IL1B and comprises a targeting domain encoded by at least one of SEQ ID NOs: 21-24, or wherein the at least one guide RNA targets IL1R2 and comprises a targeting domain encoded by at least one of SEQ ID NOs: 25-28, or wherein the at least one guide RNA targets IL1RN and comprises a targeting domain encoded by at least one of SEQ ID NOs: 29-32, or wherein the at least one guide RNA targets HBG1 or HBG2 and comprises a targeting domain encoded by at least one of SEQ ID NOs: 33-36, or wherein the at least one guide RNA targets MYOD1 and comprises a targeting domain encoded by at least one of SEQ ID NOs: 37-40, or wherein the at least one guide RNA targets BRN2 and targets a sequence comprising at least one of SEQ ID NOs: 516-563, or wherein the at least one guide RNA targets HS2 and targets a sequence comprising at least one of SEQ ID NOs: 564-605, or wherein the at least one guide RNA targets dystrophin and comprises a targeting domain encoded by at least one of SEQ ID NOs: 65, 67, 69, 70, 100, or 679-750, or targets a sequence comprising at least one of SEQ ID NOs: 66, 68, 71-99, 104-144, or 628-632.

9. A polynucleotide encoding the DNA targeting composition according to any one of claims 1 to 8.

10. A vector comprising the polynucleotide of claim 9.

11. A composition for regulating gene expression in a cell, wherein the gene is selected from ASCL1, BRN2, MYT1L, NANOG, VEGFA, TERT, IL1B, IL1R2, IL1RN, HBG1, HBG2, MYOD1, HS2 and dystrophin, the composition comprising the DNA targeting composition of any one of claims 1 to 8, a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, or a vector comprising a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8.

12. The composition of claim 11, wherein the second polypeptide domain comprises a VP64 domain or a p300 domain and expression of the gene is increased.

13. The composition of claim 11, wherein the second polypeptide domain comprises a KRAB domain and expression of the gene is reduced.

14. A composition for inducing gene expression in a cell, the composition comprising the DNA targeting composition of any one of claims 1 to 8, a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, or a vector comprising a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, wherein the second polypeptide domain comprises a VP64 domain or a p300 domain.

15. A composition for regulating or editing a gene in a subject, the composition comprising the DNA targeting composition of any one of claims 1 to 8, a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, or a vector comprising a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8.

16. The composition of claim 15, wherein the second polypeptide domain comprises a VP64 domain or a p300 domain and expression of the gene is increased.

17. The composition of claim 15, wherein the second polypeptide domain comprises a KRAB domain and expression of the gene is reduced.

18. A cell comprising the DNA targeting composition of any one of claims 1 to 8, a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, a vector comprising a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, or the composition of any one of claims 11 to 17.

19. A kit comprising the DNA targeting composition of any one of claims 1 to 8, a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, a vector comprising a polynucleotide encoding the DNA targeting composition of any one of claims 1 to 8, a composition of any one of claims 11 to 17, or a cell of claim 18.

20. Use of the DNA targeting composition of any one of claims 1 to 8, the polynucleotide of claim 9, the vector of claim 10, or the composition of any one of claims 11 to 17 in the preparation of a medicament for modulating the expression of at least one endogenous gene in a cell, wherein the cell has a disease or condition selected from the group consisting of a skeletal muscle condition, or a cardiac muscle condition, or a combination thereof.

21. Use of the DNA targeting composition of any one of claims 1 to 8, the polynucleotide of claim 9, the vector of claim 10, or the composition of any one of claims 11 to 17 in the preparation of a medicament for activating an endogenous gene in a cell, wherein the second polypeptide domain comprises a VP64 domain or a p300 domain, wherein the cell has a disease or condition selected from the group consisting of a skeletal muscle condition, a myocardial condition, or a combination thereof.

22. Use of the DNA targeting composition of any one of claims 1 to 8, the polynucleotide of claim 9, the vector of claim 10, or the composition of any one of claims 11 to 17 in the preparation of a medicament for transdifferentiating or inducing cell differentiation, wherein the cell has a disease or condition selected from the group consisting of a skeletal muscle condition, or a cardiac muscle condition, or a combination thereof.

23. Use of the DNA targeting composition of any one of claims 1-8, the polynucleotide of claim 9, the vector of claim 10, or the composition of any one of claims 11-17 in the preparation of a medicament for performing multiple gene editing in a cell, wherein the cell has a disease or condition selected from the following: a skeletal muscle condition, or a myocardial condition, or a combination thereof.

24. Use of the DNA targeting composition of any one of claims 1 to 8, the polynucleotide of claim 9, the vector of claim 10, or the composition of any one of claims 11 to 17 in the preparation of a medicament for correcting a mutant gene in a cell, wherein the cell has a disease or condition selected from the group consisting of a skeletal muscle condition, or a cardiac muscle condition, or a combination thereof.

25. Use of the DNA targeting composition of any one of claims 1-8, the polynucleotide of claim 9, the vector of claim 10, or the composition of any one of claims 11-17 in the preparation of a medicament for genome editing in muscle of a subject, wherein the muscle is skeletal muscle or cardiac muscle, and wherein the subject has a disease or condition selected from the group consisting of a skeletal muscle condition, or a cardiac muscle condition, or a combination thereof.

26. Use of the DNA targeting composition of any one of claims 1-8, the polynucleotide of claim 9, the vector of claim 10, or the composition of any one of claims 11-17 in the preparation of a medicament for treating a disease or condition in a subject, wherein the subject has a disease or condition selected from the group consisting of a skeletal muscle condition, or a cardiac muscle condition, or a combination thereof.

Images