COMPOSITIONS AND METHODS FOR CORRECTING DYSTROPHIN MUTATIONS IN HUMAN CARDIOMYOCYTES

Abstract
The disclosure provides a method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a Cas9 nuclease or a sequence encoding a Cas9 nuclease, and a gRNA or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene. The administering restores dystrophin expression in at least a subset of the subjects cardiomyocytes, and may at least partially or fully restore cardiac contractility.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 31, 2019, is named UTFDP0002WO.txt and is 1,722,119 bytes in size.


FIELD OF THE DISCLOSURE

The present disclosure relates to the fields of molecular biology, medicine and genetics. More particularly, the disclosure relates to compositions and uses thereof for genome editing to correct mutations in vivo using an exon-skipping approach.


BACKGROUND

Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Duchenne muscular dystrophy (DMD) is one of the most severe forms of MD that affects approximately 1 in 5000 boys and is characterized by progressive muscle weakness and premature death. Cardiomyopathy and heart failure are common, incurable and lethal features of DMD. The disease is caused by mutations in the gene encoding dystrophin (DMD), a large intracellular protein that links the dystroglycan complex at the cell surface with the underlying cytoskeleton, thereby maintaining integrity of the muscle cell membrane during contraction. Mutations in the dystrophin gene result in loss of expression of dystrophin, causing muscle membrane fragility and progressive muscle wasting.


SUMMARY

Genomic editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (DMD). Most of these mutations are clustered in “hotspots.” As described in the Examples herein, a screen was performed for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots. The correction of DMD mutations by exon skipping is referred to herein as “myoediting.” In proof-of-concept studies, myoediting was performed in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. Thus, it is shown that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allows correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.


Thus, in some embodiments, the disclosure provides a method for editing a mutant dystrophin gene in a cardiomyocyte, the method comprising contacting the cardiomyocyte with a Cas9 nuclease or a sequence encoding a Cas9 nuclease, and a gRNA or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene.


The disclosure also provides a method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a Cas9 nuclease or a sequence encoding a Cas9 nuclease, and a gRNA or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene; wherein the administering restores dystrophin expression in at least 10% of the subject's cardiomyocytes.


The disclosure also provides a method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising contacting an induced pluripotent stem cell (iPSC) with a Cas9 nuclease or a sequence encoding a Cas9 nuclease, and a gRNA, or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene; differentiating the iPSC into a cardiomyocyte; and administering the cardiomyocyte to a the subject.


Also provided is a cell (such as an induced pluripotent stem cell (iPSC) or cardiomyocyte) produced according to the methods of the disclosure, and compositions thereof. In some embodiments, the cell expresses a dystrophin protein.


Also provided is an induced pluripotent stem cell (iPSC) comprising a Cas9 nuclease, or a sequence encoding a Cas9 nuclease, and a gRNA, or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene.


As used in the specification, “a” or “an” may mean one or more. As used in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.


The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.


Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, for the method being employed to determine the value, or that exists among the study subjects. Such an inherent variation may be a variation of ±10% of the stated value.


Throughout this application, nucleotide sequences are listed in the 5′ to 3′ direction, and amino acid sequences are listed in the N-terminal to C-terminal direction, unless indicated otherwise.


Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.





BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.



FIG. 1A-1C. Myoediting strategy and identification of optimal guide RNAs to target the top 12 exons in DMD. (FIG. 1A) Conserved splice sites contain multiple NAG and NGG sequences, which enable cleavage by SpCas9. The numbers indicate the frequency of occurrence (%). (FIG. 1B) Human DMD exon structure. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. Red arrowheads indicate the top 12 targeted exons. The numbers indicate the order of the exons. (FIG. 1C) T7E1 assays in human 293 cells transfected with plasmids expressing the corresponding guide RNA (gRNA), SpCas9, and GFP for the top 12 exons. The PCR products from GFP+ and GFP− cells were cut with T7 endonuclease I (T7E1), which is specific to heteroduplex DNA caused by CRISPR/Cas9-mediated genome editing. Red arrowhead indicates cleavage bands of T7E1. M denotes size marker lane. bp indicates the base pair length of the marker bands.



FIG. 2A-2J. Rescue of dystrophin mRNA expression in iPSC-derived cardiomyocytes with diverse mutations by myoediting. (FIG. 2A) Schematic of the myoediting of DMD iPSCs and 3D-EHMs-based functional assay. (FIG. 2B) Myoediting targets the exon 51 splice acceptor site in Del DMD iPSCs. A deletion (exons 48 to 50) in a DMD patient creates a frameshift mutation in exon 51. The red box indicates out-of-frame exon 51 with a stop codon. Destruction of the exon 51 splice acceptor in DMD iPSCs allows splicing from exons 47 to 52 and restoration of the dystrophin open reading frame. (FIG. 2C) Using the guide RNA library, three guide RNAs (Ex51-g1, Ex51-g2, and Ex51-g3) that target sequences 5′ of exon 51 were selected. FIG. 2C discloses SEQ ID NO: 2481. (FIG. 2D) RT-PCR of cardiomyocytes differentiated from uncorrected DMD (Del), corrected DMD iPSCs (Del-Cor.), and WT. Skipping of exon 51 allows splicing from exons 47 to 52 (lower band) and restoration of the DMD open reading frame. (FIG. 2E) Myoediting strategy for pseudo-exon 47A (pEx). DMD exons are represented as blue boxes. Pseudo-exon 47A (red) with stop codon is marked by a stop sign. The black box indicates myoediting-mediated indel. (FIG. 2F) Sequence of guide RNAs for pseudo-exon 47A of pEx. DMD exons are represented as blue boxes, and pseudo-exons are represented as red boxes (47A). sgRNA, single-guide RNA. FIG. 2F discloses SEQ ID NOS 2482-2484, respectively, in order of appearance. (FIG. 2G) RT-PCR of human cardiomyocytes differentiated from WT, uncorrected DMD (pEx), and corrected DMD iPSCs (pEx-Cor.) by guide RNAs In47A-g1 and In47A-g2. Skipping of pseudo-exon 47A allows splicing from exons 47 to 48 (lower band) and restoration of the DMD open reading frame. (FIG. 2H) Myoediting strategy for the duplication (Dup) of exons 55 to 59. DMD exons are represented as blue boxes. Duplicated exons are represented as red boxes. The black box indicates myoediting-mediated indel. (FIG. 2I) Sequence of guide RNAs for intron 54 of Dup (In54-g1, In54-g2, and In54-g3). FIG. 2I discloses SEQ ID NOS 2485-2487, respectively, in order of appearance. (FIG. 2J) RT-PCR of human cardiomyocytes differentiated from WT, uncorrected DMD (Dup), and corrected DMD iPSCs (Dup-Cor.). Skipping of duplicated exons 55 to 59 allows splicing from exons 54 to 55 and restoration of the DMD open reading frame. RT-PCR of RNA was performed with the indicated sets of primers (F and R) (Table 4).



FIG. 3A-3F. Immunocytochemistry and Western blot analysis show dystrophin protein expression rescued by myoediting. (FIG. 3A to 3C) Immunocytochemistry of dystrophin expression (green) shows DMD iPSC cardiomyocytes lacking dystrophin expression. Following successful myoediting, the corrected DMD iPSC cardiomyocytes express dystrophin. Immunofluorescence (red) detects cardiac marker troponin-I. Nuclei are labeled by Hoechst dye (blue). (FIG. 3D to 3F) Western blot analysis of WT (100 and 50%), uncorrected (Del, pEx, and Dup) and corrected DMD (Del-Cor #27, pEx-Cor #19, and Dup-Cor #6.) iCM. Red arrowhead (above 250 kD) indicates the immunoreactive bands of dystrophin. Blue arrowhead (above 150 kD) indicates the immunoreactive bands of MyHC loading controls. kD indicates protein molecular weight. Scale bar, 100 mm.



FIG. 4A-4F. Rescued DMD cardiomyocyte-derived EHM showed enhanced FOC (force of contraction). (FIG. 4A) Experimental setup for EHM preparation, culture, and analysis of contractile function. (FIG. 4B to 4D) Contractile dysfunction in DMD EHM can be rescued by myoediting. FOC normalized to muscle content of each individual EHM in response to increasing extracellular calcium concentrations; n=8/8/6/4/6/6/4/4; *P<0.05 by two-way analysis of variance (ANOVA) and Tukey's multiple comparison test. WT EHM data are pooled from parallel experiments with indicated DMD lines and applied to FIG. 4 (B to D). (FIG. 4E) Maximal cardiomyocyte FOC normalized to WT. n=8/8/6/4/6/6/4/4; *P<0.05 by one-way ANOVA and Tukey's multiple comparison test. (FIG. 4F) Titration of corrected cardiomyocytes revealed that 30% of cardio-myocytes needed to be repaired to partially rescue the phenotype, and 50% of cardiomyocytes needed to be repaired to fully rescue the phenotype (100% Del-Cor.) in EHMs. WT, Del, and 100% Del-Cor. are pooled data, as displayed in FIG. 4.



FIG. 5A-5B. Genome editing of DMD top 12 exons by CRISPR/Cas9. (FIG. 5A) DNA sequences of DMD top 12 exons (51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8 and 55) from GPF+ human 293 cells edited by SpCas9 using the corresponding guide RNAs (Table 5). PCR products from genomic DNA of each sample were subcloned into pCRII-TOPO vector and individual clones were picked and sequenced. Unedited wild type (WT) sequences are on the top and representative edited sequences are on the bottom. Deleted sequences are replaced by black dashes. Red lower case letters (ag) indicate the splice acceptor sites (SA, 3′ end of the intron). Blue lower case letters (gt) indicate the splice donor sites (SD, 5′ end of the intron). FIG. 5A discloses SEQ ID NOS 2488-2526 in the left column and SEQ ID NOS 2427-2546 in the right column, all respectively, in order of appearance. (FIG. 5B) RT-PCR of RNA from edited 293 cells indicate deletion of targeted DMD Dp140 isoform exons (51, 53, 46, 52, 50 and 55). Black arrows indicate the RT-PCR products with exon deletions. M denotes size marker lane. bp indicates the length of the marker bands. Sequence of the RT-PCR products of exon deletion bands contained the two flanking exons, but skipped the targeted exon. For example, sequence of the RT-PCR products of ΔEx51 band confirmed that exon 50 spliced directly to exon 52, excluding exon 51. FIG. 5B discloses “GAGCCTGCAACA” as SEQ ID NO: 2547, “ATCGAACAGTTG” as SEQ ID NO: 2548, “AAAGAGTTACTG” as SEQ ID NO: 2549, “CAGAAGTTGAAA” as SEQ ID NO: 2550, “GTGAAGCTCCTA” as SEQ ID NO: 2551 and “TAAAAGGACCTC” as SEQ ID NO: 2552.



FIG. 6A-6D. Correction of a large deletion mutation (Del. Ex47-50) in DMD iPSCs and iPSC-derived cardiomyocytes. (FIG. 6A) T7E1 assay using human 293 cells transfected with plasmid expressing SpCas9, gRNAs (Ex51-g1, g2 and g3), and GFP show genome cleavage at DMD exon 51. Red arrowheads point to cleavage products. M, marker; bp, base pair. (FIG. 6B) DNA sequences of DMD exon 51 from GPF+ DMD Del iPSCs edited by SpCas9 and the guide RNA Ex51 g3. PCR products from genomic DNA of a mixture of myoedited DMD iPSCs were subcloned into pCRII-TOPO vector and sequenced as described above. Uncorrected exon51 sequence is on the top and representative edited sequences are on the bottom. Deleted sequences are replaced by black dashes. Red lower-case letters (ag) indicate the splice acceptor sites. The number of deleted nucleotides is indicated by (-). FIG. 6B discloses SEQ ID NOS 2553-2561, respectively, in order of appearance. (FIG. 6C) Sequence of the lower RT-PCR band from FIG. 2D (Del-Cor. lane) confirms skipping of exon 51, which reframed the DMD ORF (dystrophin transcript from exons 47 to 52). FIG. 6C discloses SEQ ID NO: 2562. (FIG. 6D) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures (Del-Cor.) and single colony (Del-Cor-SC) following SpCas9-mediated exon skipping with guide RNA Ex51-g3 compared to WT and uncorrected cardiomyocyte (Del). Green, dystrophin staining; red, troponin I staining; blue, nuclei staining. Scale bar=100 μm.



FIG. 7A-7D. Correction of a pseudo-exon mutation (pEx47A) in DMD iPSCs and iPSC-derived cardiomyocytes. (FIG. 7A) T7E1 assay using DMD pEx47A iPSCs nucleofected with vector expressing SpCas9, gRNAs (pEx47A-g1 and g2), and GFP show genome cleavage at DMD pseudo-exon 47A. Red arrowheads point to cleavage products. M, marker; bp, base pair. (FIG. 7B) DNA sequences of DMD pseudo-exon 47A from GPF+ DMD Del iPSCs edited by SpCas9 and the guide RNA pEx47A-g1 and g2. PCR products from genomic DNA of a mixture of myoedited DMD iPSCs were subcloned and sequenced as described above. Uncorrected pseudo-exon 47A sequence is on the top and representative edited sequences are on the bottom. Deleted sequences are replaced by black dashes. Red lower case letter (g) indicate point mutation in the cryptic splice acceptor site. The number of deleted nucleotides is indicated by (-). FIG. 7B discloses SEQ ID NOS 2563-2567, respectively, in order of appearance. (FIG. 7C) Sequence of the lower RT-PCR bands from FIG. 2G (pEx and pEx-Cor. lanes) confirms skipping of pseudo-exon 47A, which reframed the DMD ORF (dystrophin transcript from exons 47 to 48). FIG. 7C discloses SEQ ID NOS 2568-2569, respectively, in order of appearance. (FIG. 7D) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures (pEx-Cor.) and single colony (pEx-Cor-SC) following SpCas9-mediated exon skipping with guide RNA pEx47A-g2 compared to WT and uncorrected cardiomyocyte (pEx). Green, dystrophin staining; red, troponin I staining; blue, nuclei staining. Scale bar=100 μm.



FIG. 8A-8E. Correction of a large duplication mutation (Dup. Ex55-59) in DMD iPSCs and iPSC-derived cardiomyocytes. (FIG. 8A) This insertion site (In59-In54 junction) was confirmed by PCR using a forward primer targeting intron 59 (F2) and a reverse primer targeting intron 54 (F1) (FIG. 2H and Table 4). The duplication-specific PCR band was absent in WT cells and was presented in Dup cells. (FIG. 8B) T7E1 assays using 293 cells with vector expressing SpCas9, gRNAs (In54-g1, g2 and g3), and GFP show genome cleavage at DMD intron 54. Red arrowheads point to cleavage products. M, marker; bp, base pair. (FIG. 8C) mRNA with duplicated exons was semi-quantified by RT-PCR using the primers flanking the duplication borders exon 53 and exon 55 (Ex53F, a forward primer in exon 53 and Ex59R, a reverse primer in exon 59). Similarly, duplicated exons was semi-quantified by RT-PCR using the primers flanking the duplication borders exon 59 and exon 60 (Ex59F, a forward primer in exon 59 and Ex60R, a reverse primer in exon 60). The duplication-specific RT-PCR upper bands (red arrowhead) were absent in WT cells and were decreased dramatically in Dup-Cor. cells. (FIG. 8D) PCR results of three representative corrected single colonies (Dup-Cor-SC #4, 6 and 26) and the uncorrected control (Dup). The absence of a duplication-specific PCR band (F2-R1) in colonies 4, 6 and 26 confirmed the deletion of the duplicated DNA region. M denotes size marker lane. bp indicates the length of the marker bands. (FIG. 8E) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures (Dup-Cor.) and single colony (Dup-Cor-SC #6) following SpCas9-mediated exon skipping with guide RNA In54-g1 compared to WT and uncorrected cardiomyocyte (Dup). Green, dystrophin staining; red, troponin I staining; blue, nuclei staining. Scale bar=100 μm.





DETAILED DESCRIPTION

DMD is a new mutation syndrome with more than 4,000 independent mutations that have been identified in humans (world-wide web at dmd.nl). The majority of patient mutations include deletions that cluster in a hotspot, and thus a therapeutic approach for skipping certain exon applies to large group of patients. The rationale of the exon skipping approach is based on the genetic difference between DMD and Becker muscular dystrophy (BMD) patients. In DMD patients, the reading frame of dystrophin mRNA is disrupted resulting in prematurely truncated, non-functional dystrophin proteins. BMD patients have mutations in the DMD gene that maintain the reading frame allowing the production of internally deleted, but partially functional dystrophins leading to much milder disease symptoms compared to DMD patients.


Duchenne muscular dystrophy (DMD) afflicts ˜1 in 5000 males and is caused by mutations in the X-linked dystrophin gene (DMD). These mutations include large deletions, large duplications, point mutations, and other small mutations. The rod-shaped dystrophin protein links the cytoskeleton and the extracellular matrix of muscle cells and maintains the integrity of the plasma membrane. In its absence, muscle cells degenerate. Although DMD causes many severe symptoms, dilated cardiomyopathy is a leading cause of death of DMD patients.


CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing is emerging as a promising tool for correction of genetic disorders. Briefly, an engineered RNA-guided nuclease, such as Cas9 or Cpf1, generates a double-strand break (DSB) at the targeted genomic locus adjacent to a short protospacer adjacent motif (PAM) sequence. There are three primary pathways to repair the DSB: (i) Nonhomologous end joining (NHEJ) directly ligates two DNA ends and leads to imprecise insertion/deletion (indel) mutations. (ii) Homology-directed repair (HDR) uses sister chromatid or exogenous DNA as a repair template and generates a precise modification at the target sites. (iii) Microhomology-mediated end joining (MMEJ) uses short sequences of nucleotide homology (5 to 25 base pairs) flanking the original DSB to ligate the broken ends and deletes the region between the microhomologies. Although NHEJ can effectively generate indel mutations in most cell types, HDR- or MMEJ-mediated editing is generally thought to be restricted to proliferating cells.


Internal in-frame deletions of dystrophin are associated with Becker muscular dystrophy (BMD), a relatively mild form of muscular dystrophy. Inspired by the attenuated clinical severity of BMD versus DMD, exon skipping has been advanced as a therapeutic strategy to bypass mutations that disrupt the dystrophin open reading frame by modulating splicing patterns of the DMD gene. Several recent studies used CRISPR/Cas9-mediated genome editing to correct various types of DMD mutations in human cells and mice. Some have deployed pairs of guide RNAs to correct the mutation, which requires simultaneous cutting of DNA and excision of large intervening genomic sequences (23 to 725 kb). Fortuitously, the PAM sequence for Streptococcus pyogenes Cas9 (SpCas9), the first and most widely used form of Cas9, contains NAG or NGG, corresponding to the universal splice acceptor sequence (AG) and most of the donor sequences (GG). Thus, in principle, directing Cas9 to splice junctions and the elimination of these consensus sequences by indels can allow for efficient exon skipping. In addition, only a single cleavage of DNA, which disrupts the splice site, can enable skipping of an entire exon.


Given the thousands of individual DMD mutations that have been identified in humans, an obvious question is how such a large number of mutations might be corrected by CRISPR/Cas9-mediated genome editing. Human DMD mutations are clustered in specific “hotspot” areas of the gene (exons 45 to 55 and exons 2 to 10) such that skipping 1 or 2 of 12 targeted exons within or nearby the hotspots (termed “top 12 exons”) can, in principle, rescue dystrophin function in a majority (˜60%) of DMD patients. Here, CRISPR/Cas9 is used with single-guide RNAs to destroy the conserved splice acceptor or donor sites preceding DMD mutations or to bypass mutant or out-of-frame exons, thereby allowing splicing between surrounding exons to recreate in-frame dystrophin proteins lacking the mutations. This approach was first tested by screening for optimal guide RNAs capable of inducing skipping of the DMD 12 exons that would potentially allow skipping of the most commonly mutated or out-of-frame exons within nearby mutational hotspots. As examples of this approach, the restoration of dystrophin expression is demonstrated in induced pluripotent stem cell (iPSC)-derived cardiomyocytes harboring exon deletions and a pseudo-exon point mutation. Finally, human iPSC-derived three-dimensional (3D) engineered heart muscle (EHM) was used to test the efficacy of gene editing to overcome abnormalities in cardiac contractility associated with DMD. Contractile dysfunction was observed in DMD EHM, recapitulating the dilated cardiomyopathy (DCM) clinical phenotype of DMD patients, and contractile function was effectively restored in corrected DMD EHM. Thus, genome editing represents a powerful means of eliminating the genetic cause and correcting the muscle and cardiac abnormalities associated with DMD.


These and other aspects of the disclosure are described in further detail below.


CRISPR Systems

CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a virus. CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with Cas genes that code for proteins related to CRISPRs. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.


CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote's genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.


Guide RNA (gRNA).


As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because Eukaryotic systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type III promoter U6. Synthetic gRNAs are slightly over 100 bp at the minimum length and contain a portion which is targets the 20 protospacer nucleotides immediately preceding the PAM sequence NGG; gRNAs do not contain a PAM sequence.


In some embodiments, the gRNA targets a site within a wildtype dystrophin gene. An exemplary wildtype dystrophin gene includes the human sequence (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5), the sequence of which is reproduced below:











   1
MLWWEEVEDC YEREDVQKKT FTKWVNAQFS KFGKQHIENL FSDLQDGRRL LDLLEGLTGQ






  61
KLPKEKGSTR VHALNNVNKA LRVLQNNNVD LVNIGSTDIV DGNHKLTLGL IWNIILHWQV





 121
KNVMKNIMAG LQQTNSEKIL LSWVRQSTRN YPQVNVINFT TSWSDGLALN ALIHSHRPDL





 181
FDWNSVVCQQ SATQRLEHAF NIARYQLGIE KLLDPEDVDT TYPDKKSILM YITSLFQVLP





 241
QQVSIEAIQE VEMLPRPPKV TKEEHFQLHH QMHYSQQITV SLAQGYERTS SPKPRFKSYA





 301
YTQAAYVTTS DPTRSPFPSQ HLEAPEDKSF GSSLMESEVN LDRYQTALEE VLSWLLSAED





 361
TLQAQGEISN DVEVVKDQFH THEGYMMDLT AHQGRVGNIL QLGSKLIGTG KLSEDEETEV





 421
QEQMNLLNSR WECLRVASME KQSNLHRVLM DLQNQKLKEL NDWLTKTEER TRKMEEEPLG





 481
PDLEDLKRQV QQHKVLQEDL EQEQVRVNSL THMVVVVDES SGDHATAALE EQLKVLGDRW





 541
ANICRWTEDR WVLLQDILLK WQRLTEEQCL FSAWLSEKED AVNKIHTTGF KDQNEMLSSL





 601
QKLAVLKADL EKKKQSMGKL YSLKQDLLST LKNKSVTQKT EAWLDNFARC WDNLVQKLEK





 661
STAQISQAVT TTQPSLTQTT VMETVTTVTT REQILVKHAQ EELPPPPPQK KRQITVDSEI





 721
RKRLDVDITE LHSWITRSEA VLQSPEFAIF RKEGNFSDLK EKVNAIEREK AEKFRKLQDA





 781
SRSAQALVEQ MVNEGVNADS IKQASEQLNS RWIEFCQLLS ERLNWLEYQN NIIAFYNQLQ





 841
QLEQMTTTAE NWLKIQPTTP SEPTAIKSQL KICKDEVNRL SGLQPQIERL KIQSIALKEK





 901
GQGPMFLDAD FVAFTNHFKQ VFSDVQAREK ELQTIFDTLP PMRYQETMSA IRTWVQQSET





 961
KLSIPQLSVT DYEIMEQRLG ELQALQSSLQ EQQSGLYYLS TTVKEMSKKA PSEISRKYQS





1021
EFEEIEGRWK KLSSQLVEHC QKLEEQMNKL RKIQNHIQTL KKWMAEVDVF LKEEWPALGD





1081
SEILKKQLKQ CRLLVSDIQT IQPSLNSVNE GGQKIKNEAE PEFASRLETE LKELNTQWDH





1141
MCQQVYARKE ALKGGLEKTV SLQKDLSEMH EWMTQAEEEY LERDFEYKTP DELQKAVEEM





1201
KRAKEEAQQK EAKVKLLTES VNSVIAQAPP VAQEALKKEL ETLTTNYQWL CTRLNGKCKT





1261
LEEVWACWHE LLSYLEKANK WLNEVEFKLK TTENIPGGAE EISEVLDSLE NLMRHSEDNP





1321
NQIRILAQTL TDGGVMDELI NEELETFNSR WRELHEEAVR RQKLLEQSIQ SAQETEKSLH





1381
LIQESLTFID KQLAAYIADK VDAAQMPQEA QKIQSDLTSH EISLEEMKKH NQGKEAAQRV





1441
LSQIDVAQKK LQDVSMKFRL FQKPANFELR LQESKMILDE VKMHLPALET KSVEQEVVQS





1501
QLNHCVNLYK SLSEVKSEVE MVIKTGRQIV QKKQTENPKE LDERVTALKL HYNELGAKVT





1561
ERKQQLEKCL KLSRKMRKEM NVLTEWLAAT DMELTKRSAV EGMPSNLDSE VAWGKATQKE





1621
IEKQKVHLKS ITEVGEALKT VLGKKETLVE DKLSLLNSNW IAVTSRAEEW LNLLLEYQKH





1681
METFDQNVDH ITKWIIQADT LLDESEKKKP QQKEDVLKRL KAELNDIRPK VDSTRDQAAN





1741
LMANRGDHCR KLVEPQISEL NHRFAAISHR IKTGKASIPL KELEQFNSDI QKLLEPLEAE





1801
IQQGVNLKEE DFNKDMNEDN EGTVKELLQR GDNLQQRITD ERKREEIKIK QQLLQTKHNA





1861
LKDLRSQRRK KALEISHQWY QYKRQADDLL KCLDDIEKKL ASLPEPRDER KIKEIDRELQ





1921
KKKEELNAVR RQAEGLSEDG AAMAVEPTQI QLSKRWREIE SKFAQFRRLN FAQIHTVREE





1981
TMMVMTEDMP LEISYVPSTY LTEITHVSQA LLEVEQLLNA PDLCAKDFED LFKQEESLKN





2041
IKDSLQQSSG RIDIIHSKKT AALQSATPVE RVKLQEALSQ LDFQWEKVNK MYKDRQGRFD





2101
RSVEKWRRFH YDIKIFNQWL TEAEQFLRKT QIPENWEHAK YKWYLKELQD GIGQRQTVVR





2161
TLNATGEEII QQSSKTDASI LQEKLGSLNL RWQEVCKQLS DRKKRLEEQK NILSEFQRDL





2221
NEFVLWLEEA DNIASIPLEP GKEQQLKEKL EQVKLLVEEL PLRQGILKQL NETGGPVLVS





2281
APISPEEQDK LENKLKQTNL QWIKVSRALP EKQGEIEAQI KDLGQLEKKL EDLEEQLNHL





2341
LLWLSPIRNQ LEIYNQPNQE GPFDVQETEI AVQAKQPDVE EILSKGQHLY KEKPATQPVK





2401
RKLEDLSSEW KAVNRLLQEL RAKQPDLAPG LTTIGASPTQ TVTLVTQPVV TKETAISKLE





2461
MPSSLMLEVP ALADFNRAWT ELTDWLSLLD QVIKSQRVMV GDLEDINEMI IKQKATMQDL





2521
EQRRPQLEEL ITAAQNLKNK TSNQEARTII TDRIERIQNQ WDEVQEHLQN RRQQLNEMLK





2581
DSTQWLEAKE EAEQVLGQAR AKLESWKEGP YTVDAIQKKI TETKQLAKDL RQWQTNVDVA





2641
NDLALKLLRD YSADDTRKVH MITENINASW RSIHKRVSER EAALEETHRL LQQFPLDLEK





2701
FLAWLTEAET TANVLQDATR KERLLEDSKG VKELMKQWQD LQGEIEAHTD VYHNLDENSQ





2761
KILRSLEGSD DAVLLQRRLD NMNFKWSELR KKSLNIRSHL EASSDQWKRL HLSLQELLVW





2821
LQLKDDELSR QAPIGGDFPA VQKQNDVHRA FKRELKTKEP VIMSTLETVR IFLTEQPLEG





2881
LEKLYQEPRE LPPEERAQNV TRLLRKQAEE VNTEWEKLNL HSADWQRKID ETLERLQELQ





2941
EATDELDLKL RQAEVIKGSW QPVGDLLIDS LQDHLEKVKA LRGEIAPLKE NVSHVNDLAR





3001
QLTTLGIQLS PYNLSTLEDL NTRWKLLQVA VEDRVRQLHE AHRDFGPASQ HFLSTSVQGP





3061
WERAISPNKV PYYINHETQT TCWDHPKMTE LYQSLADLNN VRFSAYRTAM KLRRLQKALC





3121
LDLLSLSAAC DALDQHNLKQ NDQPMDILQI INCLTTIYDR LEQEHNNLVN VPLCVDMCLN





3181
WLLNVYDTGR TGRIRVLSFK TGIISLCKAH LEDKYRYLFK QVASSTGFCD QRRLGLLLHD





3241
SIQIPRQLGE VASFGGSNIE PSVRSCFQFA NNKPEIEAAL FLDWMRLEPQ SMVWLPVLHR





3301
VAAAETAKHQ AKCNICKECP IIGFRYRSLK HFNYDICQSC FFSGRVAKGH KMHYPMVEYC





3361
TPTTSGEDVR DFAKVLKNKF RTKRYFAKHP RMGYLPVQTV LEGDNMETPV TLINFWPVDS





3421
APASSPQLSH DDTHSRIEHY ASRLAEMENS NGSYLNDSIS PNESIDDEHL LIQHYCQSLN





3481
QDSPLSQPRS PAQILISLES EERGELERIL ADLEEENRNL QAEYDRLKQQ HEHKGLSPLP





3541
SPPEMMPTSP QSPRDAELIA EAKLLRQHKG RLEARMQILE DHNKQLESQL HRLRQLLEQP





3601
QAEAKVNGTT VSSPSTSLQR SDSSQPMLLR VVGSQTSDSM GEEDLLSPPQ DTSTGLEEVM





3661
EQLNNSFPSS RGRNTPGKPM REDTM.






In some embodiments, the gRNA targets a site within a mutant dystrophin gene. In some embodiments, the gRNA targets a dystrophin intron. In some embodiments, the gRNA targets a dystrophin exon. In some embodiments, the gRNA targets a site in a dystrophin exon that is expressed and is present in one or more of the dystrophin isoforms shown in Table 1. In embodiments, the gRNA targets a dystrophin splice site. In some embodiments, the gRNA targets a splice donor site on the dystrophin gene. In embodiments, the gRNA targets a splice acceptor site on the dystrophin gene.









TABLE 1







Dystrophin isoforms
















Pro-





Nucleic

tein





Acid
Protein
SEQ



Sequence
Nucleic Acid
SEQ ID
Accession
ID



Name
Accession No.
NO:
No.
NO:
Description





DMD
NC_000023.11
None
None
None
Sequence from Human X Chromosome (at


Genomic
(positions



positions Xp21.2 to p21.1) from Assembly


Sequence
31119219 to



GRCh38.p7 (GCF_000001405.33)



33339609)









Dystrophin
NM_000109.3
  6
NP_000100.2
  7
Transcript Variant: transcript Dp427c is


Dp427c




expressed predominantly in neurons of the 


isoform




cortex and the CA regions of the hippocampus. 







It uses a unique promoter/exon 1 located about







130 kb upstream of the Dp427m transcript 







promoter. The transcript includes the common 







exon 2 of transcript Dp427m and has a similar 







length of 14 kb. The Dp427c isoform contains a







unique N-terminal MED sequence, instead of the







MLWWEEVEDCY sequence (SEQ ID NO: 2476) of 







isoform Dp427m. The remainder of isoform 







Dp427c is identical to isoform Dp427m.





Dystrophin
NM_004006.2
  8
NP_003997.1
  9
Transcript Variant: transcript Dp427m encodes 


Dp427m




the main dystrophin protein found in muscle. 


isoform




As a result of alternative promoter use, exon 







1 encodes a unique N-terminal MLWWEEVEDCY 







(SEQ ID NO: 2476) aa sequence.





Dystrophin
NM_004009.3
 10
NP_004000.1
 11
Transcript Variant: transcript Dp427p1 


Dp427p1




initiates from a unique promoter/exon 1 


isoform




located in what corresponds to the first 







intron of transcript Dp427m. The transcript 







adds the common exon 2 of Dp427m and has a 







similar length (14 kb). The Dp427p1 isoform 







replaces the MLWWEEVEDCY (SEQ ID NO: 2476)-







start of Dp427m with a unique N-terminal 







MSEVSSD (SEQ ID NO: 2477) aa sequence.





Dystrophin
NM_004011.3
 12
NP_004002.2
 13
Transcript Variant: transcript Dp260-1 uses 


Dp260-1




exons 30-79, and originates from a 


isoform




promoter/exon 1 sequence located in intron 29







of the dystrophin gene. As a result, Dp260-1 







contains a 95 bp exon 1 encoding a unique N-







terminal 16 aa MTEIILLIFFPAYFLN-sequence (SEQ







ID NO: 2478) that replaces amino acids 1-1357







of the full-length dystrophin product (Dp427m







isoform).





Dystrophin
NM_004012.3
 14
NP_004003.1
 15
Transcript Variant: transcript Dp260-2 uses 


Dp260-2




exons 30-79, starting from a promoter/exon 1 


isoform




sequence located in intron 29 of the 







dystrophin gene that is alternatively spliced







and lacks N-terminal amino acids 1-1357 of the







full length dystrophin (Dp427m isoform). The 







Dp260-2 transcript encodes a unique N-terminal







MSARKLRNLSYKK sequence (SEQ ID NO: 2479).





Dystrophin
NM_004013.2
 16
NP_004004.1
 17
Transcript Variant: Dp140 transcripts use exons


Dp140




45-79, starting at a promoter/exon 1 located in


isoform




intron 44. Dp140 transcripts have a long (1 kb)







5′ UTR since translation is initiated in exon 







51 (corresponding to aa 2461 of dystrophin). In







addition to the alternative promoter and exon 







1, differential splicing of exons 71-74 and 78







produces at least five Dp140 isoforms. Of 







these, this transcript (Dp140) contains all of







the exons.





Dystrophin
NM_004014.2
 18
NP_004005.1
 19
Transcript Variant: transcript Dp116 uses exons


Dp116




56-79, starting from a promoter/exon 1 within 


isoform




intron 55. As a result, the Dp116 isoform 







contains a unique N-terminal MLHRKTYHVK aa 







sequence (SEQ ID NO: 2480), instead of aa 1-







2739 of dystrophin. Differential splicing 







produces several Dp116-subtypes. The Dp116 







isoform is also known as S-dystrophin or apo-







dystrophin-2.





Dystrophin
NM_004015.2
 20
NP_004006.1
 21
Transcript Variant: Dp71 transcripts use exons 


Dp71




63-79 with a novel 80- to 100-nt exon 


isoform




containing an ATG start site for a new coding 







sequence of 17 nt. The short coding sequence is







in-frame with the consecutive dystrophin 







sequence from exon 63. Differential splicing of







exons 71 and 78 produces at least four Dp71 







isoforms. Of these, this transcript (Dp71) 







includes both exons 71 and 78.





Dystrophin
NM_004016.2
 22
NP_004007.1
 23
Transcript Variant: Dp71 transcripts use exons 


Dp71b




63-79 with a novel 80- to 100-nt exon 


isoform




containing an ATG start site for a new coding 







sequence of 17 nt. The short coding sequence is







in-frame with the consecutive dystrophin 







sequence from exon 63. Differential splicing of







exons 71 and 78 produces at least four Dp71 







isoforms. Of these, this transcript (Dp71b) 







lacks exon 78 and encodes a protein with a 







different C-terminus than Dp71 and Dp71a







isoforms.





Dystrophin
NM_004017.2
 24
NP_004008.1
 25
Transcript Variant: Dp71 transcripts use exons 


Dp71a




63-79 with a novel 80- to 100-nt exon 


isoform




containing an ATG start site for a new coding 







sequence of 17 nt. The short coding sequence is







in-frame with the consecutive dystrophin 







sequence from exon 63. Differential splicing of







exons 71 and 78 produces at least four Dp71 







isoforms. Of these, this transcript (Dp71a) 







lacks exon 71.





Dystrophin
NM_004018.2
 26
NP_004009.1
 27
Transcript Variant: Dp71 transcripts use exons 


Dp71ab




63-79 with a novel 80- to 100-nt exon 


isoform




containing an ATG start site for a new coding 







sequence of 17 nt. The short coding sequence is







in-frame with the consecutive dystrophin 







sequence from exon 63. Differential splicing of







exons 71 and 78 produces at least four Dp71 







isoforms. Of these, this transcript (Dp71ab) 







lacks both exons 71 and 78 and encodes a







protein with a C-terminus like isoform Dp71b.





Dystrophin
NM_004019.2
 28
NP_004010.1
 29
Transcript Variant: transcript Dp40 uses exons 


Dp40




63-70. The 5′ UTR and encoded first 7 aa are 


isoform




identical to that in transcript Dp71, but the 







stop codon lies at the splice junction of the 







exon/intron 70. The 3′ UTR includes nt from 







intron 70 which includes an alternative 







polyadenylation site. The Dp40 isoform lacks 







the normal C-terminal end of full-length 







dystrophin (aa 3409-3685).





Dystrophin
NM_004020.3
 30
NP_004011.2
 31
Transcript Variant: Dp140 transcripts use exons


Dp140c




45-79, starting at a promoter/exon 1 located in


isoform




intron 44. Dp140 transcripts have a long (1 kb) 







5′ UTR since translation is initiated in exon 







51 (corresponding to aa 2461 of dystrophin). In







addition to the alternative promoter and exon 







1, differential splicing of exons 71-74 and 78







produces at least five Dp140 isoforms. Of 







these, this transcript (Dp140c) lacks exons 







71-74.





Dystrophin
NM_004021.2
 32
NP_004012.1
 33
Transcript Variant: Dp140 transcripts use exons


Dp140b




45-79, starting at a promoter/exon 1 located in


isoform




intron 44. Dp140 transcripts have a long (1 kb) 







5′ UTR since translation is initiated in exon 







51 (corresponding to aa 2461 of dystrophin). In







addition to the alternative promoter and exon 







1, differential splicing of exons 71-74 and 78







produces at least five Dp140 isoforms. Of 







these, this transcript (Dp140b) lacks exon 78 







and encodes a protein with a unique C-terminus.





Dystrophin
NM_004022.2
 34
NP_004013.1
 35
Transcript Variant: Dp140 transcripts use exons


Dp140ab




45-79, starting at a promoter/exon 1 located in


isoform




intron 44. Dp140 transcripts have a long (1 kb)







5′ UTR since translation is initiated in exon 







51 (corresponding to aa 2461 of dystrophin). In







addition to the alternative promoter and exon 







1, differential splicing of exons 71-74 and 78







produces at least five Dp140 isoforms. Of 







these, this transcript (Dp140ab) lacks exons 71







and 78 and encodes a protein with a unique 







C-terminus.





Dystrophin
NM_004023.2
 36
NP_004014.1
 37
Transcript Variant: Dp140 transcripts use exons


Dp140bc




45-79, starting at a promoter/exon 1 located in


isoform




intron 44. Dp140 transcripts have a long (1 kb)







5′ UTR since translation is initiated in exon 







51 (corresponding to aa 2461 of dystrophin). In







addition to the alternative promoter and exon 







1, differential splicing of exons 71-74 and 78







produces at least five Dp140 isoforms. Of 







these, this transcript (Dp140bc) lacks exons 







71-74 and 78 and encodes a protein with a 







unique C-terminus.





Dystrophin
XM_006724469.3
 38
XP_006724532.1
 39



isoform X2










Dystrophin
XM_011545467.1
 40
XP_011543769.1
 41



isoform X5










Dystrophin
XM_006724473.2
 42
XP_006724536.1
 43



isoform X6










Dystrophin
XM_006724475.2
 44
XP_006724538.1
 45



isoform X8










Dystrophin
XM_017029328.1
 46
XP_016884817.1
 47



isoform X4










Dystrophin
XM_006724468.2
 48
XP_006724531.1
 49



isoform X1










Dystrophin
XM_017029331.1
 50
XP_016884820.1
 51



isoform







X13










Dystrophin
XM_006724470.3
 52
XP_006724533.1
 53



isoform X3










Dystrophin
XM_006724474.3
 54
XP_006724537.1
 55



isoform X7










Dystrophin
XM_011545468.2
 56
XP_011543770.1
 57



isoform X9










Dystrophin
XM_017029330.1
 58
XP_016884819.1
 59



isoform







X11










Dystrophin
XM_017029329.1
865
XP_016884818.1
866



isoform







X10










Dystrophin
XM_011545469.1
867
XP_011543771.1
868



isoform







X12









In embodiments, the guide RNA targets a mutant DMD exon. In some embodiments, the mutant exon is exon 23 or 51. In some embodiments, the guide RNA targets at least one of exons 1, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 of the dystrophin gene. In embodiments, the guide RNA targets at least one of introns 44, 45, 50, 51, 52, 53, 54, or 55 of the dystrophin gene. In preferred embodiments, the guide RNAs are designed to induce skipping of exon 51 or exon 23. In embodiments, the gRNA is targeted to a splice acceptor site of exon 51 or exon 23.


Suitable gRNAs and genomic target sequences for use in various compositions and methods disclosed herein are provided as SEQ ID NOs: 60-705, 712-862, and 947-2377.


In some embodiments, the gRNA or gRNA target site has a sequence of any one of the gRNAs or gRNA target sites shown in Tables 5-19.


In some embodiments, gRNAs of the disclosure comprise a sequence that is complementary to a target sequence within a coding sequence or a non-coding sequence corresponding to the DMD gene, and, therefore, hybridize to the target sequence. In some embodiments, gRNAs for Cpf1 comprise a single crRNA containing a direct repeat scaffold sequence followed by 24 nucleotides of guide sequence. In some embodiments, a “guide” sequence of the crRNA comprises a sequence of the gRNA that is complementary to a target sequence. In some embodiments, crRNA of the disclosure comprises a sequence of the gRNA that is not complementary to a target sequence. “Scaffold” sequences of the disclosure link the gRNA to the Cpf1 polypeptide. “Scaffold” sequences of the disclosure are not equivalent to a tracrRNA sequence of a gRNA-Cas9 construct.


In some embodiments, a nucleic acid may comprise one or more sequences encoding a gRNA. In some embodiments, a nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sequences encoding a gRNA. In some embodiments, all of the sequences encode the same gRNA. In some embodiments, all of the sequences encode different gRNAs. In some embodiments, at least 2 of the sequences encode the same gRNA, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encode the same gRNA.


Nucleases

Cas Nucleases.


CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.


Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (˜30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype E coli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.


Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. One or both sites may be deactivated while preserving Cas9's ability to locate its target DNA. Jinek et al. (2012) combined tracrRNA and spacer RNA into a “single-guide RNA” molecule that, mixed with Cas9, can find and cut the correct DNA targets and such synthetic guide RNAs are used for gene editing.


Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. It has been shown that coinjection of Cas9 mRNA and sgRNAs into the germline (zygotes) can be used to generate mice with mutations. Delivery of Cas9 DNA sequences also is contemplated.


The CRISPR/Cas systems are separated into three classes. Class 1 uses several Cas proteins together with the CRISPR RNAs (crRNA) to build a functional endonuclease. Class 2 CRISPR systems use a single Cas protein with a crRNA. Cpf1 has been recently identified as a Class II, Type V CRISPR/Cas system containing a 1,300 amino acid protein. See also U.S. Patent Publication 2014/0068797, which is incorporated by reference in its entirety.


In some embodiments, the compositions of the disclosure include a small version of a Cas9 from the bacterium Staphylococcus aureus (UniProt Accession No. J7RUA5). The small version of the Cas9 provides advantages over wildtype or full length Cas9. In some embodiments the Cas9 is a Streptococcus pyogenes (spCas9).


Cpf1 Nucleases.


Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technology which shares some similarities with the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations.


Cpf1 appears in many bacterial species. The ultimate Cpf1 endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it.


In embodiments, the Cpf1 is a Cpf1 enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6; SEQ ID NO: 870), having the sequence set forth below:











   1
MTQFEGFTNL YQVSKTLRFE LIPQGKTLKH IQEQGFIEED KARNDHYKEL KPIIDRIYKT






  61
YADQCLQLVQ LDWENLSAAI DSYRKEKTEE TRNALIEEQA TYRNAIHDYF IGRTDNLTDA





 121
INKRHAEIYK GLFKAELFNG KVLKQLGTVT TTEHENALLR SFDKFTTYFS GFYENRKNVF





 181
SAEDISTAIP HRIVQDNFPK FKENCHIFTR LITAVPSLRE HFENVKKAIG IFVSTSIEEV





 241
FSFPFYNQLL TQTQIDLYNQ LLGGISREAG TEKIKGLNEV LNLAIQKNDE TAHIIASLPH





 301
RFIPLFKQIL SDRNTLSFIL EEFKSDEEVI QSFCKYKTLL RNENVLETAE ALFNELNSID





 361
LTHIFISHKK LETISSALCD HWDTLRNALY ERRISELTGK ITKSAKEKVQ RSLKHEDINL





 421
QEIISAAGKE LSEAFKQKTS EILSHAHAAL DQPLPTTLKK QEEKEILKSQ LDSLLGLYHL





 481
LDWFAVDESN EVDPEFSARL TGIKLEMEPS LSFYNKARNY ATKKPYSVEK FKLNFQMPTL





 541
ASGWDVNKEK NNGAILFVKN GLYYLGIMPK QKGRYKALSF EPTEKTSEGF DKMYYDYFPD





 601
AAKMIPKCST QLKAVTAHFQ THTTPILLSN NFIEPLEITK EIYDLNNPEK EPKKFQTAYA





 661
KKTGDQKGYR EALCKWIDFT RDFLSKYTKT TSIDLSSLRP SSQYKDLGEY YAELNPLLYH





 721
ISFQRIAEKE IMDAVETGKL YLFQIYNKDF AKGHHGKPNL HTLYWTGLFS PENLAKTSIK





 781
LNGQAELFYR PKSRMKRMAH RLGEKMLNKK LKDQKTPIPD TLYQELYDYV NHRLSHDLSD





 841
EARALLPNVI TKEVSHEIIK DRRFTSDKFF FHVPITLNYQ AANSPSKFNQ RVNAYLKEHP





 901
ETPIIGIDRG ERNLIYITVI DSTGKILEQR SLNTIQQFDY QKKLDNREKE RVAARQAWSV





 961
VGTIKDLKQG YLSQVIHEIV DLMIHYQAVV VLENLNFGFK SKRTGIAEKA VYQQFEKMLI





1021
DKLNCLVLKD YPAEKVGGVL NPYQLTDQFT SFAKMGTQSG FLFYVPAPYT SKIDPLTGFV





1081
DPFVWKTIKN HESRKHFLEG FDFLHYDVKT GDFILHFKMN RNLSFQRGLP GFMPAWDIVF





1141
EKNETQFDAK GTPFIAGKRI VPVIENHRFT GRYRDLYPAN ELIALLEEKG IVFRDGSNIL





1201
PKLLENDDSH AIDTMVALIR SVLQMRNSNA ATGEDYINSP VRDLNGVCFD SRFQNPEWPM





1261
DADANGAYHI ALKGQLLLNH LKESKDLKLQ NGISNQDWLA YIQELRN






In some embodiments, the Cpf1 is a Cpf1 enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3; SEQ ID NO: 871), having the sequence set forth below:











   1
AASKLEKFTN CYSLSKTLRF KAIPVGKTQE NIDNKRLLVE DEKRAEDYKG VKKLLDRYYL






  61
SFINDVLHSI KLKNLNNYIS LFRKKTRTEK ENKELENLEI NLRKEIAKAF KGAAGYKSLF





 121
KKDIIETILP EAADDKDEIA LVNSFNGFTT AFTGFFDNRE NMFSEEAKST SIAFRCINEN





 181
LTRYISNMDI FEKVDAIFDK HEVQEIKEKI LNSDYDVEDF FEGEFFNFVL TQEGIDVYNA





 241
IIGGFVTESG EKIKGLNEYI NLYNAKTKQA LPKFKPLYKQ VLSDRESLSF YGEGYTSDEE





 301
VLEVFRNTLN KNSEIFSSIK KLEKLFKNFD EYSSAGIFVK NGPAISTISK DIFGEWNLIR





 361
DKWNAEYDDI HLKKKAVVTE KYEDDRRKSF KKIGSFSLEQ LQEYADADLS VVEKLKEIII





 421
QKVDEIYKVY GSSEKLFDAD FVLEKSLKKN DAVVAIMKDL LDSVKSFENY IKAFFGEGKE





 481
TNRDESFYGD FVLAYDILLK VDHIYDAIRN YVTQKPYSKD KFKLYFQNPQ FMGGWDKDKE





 541
TDYRATILRY GSKYYLAIMD KKYAKCLQKI DKDDVNGNYE KINYKLLPGP NKMLPKVFFS





 601
KKWMAYYNPS EDIQKIYKNG TFKKGDMFNL NDCHKLIDFF KDSISRYPKW SNAYDFNFSE





 661
TEKYKDIAGF YREVEEQGYK VSFESASKKE VDKLVEEGKL YMFQIYNKDF SDKSHGTPNL





 721
HTMYFKLLFD ENNHGQIRLS GGAELFMRRA SLKKEELVVH PANSPIANKN PDNPKKTTTL





 781
SYDVYKDKRF SEDQYELHIP IAINKCPKNI FKINTEVRVL LKHDDNPYVI GIDRGERNLL





 841
YIVVVDGKGN IVEQYSLNEI INNFNGIRIK TDYHSLLDKK EKERFEARQN WTSIENIKEL





 901
KAGYISQVVH KICELVEKYD AVIALEDLNS GFKNSRVKVE KQVYQKFEKM LIDKLNYMVD





 961
KKSNPCATGG ALKGYQITNK FESFKSMSTQ NGFIFYIPAW LTSKIDPSTG FVNLLKTKYT





1021
SIADSKKFIS SFDRIMYVPE EDLFEFALDY KNFSRTDADY IKKWKLYSYG NRIRIFAAAK





1081
KNNVFAWEEV CLTSAYKELF NKYGINYQQG DIRALLCEQS DKAFYSSFMA LMSLMLQMRN





1141
SITGRTDVDF LISPVKNSDG IFYDSRNYEA QENAILPKNA DANGAYNIAR KVLWAIGQFK





1201
KAEDEKLDKV KIAISNKEWL EYAQTSVK






In some embodiments, the Cpf1 is codon optimized for expression in mammalian cells. In some embodiments, the Cpf1 is codon optimized for expression in human cells or mouse cells.


The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9.


Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2 and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the abundance of Cpf1-family proteins in many bacterial species.


Functional Cpf1 does not require a tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (approximately half as many nucleotides as Cas9).


The Cpf1-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5′-YTN-3′ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5′-TTN-3′, in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double-stranded break of 4 or 5 nucleotides overhang.


The CRISPR/Cpf1 system consist of a Cpf1 enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR/Cpf1 systems activity has three stages:

    • Adaptation, during which Cas1 and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array;
    • Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein; and
    • Interference, in which the Cpf1 is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence.


Cas9 Versus Cpf1.


Cas9 requires two RNA molecules to cut DNA while Cpf1 needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind ‘blunt’ ends. Cpf1 leaves one strand longer than the other, creating ‘sticky’ ends that are easier to work with. Cpf1 appears to be more able to insert new sequences at the cut site, compared to Cas9. Although the CRISPR/Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in. Cpf1 lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB.


In summary, important differences between Cpf1 and Cas9 systems are that Cpf1 recognizes different PAMs, enabling new targeting possibilities, creates 4-5 nt long sticky ends, instead of blunt ends produced by Cas9, enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR, and cuts target DNA further away from PAM, further away from the Cas9 cutting site, enabling new possibilities for cleaving the DNA.









TABLE 2







Differences between Cas9 and Cpf1









Feature
Cas9
Cpf1





Structure
Two RNA required
One RNA required



(Or 1 fusion transcript



(crRNA + tracrRNA = gRNA))


Cutting
Blunt end cuts
Staggered end cuts


mechanism


Cutting site
Proximal to recognition site
Distal from




recognition site


Target sites
G-rich PAM
T-rich PAM









Other Nucleases.


In some embodiments, the nuclease is a Cas9 or a Cpf1 nuclease. In addition to Cas9 nucleases and Cpf1 nucleases, other nucleases may be used in the compositions and methods of the disclosure. For example, in some embodiments, the nuclease is a Type II, Type V-A, Type V-B, Type V-C, Type V-U, Type VI-B nuclease. In some embodiments, the nuclease is a Cas9, Cas12a, Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), or Cas13b nuclease. In some embodiments, the nuclease is a TAL nuclease, a meganuclease, or a zinc-finger nuclease.


CRISPR-Mediated Gene Editing.


The first step in editing the DMD gene using CRISPR/Cpf1 or CRISPR/Cas9 (or another nuclease) is to identify the genomic target sequence. The genomic target for the gRNAs of the disclosure can be any approximately 24 nucleotide DNA sequence, provided that the sequence is unique compared to the rest of the genome. In some embodiments, the genomic target sequence corresponds to a sequence within exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence is a 5′ or 3′ splice site of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence corresponds to a sequence within an intron immediately upstream or downstream of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. Exemplary genomic target sequences can be found in Tables 2, 6, 8, 10, 12, 14 and 19.


The next step in editing the DMD gene is to identify all Protospacer Adjacent Motif (PAM) sequences within the genetic region to be targeted. The target sequence must be immediately upstream of a PAM. Once all possible PAM sequences and putative target sites have been identified, the next step is to choose which site is likely to result in the most efficient on-target cleavage. The gRNA targeting sequence needs to match the target sequence, and the gRNA targeting sequence must not match additional sites within the genome. In preferred embodiments, the gRNA targeting sequence has perfect homology to the target with no homology elsewhere in the genome. In some embodiments, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called “off-targets” and should be considered when designing a gRNA. In general, off-target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity. In addition to “off-target activity”, factors that maximize cleavage of the desired target sequence (“on-target activity”) must be considered. It is known to those of skill in the art that two gRNA targeting sequences, each having 100% homology to the target DNA may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. Close examination of predicted on-target and off-target activity of each potential gRNA targeting sequence is necessary to design the best gRNA. Several gRNA design programs have been developed that are capable of locating potential PAM and target sequences and ranking the associated gRNAs based on their predicted on-target and off-target activity (e.g. CRISPRdirect, available at www.crispr.dbcls.jp).


The next step is to synthesize and clone desired gRNAs. Targeting oligos can be synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector that is chosen. The gRNAs for Cpf1 are notably simpler than the gRNAs for Cas9, and only consist of a single crRNA containing direct repeat scaffold sequence followed by approximately 24 nucleotides of guide sequence.


Each gRNA should then be validated in one or more target cell lines. For example, after the Cas9 or Cpf1 and the gRNA are delivered to the cell, the genomic target region may be amplified using PCR and sequenced according to methods known to those of skill in the art.


In some embodiments, gene editing may be performed in vitro or ex vivo. In some embodiments, cells are contacted in vitro or ex vivo with a Cas9 or a Cpf1 and a gRNA that targets a dystrophin splice site. In some embodiments, the cells are contacted with one or more nucleic acids encoding the Cas9 or Cpf1 and the guide RNA. In some embodiments, the one or more nucleic acids are introduced into the cells using, for example, lipofection or electroporation. Gene editing may also be performed in zygotes. In embodiments, zygotes may be injected with one or more nucleic acids encoding Cas9 or Cpf1 and a gRNA that targets a dystrophin splice site. The zygotes may subsequently be injected into a host.


In embodiments, the Cas9 or Cpf1 is provided on a vector. In embodiments, the vector contains a Cas9 derived from S. pyogenes (SpCas9, SEQ ID NO. 872). In embodiments, the vector contains a Cas9 derived from S. aureus (SaCas9, SEQ ID NO. 873). In embodiments, the vector contains a Cpf1 sequence derived from a Lachnospiraceae bacterium. See, for example, Uniprot Accession No. A0A182DWE3; SEQ ID NO. 871. In embodiments, the vector contains a Cpf1 sequence derived from an Acidaminococcus bacterium. See, for example, Uniprot Accession No. U2UMQ6; SEQ ID NO. 870. In some embodiments, the Cas9 or Cpf1 sequence is codon optimized for expression in human cells or mouse cells. In some embodiments, the vector further contains a sequence encoding a fluorescent protein, such as GFP, which allows Cas9 or Cpf1-expressing cells to be sorted using fluorescence activated cell sorting (FACS). In some embodiments, the vector is a viral vector such as an adeno-associated viral vector.


In embodiments, the gRNA is provided on a vector. In some embodiments, the vector is a viral vector such as an adeno-associated viral vector. In embodiments, the Cas9 or Cpf1 and the guide RNA are provided on the same vector. In embodiments, the Cas9 or Cpf1 and the guide RNA are provided on different vectors.


In some embodiments, the cells are additionally contacted with a single-stranded DMD oligonucleotide to effect homology directed repair. In some embodiments, small INDELs restore the protein reading frame of dystrophin (“reframing” strategy). When the reframing strategy is used, the cells may be contacted with a single gRNA. In embodiments, a splice donor or splice acceptor site is disrupted, which results in exon skipping and restoration of the protein reading frame (“exon skipping” strategy). When the exon skipping strategy is used, the cells may be contacted with two or more gRNAs.


Efficiency of in vitro or ex vivo Cas9 or Cpf1-mediated DNA cleavage may be assessed using techniques known to those of skill in the art, such as the T7 E1 assay. Restoration of DMD expression may be confirmed using techniques known to those of skill in the art, such as RT-PCR, western blotting, and immunocytochemistry.


In some embodiments, in vitro or ex vivo gene editing is performed in a muscle or satellite cell. In some embodiments, gene editing is performed in iPSC or iCM cells. In embodiments, the iPSC cells are differentiated after gene editing. For example, the iPSC cells may be differentiated into a muscle cell or a satellite cell after editing. In embodiments, the iPSC cells are differentiated into cardiac muscle cells, skeletal muscle cells, or smooth muscle cells. In embodiments, the iPSC cells are differentiated into cardiomyocytes. iPSC cells may be induced to differentiate according to methods known to those of skill in the art.


In some embodiments, contacting the cell with the Cas9 or the Cpf1 and the gRNA restores dystrophin expression. In embodiments, cells which have been edited in vitro or ex vivo, or cells derived therefrom, show levels of dystrophin protein that is comparable to wildtype cells. In embodiments, the edited cells, or cells derived therefrom, express dystrophin at a level that is 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between of wildtype dystrophin expression levels. In embodiments, the cells which have been edited in vitro or ex vivo, or cells derived therefrom, have a mitochondrial number that is comparable to that of wildtype cells. In embodiments the edited cells, or cells derived therefrom, have 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between as many mitochondria as wildtype cells. In embodiments, the edited cells, or cells derived therefrom, show an increase in oxygen consumption rate (OCR) compared to non-edited cells at baseline.


Nucleic Acid Expression Vectors.


As discussed above, in certain embodiments, expression cassettes are employed to express a transcription factor product, either for subsequent purification and delivery to a cell/subject, or for use directly in a genetic-based delivery approach. Provided herein are expression vectors which contain one or more nucleic acids encoding Cas9 or Cpf1 and at least one DMD guide RNA that targets a dystrophin splice site. In some embodiments, a nucleic acid encoding Cas9 or Cpf1 and a nucleic acid encoding at least one guide RNA are provided on the same vector. In further embodiments, a nucleic acid encoding Cas9 or Cpf1 and a nucleic acid encoding least one guide RNA are provided on separate vectors.


Expression requires that appropriate signals be provided in the vectors, and include various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.


Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.


Regulatory Elements.


The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.


At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.


RNA Polymerase and Pol III Promoters.


In eukaryotes, RNA polymerase III (also called Pol III) transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small


RNAs. The genes transcribed by RNA Pol III fall in the category of “housekeeping” genes whose expression is required in all cell types and most environmental conditions. Therefore, the regulation of Pol III transcription is primarily tied to the regulation of cell growth and the cell cycle, thus requiring fewer regulatory proteins than RNA polymerase II. Under stress conditions however, the protein Maf1 represses Pol III activity.


In the process of transcription (by any polymerase) there are three main stages: (i) initiation, requiring construction of the RNA polymerase complex on the gene's promoter; (ii) elongation, the synthesis of the RNA transcript; and (iii) termination, the finishing of RNA transcription and disassembly of the RNA polymerase complex.


Promoters under the control of RNA Pol III include those for ribosomal 5S rRNA, tRNA and few other small RNAs such as U6 spliceosomal RNA, RNase P and RNase MRP RNA, 7SL RNA (the RNA component of the signal recognition particles), Vault RNAs, Y RNA, SINEs (short interspersed repetitive elements), 7SK RNA, two microRNAs, several small nucleolar RNAs and several few regulatory antisense RNAs.


Additional Promoters and Elements

In some embodiments, the Cas9 or Cpf1 constructs of the disclosure are expressed by a muscle-cell specific promoter. This muscle-cell specific promoter may be constitutively active or may be an inducible promoter.


Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.


In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.


Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.


Below is a list of promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.


The promoter and/or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), α1-antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), Duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus.


In some embodiments, inducible elements may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor virus), β-interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, α-2-macroglobulin, vimentin, MHC class I gene H-2κb, HSP70, proliferin, tumor necrosis factor, and/or thyroid stimulating hormone a gene. In some embodiments, the inducer is phorbol ester (TFA), heavy metals, glucocorticoids, poly(rI)x, poly(rc), E1A, phorbol ester (TPA), interferon, Newcastle Disease Virus, A23187, IL-6, serum, interferon, SV40 large T antigen, PMA, and/or thyroid hormone. Any of the inducible elements described herein may be used with any of the inducers described herein.


Of particular interest are muscle specific promoters. These include the myosin light chain-2 promoter, the α-actin promoter, the troponin 1 promoter; the Na+/Ca2+ exchanger promoter, the dystrophin promoter, the α7 integrin promoter, the brain natriuretic peptide promoter and the αB-crystallin/small heat shock protein promoter, α-myosin heavy chain promoter and the ANF promoter. In some embodiments, the muscle specific promoter is the CK8 promoter. The CK8 promoter has the following sequence (SEQ ID NO. 874):











  1
CTAGACTAGC ATGCTGCCCA TGTAAGGAGG CAAGGCCTGG GGACACCCGA GATGCCTGGT






 61
TATAATTAAC CCAGACATGT GGCTGCCCCC CCCCCCCCAA CACCTGCTGC CTCTAAAAAT





121
AACCCTGCAT GCCATGTTCC CGGCGAAGGG CCAGCTGTCC CCCGCCAGCT AGACTCAGCA





181
CTTAGTTTAG GAACCAGTGA GCAAGTCAGC CCTTGGGGCA GCCCATACAA GGCCATGGGG





241
CTGGGCAAGC TGCACGCCTG GGTCCGGGGT GGGCACGGTG CCCGGGCAAC GAGCTGAAAG





301
CTCATCTGCT CTCAGGGGCC CCTCCCTGGG GACAGCCCCT CCTGGCTAGT CACACCCTGT





361
AGGCTCCTCT ATATAACCCA GGGGCACAGG GGCTGCCCTC ATTCTACCAC CACCTCCACA





421
GCACAGACAG ACACTCAGGA GCCAGCCAGC






In some embodiments, the muscle-cell cell specific promoter is a variant of the CK8 promoter, called CK8e. The CK8e promoter has the following sequence (SEQ ID NO. 875):











  1
TGCCCATGTA AGGAGGCAAG GCCTGGGGAC ACCCGAGATG CCTGGTTATA ATTAACCCAG






 61
ACATGTGGCT GCCCCCCCCC CCCCAACACC TGCTGCCTCT AAAAATAACC CTGCATGCCA





121
TGTTCCCGGC GAAGGGCCAG CTGTCCCCCG CCAGCTAGAC TCAGCACTTA GTTTAGGAAC





181
CAGTGAGCAA GTCAGCCCTT GGGGCAGCCC ATACAAGGCC ATGGGGCTGG GCAAGCTGCA





241
CGCCTGGGTC CGGGGTGGGC ACGGTGCCCG GGCAACGAGC TGAAAGCTCA TCTGCTCTCA





301
GGGGCCCCTC CCTGGGGACA GCCCCTCCTG GCTAGTCACA CCCTGTAGGC TCCTCTATAT





361
AACCCAGGGG CACAGGGGCT GCCCTCATTC TACCACCACC TCCACAGCAC AGACAGACAC





421
TCAGGAGCCA GCCAGC






Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.


Therapeutic Compositions
AAV-Cas9 Vectors

In some embodiments, a Cas9 may be packaged into an AAV vector. In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV or any combination thereof.


Exemplary AAV-Cas9 vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the Cas9 sequence. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype.


In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110±10 base pairs. In some embodiments, the ITRs have a length of 120±10 base pairs. In some embodiments, the ITRs have a length of 130±10 base pairs. In some embodiments, the ITRs have a length of 140±10 base pairs. In some embodiments, the ITRs have a length of 150±10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs.


In some embodiments, the AAV-Cas9 vector may contain one or more nuclear localization signals (NLS). In some embodiments, the AAV-Cas9 vector contains 1, 2, 3, 4, or 5 nuclear localization signals. Exemplary NLS include the c-myc NLS (SEQ ID NO: 884), the SV40 NLS (SEQ ID NO: 885), the hnRNPAI M9 NLS (SEQ ID NO: 886), the nucleoplasmin NLS (SEQ ID NO: 887), the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 888) of the IBB domain from importin-alpha, the sequences VSRKRPRP (SEQ ID NO: 889) and PPKKARED (SEQ ID NO: 890) of the myoma T protein, the sequence PQPKKKPL (SEQ ID NO: 891) of human p53, the sequence SALIKKKKMAP (SEQ ID NO: 892) of mouse c-abl IV, the sequences DRLRR (SEQ ID NO: 893) and KQKKRK (SEQ ID NO: 894) of the influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO: 895) of the Hepatitis virus delta antigen and the sequence REKKKFLKRR (SEQ ID NO: 896) of the mouse Mx1 protein. Further acceptable nuclear localization signals include bipartite nuclear localization sequences such as the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 897) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 898) of the steroid hormone receptors (human) glucocorticoid.


In some embodiments, the AAV-Cas9 vector may comprise additional elements to facilitate packaging of the vector and expression of the Cas9. In some embodiments, the AAV-Cas9 vector may comprise a polyA sequence. In some embodiments, the polyA sequence may be a mini-polyA sequence. In some embodiments, the AAV-CAs9 vector may comprise a transposable element. In some embodiments, the AAV-Cas9 vector may comprise a regulator element. In some embodiments, the regulator element is an activator or a repressor.


In some embodiments, the AAV-Cas9 may contain one or more promoters. In some embodiments, the one or more promoters drive expression of the Cas9. In some embodiments, the one or more promoters are muscle-specific promoters. Exemplary muscle-specific promoters include myosin light chain-2 promoter, the a-actin promoter, the troponin 1 promoter, the Na+/Ca2+ exchanger promoter, the dystrophin promoter, the α7 integrin promoter, the brain natriuretic peptide promoter, the αB-crystallin/small heat shock protein promoter, α-myosin heavy chain promoter, the ANF promoter, the CK8 promoter and the CK8e promoter.


In some embodiments, the AAV-Cas9 vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a baculovirus expression system.


AAV-sgRNA Vectors

In some embodiments, at least a first sequence encoding a gRNA and a second sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, and a fourth sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, a fourth sequence encoding a gRNA, and a fifth sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, a plurality of sequences encoding a gRNA are packaged into an AAV vector. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sequences encoding a gRNA may be packaged into an AAV vector. In some embodiments, each sequence encoding a gRNA is different. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encoding a gRNA are the same. In some embodiments, all of the sequence encoding a gRNA are the same.


In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV or any combination thereof.


Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV or any combination thereof. In some embodiments, the ITRs are isolated or derived from an AAV vector of a first serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a second serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the first serotype is AAV2 and the second serotype is AAV9.


In some embodiments, a first ITR is isolated or derived from an AAV vector of a first serotype, a second ITR is isolated or derived from an AAV vector of a second serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a third serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype, the second serotype, and the third serotype are the same. In some embodiments, the first serotype, the second serotype, and the third serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV9. Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110±10 base pairs. In some embodiments, the ITRs have a length of 120±10 base pairs. In some embodiments, the ITRs have a length of 130±10 base pairs. In some embodiments, the ITRs have a length of 140±10 base pairs. In some embodiments, the ITRs have a length of 150±10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs.


In some embodiments, the AAV-sgRNA vector may comprise additional elements to facilitate packaging of the vector and expression of the sgRNA. In some embodiments, the AAV-sgRNA vector may comprise a transposable element. In some embodiments, the AAV-sgRNA vector may comprise a regulatory element. In some embodiments, the regulatory element comprises an activator or a repressor. In some embodiments, the AAV-sgRNA sequence may comprise a non-functional or “stuffer” sequence. Exemplary stuffer sequences of the disclosure may have some (a non-zero percentage of) identity or homology to a genomic sequence of a mammal (including a human). Alternatively, exemplary stuffer sequences of the disclosure may have no identity or homology to a genomic sequence of a mammal (including a human). Exemplary stuffer sequences of the disclosure may comprise or consist of naturally occurring non-coding sequences or sequences that are neither transcribed nor translated following administration of the AAV vector to a subject.


In some embodiments, the AAV-sgRNA vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-sgRNA vector may be optimized for expression in human cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in a baculovirus expression system.


In some embodiments, the AAV-sgRNA vector comprises at least one promoter. In some embodiments, the AAV-sgRNA vector comprises at least two promoters. In some embodiments, the AAV-sgRNA vector comprises at least three promoters. In some embodiments, the AAV-sgRNA vector comprises at least four promoters. In some embodiments, the AAV-sgRNA vector comprises at least five promoters. Exemplary promoters include, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), α1-antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus. Further exemplary promoters include the U6 promoter, the H1 promoter, and the 7SK promoter.


In some embodiments, the sequence encoding the gRNA or the genomic target sequence comprises a sequence selected from SEQ ID NOs. 60-705, 712-862, and 947-2377.


Pharmaceutical Compositions and Delivery Methods

Also provided herein are compositions comprising one or more vectors and/or nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.


For clinical applications, pharmaceutical compositions are prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.


Appropriate salts and buffers are used to render drugs, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is not incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions may be used. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.


In some embodiments, the active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into muscle tissue. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.


The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.


The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.


Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.


In some embodiments, the compositions of the present disclosure are formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine) and the like.


Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.


In some embodiments, the Cas9 or Cpf1 and gRNAs described herein may be delivered to the patient using adoptive cell transfer (ACT). In adoptive cell transfer, one or more expression constructs are provided ex vivo to cells which have originated from the patient (autologous) or from one or more individual(s) other than the patient (allogeneic). The cells are subsequently introduced or reintroduced into the patient. Thus, in some embodiments, one or more nucleic acids encoding Cas9 or Cpf1 and a guide RNA that targets a dystrophin splice site are provided to a cell ex vivo before the cell is introduced or reintroduced to a patient.


Cells and Cell Compositions

Also provided is a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell (e.g., a cardiomyocyte) is derived from an iPS cell.


Also provided is a cell comprising a composition comprising one or more vectors of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell (e.g., a cardiomyocyte) is derived from an iPS cell.


Also provided is a cell produced by one or more methods of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell (e.g., a cardiomyocyte) is derived from an iPS cell.


Also provided is a composition comprising a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.


Therapeutic Methods and Uses

The disclosure also provides methods for editing a dystrophin gene, such as a mutant dystrophin gene, in a cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the cell (e.g., a cardiomyocyte) is derived from an iPS cell.


In some embodiments, the disclosure provides a method for editing a mutant dystrophin gene in a cardiomyocyte, the method comprising contacting the cardiomyocyte with a Cas9 nuclease, or a sequence encoding a Cas9 nuclease, and a gRNA, or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene. The mutant dystrophin gene may comprise one or more mutations, such as a point mutation (e.g., a pseudo-exon mutation), a deletion, and/or a duplication mutation. A deletion may be a deletion of at least 20, at least 50, at least 100, at least 500, at least 1000, at least 3000 nucleotides, at least 5000 nucleotides or at least 10,000 nucleotides. In some embodiments, the deletion comprises a deletion of one or more exons, one or more introns, or at least a portion of one intron and one exon.


In some embodiments, the disclosure provides a method for treating or preventing Duchenne Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a Cas9 nuclease, or a sequence encoding a Cas9 nuclease, and a gRNA, or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene, wherein the administering restores dystrophin expression in at least 10% of the subject's cardiomyocytes. In some embodiments, the administering restores dystrophin expression in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the subject's cardiomyocytes. The average human heart has approximately 2 to 3 billion cardiomyocytes. Accordingly, in some embodiments, the administering restores dystrophin expression in at least 2×108, at least 3×108, at least 4×108, at least 5×108, at least 6×108, at least 7×108, at least 8×108, at least 9×108, at least 10×108, at least 11×108, at least 12×108, at least 13×108, at least 14×108, at least 15×108, at least 16×108, at least 17×108, at least 18×108, at least 19×108, at least 20×108, at least 21×108, at least 22×108, at least 23×108, at least 24×108, at least 25×108, at least 26×108, at least 27×108, at least 28×108, at least 29×108, at least 30×108 of the subject's cardiomyocytes. In some embodiments, the subject suffers from dilated cardiomyopathy. In some embodiments, the administering at least partially rescues cardiac contractility, or completely rescues cardiac contractility.


In some embodiments, a method for treating or preventing Duchenne Muscular Dystrophy (DMD) in a subject in need thereof, is provided, the method comprising contacting an induced pluripotent stem cell (iPSC) with a Cas9 nuclease or a sequence encoding a Cas9 nuclease, and a gRNA or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene; differentiating the iPSC into a cardiomyocyte; and administering the cardiomyocyte to the subject. In some embodiments, at least 1×103, at least 1×104, at least 1×105, at least 1×106, at least 1×107 or at least 1×108 cardiomyocytes are administered to the patient.


The gRNA may target, for example a splice donor or splice acceptor site of exon 51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8, or 55 of the cardiomyocyte dystrophin gene. In some embodiments, the gRNA or the genomic targeting sequence has a sequence of any one of SEQ ID NOs. 60-705, 712-862, 947-2377. The cas9 nuclease may be isolated or derived from, for example, a S. pyogenes (spCas9) or a S. aureus cas9 (saCas9).


In some embodiments, a vector comprising the gRNA, or a sequence encoding the gRNA, is contacted with the cardiomyocyte. The vector may be, for example, non-viral vector such as a plasmid or a nanoparticle. In some embodiments, the vector may be a viral vector, such as an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.


In some embodiments, a single vector comprising the Cas9 nuclease, or a sequence encoding the Cas9 nuclease, and the gRNA, or a sequence encoding the gRNA, are contacted with the cardiomyocyte. In other embodiments, a first vector comprising the Cas9 nuclease, or a sequence encoding the Cas9 nuclease, and a second vector comprising the gRNA or a sequence encoding the gRNA, are contacted with the cardiomyocyte. The first and second vector may be the same or may be different. For example, the first vector and the second vector may both be AAVs, or the first vector may be an AAV and the second vector may be a plasmid.


Also provided is a method for correcting a dystrophin defect, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the guide RNA, the Cas9 protein or a nuclease domain thereof, wherein the guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one guide RNA-Cas9 complex, wherein the at least one guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping of a DMD exon and/or reframing. In some embodiments, the at least one guide RNA-Cas9 complex disrupts a dystrophin splice site and induces a reframing of a dystrophin reading frame. In some embodiments, the at least one guide RNA-Cas9 complex disrupts a dystrophin splice site and produces an insertion which restores the dystrophin protein reading frame. In some embodiments, the insertion comprises an insertion of a single adenosine.


Also provided is a method for inducing selective skipping and/or reframing of a DMD exon, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the guide RNA and the Cas9 protein or a nuclease domain thereof, wherein the guide RNA and the second guide RNA form a complex with the Cas9 protein or the nuclease domain thereof to form at least one guide RNA-Cas9 complex, wherein the at least one guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of a DMD exon.


Also provided is a method for inducing a reframing event in the dystrophin reading frame, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the guide RNA and the Cas9 protein or a nuclease domain thereof, wherein the guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one guide RNA-Cas9 complex, wherein the guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of a DMD exon. In some embodiments, the at least one guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of exon 51 of a human DMD gene.


Also provided is a method of treating or preventing muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more compositions of the disclosure. In some embodiments, the composition is administered locally. In some embodiments, the composition is administered directly to a muscle tissue. In some embodiments, the composition is administered by an intramuscular infusion or injection. In some embodiments, the muscle tissue comprises a tibialis anterior tissue, a quadriceps tissue, a soleus tissue, a diaphragm tissue, or a heart tissue. In some embodiments, the composition is administered by an intra-cardiac injection. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered by an intravenous infusion or injection. In some embodiments, following administration of the composition, the subject exhibits normal dystrophin-positive myofibers, and mosaic dystrophin-positive myofibers containing centralized nuclei, or a combination thereof. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of normal dystrophin-positive myofibers when compared to an absence or a level of abundance of normal dystrophin-positive myofibers prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei when compared to an absence or an level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits a decreased serum CK level when compared to a serum CK level prior to administration of the composition. In some embodiment, following administration of the composition, the subject exhibits improved grip strength when compared to a grip strength prior to administration of the composition. In some embodiments, the subject is a neonate, an infant, a child, a young adult, or an adult. In some embodiments, the subject has muscular dystrophy. In some embodiments, the subject is a genetic carrier for muscular dystrophy. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject appears to be asymptomatic and a genetic diagnosis reveals a mutation in one or both copies of a DMD gene that impairs function of the DMD gene product. In some embodiments, the subject presents an early sign or symptom of muscular dystrophy. In some embodiments, the early sign or symptom of muscular dystrophy comprises loss of muscle mass or proximal muscle weakness. In some embodiments, the loss of muscle mass or proximal muscle weakness occurs in one or both leg(s) and/or a pelvis, followed by one or more upper body muscle(s). In some embodiments, the early sign or symptom of muscular dystrophy further comprises pseudohypertrophy, low endurance, difficulty standing, difficulty walking, difficulty ascending a staircase or a combination thereof. In some embodiments, the subject presents a progressive sign or symptom of muscular dystrophy. In some embodiments, the progressive sign or symptom of muscular dystrophy comprises muscle tissue wasting, replacement of muscle tissue with fat, or replacement of muscle tissue with fibrotic tissue. In some embodiments, the subject presents a later sign or symptom of muscular dystrophy. In some embodiments, the later sign or symptom of muscular dystrophy comprises abnormal bone development, curvature of the spine, loss of movement, and paralysis. In some embodiments, the subject presents a neurological sign or symptom of muscular dystrophy. In some embodiments, the neurological sign or symptom of muscular dystrophy comprises intellectual impairment and paralysis. In some embodiments, administration of the composition occurs prior to the subject presenting one or more progressive, later or neurological signs or symptoms of muscular dystrophy. In some embodiments, the subject greater than 18 years old, greater than 25 years old, or greater than 30 years old. In some embodiments, the subject is less than 18 years old, less than 16 years old, less than 12 years old, less than 10 years old, less than 5 years old, or less than 2 years old. Also provided is the use of a therapeutically-effective amount of one or more compositions of the disclosure for treating muscular dystrophy in a subject in need thereof.


Delivery Vectors

There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals.


One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.


The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.


Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5′-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.


Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the E1-deleted virus is incomplete.


Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.


Improved methods for culturing 293 cells and propagating adenovirus are known in the art. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.


The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.


As stated above, the typical vector according to the present disclosure is replication defective and will not have an adenovirus E1 region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, or in the E4 region where a helper cell line or helper virus complements the E4 defect.


Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 109-1012 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.


Adenovirus vectors have been used in eukaryotic gene expression and vaccine development. Animal studies suggested that recombinant adenovirus could be used for gene therapy. Studies in administering recombinant adenovirus to different tissues include trachea instillation, muscle injection, peripheral intravenous injections and stereotactic inoculation into the brain.


The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5′ and 3′ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome.


In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells.


A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.


A different approach to targeting of recombinant retroviruses may be used, in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor are used. The antibodies are coupled via the biotin components by using streptavidin. Using antibodies against major histocompatibility complex class I and class II antigens, it has been demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro.


There are certain limitations to the use of retrovirus vectors in all aspects of the present disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes. Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact-sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (see, for example, Markowitz et al., 1988; Hersdorffer et al., 1990).


Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.


In embodiments, the AAV vector is replication-defective or conditionally replication defective. In embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV or any combination thereof.


In some embodiments, a single viral vector is used to deliver a nucleic acid encoding a Cas9 or a Cpf1 and at least one gRNA to a cell. In some embodiments, Cas9 or Cpf1 is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector.


In some embodiments, a single viral vector is used to deliver a nucleic acid encoding Cas9 or Cpf1 and at least one gRNA to a cell. In some embodiments, Cas9 or Cpf1 is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector. In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. The cell may be a muscle cell, a satellite cell, a mesangioblast, a bone marrow derived cell, a stromal cell or a mesenchymal stem cell. In embodiments, the cell is a cardiac muscle cell, a skeletal muscle cell, or a smooth muscle cell. In embodiments, the cell is a cell in the tibialis anterior, quadriceps, soleus, diaphragm or heart. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or inner cell mass cell (iCM). In further embodiments, the cell is a human iPSC or a human iCM. In some embodiments, human iPSCs or human iCMs of the disclosure may be derived from a cultured stem cell line, an adult stem cell, a placental stem cell, or from another source of adult or embryonic stem cells that does not require the destruction of a human embryo. Delivery to a cell may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.


Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.


Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.


In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.


In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.


In some embodiments, the expression construct is delivered directly to the liver, skin, and/or muscle tissue of a subject. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment.


Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.


In a further embodiment, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes.


Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. A reagent known as Lipofectamine 2000′ is widely used and commercially available.


In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.


Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific.


Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin. A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells.


Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy, affecting around 1 in 5000 boys, which results in muscle degeneration and premature death. The disorder is caused by a mutation in the gene dystrophin (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5).


In humans, dystrophin mRNA contains 79 exons. Dystrophin mRNA is known to be alternatively spliced, resulting in various isoforms. Exemplary dystrophin isoforms are listed in Table 1.


The murine dystrophin protein has the following amino acid sequence (Uniprot Accession No. P11531, SEQ. ID. NO: 869):











   1
MWWVDCYRDV KKTTKWNASK GKHDNSDDGK RDGTGKKKGS TRVHANNVNK ARVKNNVDVN






  61
GSTDVDGNHK TGWNHWVKNV MKTMAGTNSK SWVRSTRNYV NVNTSSWSDG ANAHSHRDDW





 121
NSVVSHSATR HANAKCGKDD VATTYDKKSM YTSVVSAVMR TSSKVTRHHH MHYSTVSAGY





 181
TSSSKRKSYA TAAYVATSDS TSYSHARDKS DSSMTVNDSY TAVSWSADTR AGSNDVVKHA





 241
HGMMDTSHGV GNVGSVGKGK SDAVMNNSRW CRVASMKSKH KVMDNKKDDW TKTRTKKMGD





 301
DKCVHKVDVR VNSTHMVVVV DSSGDHATAA KVGDRWANCR WTDRWVDKWH TCSTWSKDAM





 361
KNTSGKDNMM SSHKSTKDKK KTMKSSNDSA KNKSVTKMWM NARWDNTKKS SASAVTTTST





 421
TTVMTVTMVT TRMVKHAKKR TVDSRKRDVD THSWTRSAVS SAVYRKGNSD KVNAARKAKR





 481
KDASRSAAVM ANGVNASRAS NSRWTCSRVN WYTNTYNMTT TANKTSTTST AKSKCKDVNR





 541
SAKSKKGGMD ADVATNHNHD GVRAKKTDTM RYTMSSRTWS SKSVYSVTYM RGKASSKNGN





 601
YSDTVKMAKK ASCKYSGHWK KSSVSCKHMN KRKNHKTKWM AVDVKWAGDA KKKCRVGDTS





 661
NSVNGGKKSA ASRTRNTWDH CRVYTRKAKA GDKTVSKDSM HWMTAYRDYK TDTAVMKRAK





 721
AKTKVKTTVN SVAHASAAKK TTTNYWCTRN GKCKTVWACW HSYKANKWNV KKTMNVAGTV





 781
SNMHHSNNRA TTDGGVMDNT NSRWRHAVRK KSSAKSHSDK AAYTDKVDAA MAKSDTSHSM





 841
KKHNGKDANR VSDVAKKDVS MKRKANRSKM DVKMHATKSV VSSHCVNYKS SVKSVMVKTG





 901
RVKKTNKDRV TAKHYNGAKV TRKKCKSRKM RKMNVTWAAT DTTKRSAVGM SNDSVAWGKA





 961
TKKKAHKSVT GSKMVGKKTV DKSNSNWAVT SRVWNYKHMT DNTKWHADDS KKKKDKRKAM





1021
NDMRKVDSTR DAAKMANRGD HCRKVVSNRR AASHRKTGKA SKNSDKAGVN KDNKDMSDNG





1081
TVNRGDNRTD RKRKKTKHNA KDRSRRKKAS HWYYKRADDK CDKKASRDRK KDRKKKNAVR





1141
RAGSNGAAMA VTSKRWRSNA RRNAHTHTMV VTTDMDVSYV STYTSHASVD HNTCAKDDKS





1201
KNKDNSGRDH KKKTAASATS MKVKVAVAMD GKHRMYKRGR DRSVKWRHHY DMKVNWNVKK





1261
TNNWHAKYKW YKDGGRAVVR TNATGSSKTD VNKGSSRWHD CKARRKRKNV SRDNVWADNA





1321
TGDKVKARGK NTGGAVVSAR DKKKKTNWKV SRAKGVHKDR DHWSRNYNSA GDKVTVHGKA





1381
DVRSKGHYKK STVKRKDRSW AVNHRRTKDR AGSTTGASAS TVTVTSVVTK TVSKMSSVAA





1441
DNRAWTTDWS DRVKSRVMVG DDNMKKATDR RTAANKNKTS NARTTDRRWD VNRRNMKDST





1501
WAKAVGVRGK DSWKGHTVDA KKTTKAKDRR SVDVANDAKR DYSADDTRKV HMTNNTSWGN





1561
HKRVSAATHR DKSWTATTAN VDASRKKDSR GVRMKWDGTH TDYHNDNGKR SGSDARRDNM





1621
NKWSKKSNRS HASSDWKRHS VWKDDSRAGG DAVKNDHRAK RKTKVMSTTV RTGKYRRANV





1681
TRRKAVNAWD KNRSADWRKD ARAADDKRAV KGSWVGDDSD HKVKARGAKN VNRVNDAHTT





1741
GSYNSTDNTR WRVAVDRVRH AHRDGASHST SVGWRASNKV YYNHTTTCWD HKMTYSADNN





1801
VRSAYRTAMK RRKACDSSAA CDADHNKNDM DNCTTYDRHN NVNVCVDMCN WNVYDTGRTG





1861
RRVSKTGSCK AHDKYRYKVA SSTGCDRRGH DSRGVASGGS NSVRSCANNK AADWMRSMVW





1921
VHRVAAATAK HAKCNCKCGR YRSKHNYDCS CSGRVAKGHK MHYMVYCTTT SGDVRDAKVK





1981
NKRTKRYAKH RMGYVTVGDN MTVTNWVDSA ASSSHDDTHS RHYASRAMNS NGSYNDSSNS





2041
DDHHYCSNDS SRSASSRGRA DNRNAYDRKH HKGSSMMTSS RDAAAKRHKG RARMDHNKSH





2101
RRAAKVNGTT VSSSTSRSDS SMRVVGSTSS MGDSDTSTGV MNNSSSRGRN AGKMRDTM






Dystrophin is an important component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. While both sexes can carry the mutation, females are rarely affected with the skeletal muscle form of the disease.


Mutations vary in nature and frequency. Large genetic deletions are found in about 60-70% of cases, large duplications are found in about 10% of cases, and point mutants or other small changes account for about 15-30% of cases. Bladen et al. (2015), who examined some 7000 mutations, catalogued a total of 5,682 large mutations (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions, while 199 (14%) affected the splice sites. Point mutations totaled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).


DMD Subject Characteristics and Clinical Presentation.


Symptoms usually appear in boys between the ages of 2 and 3 and may be visible in early infancy. Even though symptoms do not appear until early infancy, laboratory testing can identify children who carry the active mutation at birth. Progressive proximal muscle weakness of the legs and pelvis associated with loss of muscle mass is observed first. Eventually this weakness spreads to the arms, neck, and other areas. Early signs may include pseudohypertrophy (enlargement of calf and deltoid muscles), low endurance, and difficulties in standing unaided or inability to ascend staircases. As the condition progresses, muscle tissue experiences wasting and is eventually replaced by fat and fibrotic tissue (fibrosis). By age 10, braces may be required to aid in walking but most patients are wheelchair dependent by age 12. Later symptoms may include abnormal bone development that lead to skeletal deformities, including curvature of the spine. Due to progressive deterioration of muscle, loss of movement occurs, eventually leading to paralysis. Intellectual impairment may or may not be present but if present, does not progressively worsen as the child ages. The average life expectancy for males afflicted with DMD is around 25.


The main symptom of Duchenne muscular dystrophy, a progressive neuromuscular disorder, is muscle weakness associated with muscle wasting with the voluntary muscles being first affected, especially those of the hips, pelvic area, thighs, shoulders, and calves. Muscle weakness also occurs later, in the arms, neck, and other areas. Calves are often enlarged. Symptoms usually appear before age 6 and may appear in early infancy. Other physical symptoms are:

  • 1. Awkward manner of walking, stepping, or running—(patients tend to walk on their forefeet, because of an increased calf muscle tone. Also, toe walking is a compensatory adaptation to knee extensor weakness.)
  • 2. Frequent falls.
  • 3. Fatigue.
  • 4. Difficulty with motor skills (running, hopping, jumping).
  • 5. Lumbar hyperlordosis, possibly leading to shortening of the hip-flexor muscles. This has an effect on overall posture and a manner of walking, stepping, or running.
  • 6. Muscle contractures of Achilles tendon and hamstrings impair functionality because the muscle fibers shorten and fibrose in connective tissue.
  • 7. Progressive difficulty walking.
  • 8. Muscle fiber deformities.
  • 9. Pseudohypertrophy (enlarging) of tongue and calf muscles. The muscle tissue is eventually replaced by fat and connective tissue, hence the term pseudohypertrophy.
  • 10. Higher risk of neurobehavioral disorders (e.g., ADHD), learning disorders (dyslexia), and non-progressive weaknesses in specific cognitive skills (in particular short-term verbal memory), which are believed to be the result of absent or dysfunctional dystrophin in the brain.
  • 11. Eventual loss of ability to walk (usually by the age of 12).
  • 12. Skeletal deformities (including scoliosis in some cases).
  • 13. Trouble getting up from lying or sitting position.


The condition can often be observed clinically from the moment the patient takes his first steps, and the ability to walk usually completely disintegrates between the time the boy is 9 to 12 years of age. Most men affected with DMD become essentially “paralyzed from the neck down” by the age of 21. Muscle wasting begins in the legs and pelvis, then progresses to the muscles of the shoulders and neck, followed by loss of arm muscles and respiratory muscles. Calf muscle enlargement (pseudohypertrophy) is quite obvious. Cardiomyopathy particularly (dilated cardiomyopathy) is common, but the development of congestive heart failure or arrhythmia (irregular heartbeat) is only occasional.


A positive Gowers' sign reflects the more severe impairment of the lower extremities muscles. The child helps himself to get up with upper extremities: first by rising to stand on his arms and knees, and then “walking” his hands up his legs to stand upright. Affected children usually tire more easily and have less overall strength than their peers. Creatine kinase (CPK-MM) levels in the bloodstream are extremely high. An electromyography (EMG) shows that weakness is caused by destruction of muscle tissue rather than by damage to nerves. Genetic testing can reveal genetic errors in the Xp21 gene. A muscle biopsy (immunohistochemistry or immunoblotting) or genetic test (blood test) confirms the absence of dystrophin, although improvements in genetic testing often make this unnecessary.


DMD patients may suffer from:

  • 1. Abnormal heart muscle (cardiomyopathy).
  • 2. Congestive heart failure or irregular heart rhythm (arrhythmia).
  • 3. Deformities of the chest and back (scoliosis).
  • 4. Enlarged muscles of the calves, buttocks, and shoulders (around age 4 or 5). These muscles are eventually replaced by fat and connective tissue (pseudohypertrophy).
  • 5. Loss of muscle mass (atrophy).
  • 6. Muscle contractures in the heels, legs.
  • 7. Muscle deformities.
  • 8. Respiratory disorders, including pneumonia and swallowing with food or fluid passing into the lungs (in late stages of the disease).


Duchenne muscular dystrophy (DMD) is caused by a mutation of the dystrophin gene at locus Xp21, located on the short arm of the X chromosome. Dystrophin is responsible for connecting the cytoskeleton of each muscle fiber to the underlying basal lamina (extracellular matrix), through a protein complex containing many subunits. The absence of dystrophin permits excess calcium to penetrate the sarcolemma (the cell membrane). Alterations in calcium and signaling pathways cause water to enter into the mitochondria, which then burst.


In skeletal muscle dystrophy, mitochondrial dysfunction gives rise to an amplification of stress-induced cytosolic calcium signals and an amplification of stress-induced reactive-oxygen species (ROS) production. In a complex cascading process that involves several pathways and is not clearly understood, increased oxidative stress within the cell damages the sarcolemma and eventually results in the death of the cell. Muscle fibers undergo necrosis and are ultimately replaced with adipose and connective tissue.


DMD is inherited in an X-linked recessive pattern. Females will typically be carriers for the disease while males will be affected. Typically, a female carrier will be unaware they carry a mutation until they have an affected son. The son of a carrier mother has a 50% chance of inheriting the defective gene from his mother. The daughter of a carrier mother has a 50% chance of being a carrier and a 50% chance of having two normal copies of the gene. In all cases, an unaffected father will either pass a normal Y to his son or a normal X to his daughter. Female carriers of an X-linked recessive condition, such as DMD, can show symptoms depending on their pattern of X-inactivation.


Exon deletions preceding exon 51 of the human DMD gene, which disrupt the open reading frame (ORF) by juxtaposing out of frame exons, represent the most common type of human DMD mutation. Skipping of exon 51 can, in principle, restore the DMD ORF in 13% of DMD patients with exon deletions.


Duchenne muscular dystrophy has an incidence of 1 in 5000 male infants. Mutations within the dystrophin gene can either be inherited or occur spontaneously during germline transmission.


Sequences

The following tables provide exemplary primer, gRNA and genomic targeting sequences for use in connection with the compositions and methods disclosed herein.









TABLE 4







Sequence of Primers for DMD iPSCs


PCR/T7E1 and RT-PCR primers













SEQ

SEQ


DMD

ID

ID


#
PCR/T7E1
NO:
RT-PCR
NO:





Del.
F: TTCCCTGGCAAG
2463
F: CCCAGAAGAGC
2469



GTCTGA

AAGATAAACTTGAA




R: ATCCTCAAGGTC
2464
R: CTCTGTTCCAA
2470



ACCCACC

ATCCTGCATTGT






pEx.
F: CACACCTGTTAT
2465
F: CATAAGCCCAG
2471



ATTTTTCCGTGAAG

AAGAGCAAGATAAA




R: CAAAGGAGAAGC
2466
R: ATAGGAGATAA
2472



AAAAACACATTCTA

CCACAGCAGCAGAT






Dup.
F: GTAATGTATAAC
2467
E59F: GGGAAAAA
2473



TGTATAACGTGGGGC

TTGAACCTGCAC




ACTC






R: GGTGAGTTGTT
2468
E55R: CATCAGCT
2474



GCTACAGCTCTTCC

CTTTTACTCCCTT









E53F: GGAGGGTC
2475





CCTATACAGTAG
















TABLE 5







Genomic targeting sequences of top 12 exons.













Applica-

SEQ

SEQ



bility
gRNA/PAM at
ID
gRNA/PAM at
ID


Exon
(30)
acceptor site
NO.
donor site
NO.





51
13.0%
#1:
2378






TGCAAAAACCCAA







AATATTTTAG







#2:
2379






AAAATATTTTAGC







TCCTACTCAG








#3:

2380







CAGAGTAACAGTC









TGAGTAG

GAG

*









45
 8.1%
#1:
2381






TTGCCTTTTTGGT







ATCTTACAGG







#2:
2382






TTTGCCTTTTTGG







TATCTTACAG








#3:

2383







CGCTGCCCAATGC









CATCCTG

GAG










53
 7.7%
#1:
2384

#4:

2414




ATTTATTTTTCCT


AAAGAAAATCAC






TTATTCTAG


AGAAACCA

AGG







#2:
2385
#5:
2415




TTTCCTTTTATTC

AAAATCACAGAA





TAGTTGAAAG

ACCAAGGTTAG





#3:
2386

#6:

2416




TGATTCTGAATTC


GGTATCTTTGAT






TTTCAACTAG


ACTAACCT

TGG








44
 6.2%

#1:

2387

#4:

2417





ATCCATATGCTTT



GTAATACAAATG







TACCTGC

AGG




GTATCTTA

AGG







#2:
2388






GATCCATATGCTT







TTACCTGCAG







3:
2389






CAGATCTGTCAAA







TCGCCTGCAG








46
 4.3%
#1:
2390






TTATTCTTCTTTC







TCCAGGCTAG








#2:

2391







AATTTTATTCTTC









TTTCTCC

AGG









#3:
2392






CAATTTTATTCTT







CTTTCTCCAG








52
 4.1%

#1:

2393







TAAGGGATATTTG









TTCTTAC

AGG









#2:
2394






CTAAGGGATATTT







GTTCTTACAG







#3:
2395






TGTTCTTACAGGC







AACAATGCAG








50
 4.0%

#1:

2396







TGTATGCTTTTCT









GTTAAAG

AGG









#2:
2397






ATGTGTATGCTTT







TCTGTTAAAG







#3:
2398






GTGTATGCTTTTC







TGTTAAAGAG








43
 3.8%
#1:
2399

#4:

2418




GTTTTAAAATTTT


TATGTGTTACCT






TATATTACAG


ACCCTTGT

CGG







#2:
2400
#5:
2419




TTTTATATTACAG

AAATGTACAAGG





AATATAAAAG

ACCGACAAGGG





#3:
2401

#6:

2420




ATATTACAGAATA


GTACAAGGACCG






TAAAAGATAG


ACAAGGGT

AGG








 6
 3.0%†
#1:
2402
#4:
2421




TGAAAATTTATTT

ATGCTCTCATCC





CCACATGTAG

ATAGTCATAGG





#2:
2403
#5:
2422




GAAAATTTATTTC

TCTCATCCATAG





CACATGTAGG

TCATAGGTAAG





#3:
2404
#6:
2423




TTACATTTTTGAC

CATCCATAGTCA





CTACATGTGG

TAGGTAAGAAG






 7
 3.0%†
#1:
2405






TGTGTATGTGTAT







GTGTTTTAGG







#2:
2406






TATGTGTATGTGT







TTTAGGCCAG







#3:
2407







CTATTCCAGTCAA









ATAGGTC

TGG










 8
 2.3%
#1:
2408
#4:
2424




GTGTAGTGTTAAT

TGCACTATTCTC





GTGCTTACAG

AACAGGTAAAG





#2:
2409

#5:

2425




GGACTTCTTATCT


TCAAATGCACTA






GGATAGGTGG


TTCTCAAC

AGG







#3:
2410
#6:
2426




TAGGTGGTATCAA

CTTTACACACTT





CATCTGTAAG

TACCTGTTGAG






55
 2.09%

#1:

2411







TGAACATTTGGTC









CTTTGCA

GGG









#2:
2412






TCTGAACATTTGG







TCCTTTGCAG








#3:

2413







TCTCGCTCACTCA









CCCTGCA

AAG







†Dual exon skipping (exons 6 and 7).













TABLE 6







Genomic Target Sequences












Targeted
Guide

Genomic

SEQ


gRNA Exon
#
Strand
Target Sequence*
PAM
ID NO.





Human-Exon 51
 4
 1
tctttttcttcttttttccttttt
tttt
 60





Human-Exon 51
 5
 1
ctttttcttcttttttcctttttG
tttt
 61





Human-Exon 51
 6
 1
tttttcttcttttttcctttttGC
tttc
 62





Human-Exon 51
 7
 1
tcttcttttttcctttttGCAAAA
tttt
 63





Human-Exon 51
 8
 1
cttcttttttcctttttGCAAAAA
tttt
 64





Human-Exon 51
 9
 1
ttcttttttcctttttGCAAAAAC
tttc
 65





Human-Exon 51
10
 1
ttcctttttGCAAAAACCCAAAAT
tttt
 66





Human-Exon 51
11
 1
tcctttttGCAAAAACCCAAAATA
tttt
 67





Human-Exon 51
12
 1
cctttttGCAAAAACCCAAAATAT
tttt
 68





Human-Exon 51
13
 1
ctttttGCAAAAACCCAAAATATT
tttc
 69





Human-Exon 51
14
 1
tGCAAAAACCCAAAATATTTTAGC
tttt
 70





Human-Exon 51
15
 1
GCAAAAACCCAAAATATTTTAGCT
tttt
 71





Human-Exon 51
16
 1
CAAAAACCCAAAATATTTTAGCTC
tttG
 72





Human-Exon 51
17
 1
AGCTCCTACTCAGACTGTTACTCT
TTTT
 73





Human-Exon 51
18
 1
GCTCCTACTCAGACTGTTACTCTG
TTTA
 74





Human-Exon 51
19
-1
CTTAGTAACCACAGGTTGTGTCAC
TTTC
 75





Human-Exon 51
20
-1
GAGATGGCAGTTTCCTTAGTAACC
TTTG
 76





Human-Exon 51
21
-1
TAGTTTGGAGATGGCAGTTTCCTT
TTTC
 77





Human-Exon 51
22
-1
TTCTCATACCTTCTGCTTGATGAT
TTTT
 78





Human-Exon 51
23
-1
TCATTTTTTCTCATACCTTCTGCT
TTTA
 79





Human-Exon 51
24
-1
ATCATTTTTTCTCATACCTTCTGC
TTTT
 80





Human-Exon 51
25
-1
AAGAAAAACTTCTGCCAACTTTTA
TTTA
 81





Human-Exon 51
26
-1
AAAGAAAAACTTCTGCCAACTTTT
TTTT
 82





Human-Exon 51
27
 1
TCTTTAAAATGAAGATTTTCCACC
TTTT
 83





Human-Exon 51
28
 1
CTTTAAAATGAAGATTTTCCACCA
TTTT
 84





Human-Exon 51
29
 1
TTTAAAATGAAGATTTTCCACCAA
TTTC
 85





Human-Exon 51
30
 1
AAATGAAGATTTTCCACCAATCAC
TTTA
 86





Human-Exon 51
31
 1
CCACCAATCACTTTACTCTCCTAG
TTTT
 87





Human-Exon 51
32
 1
CACCAATCACTTTACTCTCCTAGA
TTTC
 88





Human-Exon 51
33
 1
CTCTCCTAGACCATTTCCCACCAG
TTTA
 89





Human-Exon 45
 1
-1
agaaaagattaaacagtgtgctac
tttg
 90





Human-Exon 45
 2
-1
tttgagaaaagattaaacagtgtg
TTTa
 91





Human-Exon 45
 3
-1
atttgagaaaagattaaacagtgt
TTTT
 92





Human-Exon 45
 4
-1
Tatttgagaaaagattaaacagtg
TTTT
 93





Human-Exon 45
 5
 1
atcttttctcaaatAAAAAGACAT
ttta
 94





Human-Exon 45
 6
 1
ctcaaatAAAAAGACATGGGGCTT
tttt
 95





Human-Exon 45
 7
 1
tcaaatAAAAAGACATGGGGCTTC
tttc
 96





Human-Exon 45
 8
 1
TGTTTTGCCTTTTTGGTATCTTAC
TTTT
 97





Human-Exon 45
 9
 1
GTTTTGCCTTTTTGGTATCTTACA
TTTT
 98





Human-Exon 45
10
 1
TTTTGCCTTTTTGGTATCTTACAG
TTTG
 99





Human-Exon 45
11
 1
GCCTTTTTGGTATCTTACAGGAAC
TTTT
100





Human-Exon 45
12
 1
CCTTTTTGGTATCTTACAGGAACT
TTTG
101





Human-Exon 45
13
 1
TGGTATCTTACAGGAACTCCAGGA
TTTT
102





Human-Exon 45
14
 1
GGTATCTTACAGGAACTCCAGGAT
TTTT
103





Human-Exon 45
15
-1
AGGATTGCTGAATTATTTCTTCCC
TTTG
104





Human-Exon 45
16
-1
GAGGATTGCTGAATTATTTCTTCC
TTTT
105





Human-Exon 45
17
-1
TGAGGATTGCTGAATTATTTCTTC
TTTT
106





Human-Exon 45
18
-1
CTGTAGAATACTGGCATCTGTTTT
TTTC
107





Human-Exon 45
19
-1
CCTGTAGAATACTGGCATCTGTTT
TTTT
108





Human-Exon 45
20
-1
TCCTGTAGAATACTGGCATCTGTT
TTTT
109





Human-Exon 45
21
-1
CAGACCTCCTGCCACCGCAGATTC
TTTG
110





Human-Exon 45
22
-1
TGTCTGACAGCTGTTTGCAGACCT
TTTC
111





Human-Exon 45
23
-1
CTGTCTGACAGCTGTTTGCAGACC
TTTT
112





Human-Exon 45
24
-1
TCTGTCTGACAGCTGTTTGCAGAC
TTTT
113





Human-Exon 45
25
-1
TTCTGTCTGACAGCTGTTTGCAGA
TTTT
114





Human-Exon 45
26
-1
ATTCCTATTAGATCTGTCGCCCTA
TTTC
115





Human-Exon 45
27
-1
CATTCCTATTAGATCTGTCGCCCT
TTTT
116





Human-Exon 45
28
 1
AGCAGACTTTTTAAGCTTTCTTTA
TTTT
117





Human-Exon 45
29
 1
GCAGACTTTTTAAGCTTTCTTTAG
TTTA
118





Human-Exon 45
30
 1
TAAGCTTTCTTTAGAAGAATATTT
TTTT
119





Human-Exon 45
31
 1
AAGCTTTCTTTAGAAGAATATTTC
TTTT
120





Human-Exon 45
32
 1
AGCTTTCTTTAGAAGAATATTTCA
TTTA
121





Human-Exon 45
33
 1
TTTAGAAGAATATTTCATGAGAGA
TTTC
122





Human-Exon 45
34
 1
GAAGAATATTTCATGAGAGATTAT
TTTA
123





Human-Exon 44
 1
 1
TCAGTATAACCAAAAAATATACGC
TTTG
124





Human-Exon 44
 2
 1
acataatccatctatttttcttga
tttt
125





Human-Exon 44
 3
 1
cataatccatctatttttcttgat
ttta
126





Human-Exon 44
 4
 1
tcttgatccatatgcttttACCTG
tttt
127





Human-Exon 44
 5
 1
cttgatccatatgcttttACCTGC
tttt
128





Human-Exon 44
 6
 1
ttgatccatatgcttttACCTGCA
tttc
129





Human-Exon 44
 7
-1
TCAACAGATCTGTCAAATCGCCTG
TTTC
130





Human-Exon 44
 8
 1
ACCTGCAGGCGATTTGACAGATCT
tttt
131





Human-Exon 44
 9
 1
CCTGCAGGCGATTTGACAGATCTG
tttA
132





Human-Exon 44
10
 1
ACAGATCTGTTGAGAAATGGCGGC
TTTG
133





Human-Exon 44
11
-1
TATCATAATGAAAACGCCGCCATT
TTTA
134





Human-Exon 44
12
 1
CATTATGATATAAAGATATTTAAT
TTTT
135





Human-Exon 44
13
-1
TATTTAGCATGTTCCCAATTCTCA
TTTG
136





Human-Exon 44
14
-1
GAAAAAACAAATCAAAGACTTACC
TTTC
137





Human-Exon 44
15
 1
ATTTGTTTTTTCGAAATTGTATTT
TTTG
138





Human-Exon 44
16
 1
TTTTTTCGAAATTGTATTTATCTT
TTTG
139





Human-Exon 44
17
 1
TTCGAAATTGTATTTATCTTCAGC
TTTT
140





Human-Exon 44
18
 1
TCGAAATTGTATTTATCTTCAGCA
TTTT
141





Human-Exon 44
19
 1
CGAAATTGTATTTATCTTCAGCAC
TTTT
142





Human-Exon 44
20
 1
GAAATTGTATTTATCTTCAGCACA
TTTC
143





Human-Exon 44
21
-1
AGAAGTTAAAGAGTCCAGATGTGC
TTTA
144





Human-Exon 44
22
 1
TCTTCAGCACATCTGGACTCTTTA
TTTA
145





Human-Exon 44
23
-1
CATCACCCTTCAGAACCTGATCTT
TTTC
146





Human-Exon 44
24
 1
ACTTCTTAAAGATCAGGTTCTGAA
TTTA
147





Human-Exon 44
25
 1
GACTGTTGTTGTCATCATTATATT
TTTT
148





Human-Exon 44
26
 1
ACTGTTGTTGTCATCATTATATTA
TTTG
149





Human-Exon 53
 1
-1
AACTAGAATAAAAGGAAAAATAAA
TTTC
150





Human-Exon 53
 2
 1
CTACTATATATTTATTTTTCCTTT
TTTA
151





Human-Exon 53
 3
 1
TTTTTCCTTTTATTCTAGTTGAAA
TTTA
152





Human-Exon 53
 4
 1
TCCTTTTATTCTAGTTGAAAGAAT
TTTT
153





Human-Exon 53
 5
 1
CCTTTTATTCTAGTTGAAAGAATT
TTTT
154





Human-Exon 53
 6
 1
CTTTTATTCTAGTTGAAAGAATTC
TTTC
155





Human-Exon 53
 7
 1
ATTCTAGTTGAAAGAATTCAGAAT
TTTT
156





Human-Exon 53
 8
 1
TTCTAGTTGAAAGAATTCAGAATC
TTTA
157





Human-Exon 53
 9
-1
ATTCAACTGTTGCCTCCGGTTCTG
TTTC
158





Human-Exon 53
10
-1
ACATTTCATTCAACTGTTGCCTCC
TTTA
159





Human-Exon 53
11
-1
CTTTTGGATTGCATCTACTGTATA
TTTT
160





Human-Exon 53
12
-1
TGTGATTTTCTTTTGGATTGCATC
TTTC
161





Human-Exon 53
13
-1
ATACTAACCTTGGTTTCTGTGATT
TTTG
162





Human-Exon 53
14
-1
AAAAGGTATCTTTGATACTAACCT
TTTA
163





Human-Exon 53
15
-1
AAAAAGGTATCTTTGATACTAACC
TTTT
164





Human-Exon 53
16
-1
TTTTAAAAAGGTATCTTTGATACT
TTTA
165





Human-Exon 53
17
-1
ATTTTAAAAAGGTATCTTTGATAC
TTTT
166





Human-Exon 46
 1
-1
TTAATGCAAACTGGGACACAAACA
TTTG
167





Human-Exon 46
 2
 1
TAAATTGCCATGTTTGTGTCCCAG
TTTT
168





Human-Exon 46
 3
 1
AAATTGCCATGTTTGTGTCCCAGT
TTTT
169





Human-Exon 46
 4
 1
AATTGCCATGTTTGTGTCCCAGTT
TTTA
170





Human-Exon 46
 5
 1
TGTCCCAGTTTGCATTAACAAATA
TTTG
171





Human-Exon 46
 6
-1
CAACATAGTTCTCAAACTATTTGT
tttC
172





Human-Exon 46
 7
-1
CCAACATAGTTCTCAAACTATTTG
tttt
173





Human-Exon 46
 8
-1
tCCAACATAGTTCTCAAACTATTT
tttt
174





Human-Exon 46
 9
-1
tttCCAACATAGTTCTCAAACTAT
tttt
175





Human-Exon 46
10
-1
ttttCCAACATAGTTCTCAAACTA
tttt
176





Human-Exon 46
11
-1
tttttCCAACATAGTTCTCAAACT
tttt
177





Human-Exon 46
12
 1
CATTAACAAATAGTTTGAGAACTA
TTTG
178





Human-Exon 46
13
 1
AGAACTATGTTGGaaaaaaaaaTA
TTTG
179





Human-Exon 46
14
-1
GTTCTTCTAGCCTGGAGAAAGAAG
TTTT
180





Human-Exon 46
15
 1
ATTCTTCTTTCTCCAGGCTAGAAG
TTTT
181





Human-Exon 46
16
 1
TTCTTCTTTCTCCAGGCTAGAAGA
TTTA
182





Human-Exon 46
17
 1
TCCAGGCTAGAAGAACAAAAGAAT
TTTC
183





Human-Exon 46
18
-1
AAATTCTGACAAGATATTCTTTTG
TTTG
184





Human-Exon 46
19
-1
CTTTTAGTTGCTGCTCTTTTCCAG
TTTT
185





Human-Exon 46
20
-1
AGAAAATAAAATTACCTTGACTTG
TTTG
186





Human-Exon 46
21
-1
TGCAAGCAGGCCCTGGGGGATTTG
TTTA
187





Human-Exon 46
22
 1
ATTTTCTCAAATCCCCCAGGGCCT
TTTT
188





Human-Exon 46
23
 1
TTTTCTCAAATCCCCCAGGGCCTG
TTTA
189





Human-Exon 46
24
 1
CTCAAATCCCCCAGGGCCTGCTTG
TTTT
190





Human-Exon 46
25
 1
TCAAATCCCCCAGGGCCTGCTTGC
TTTC
191





Human-Exon 46
26
 1
TTAATTCAATCATTGGTTTTCTGC
TTTT
192





Human-Exon 46
27
 1
TAATTCAATCATTGGTTTTCTGCC
TTTT
193





Human-Exon 46
28
 1
AATTCAATCATTGGTTTTCTGCCC
TTTT
194





Human-Exon 46
29
 1
ATTCAATCATTGGTTTTCTGCCCA
TTTA
195





Human-Exon 46
30
-1
GCAAGGAACTATGAATAACCTAAT
TTTA
196





Human-Exon 46
31
 1
CTGCCCATTAGGTTATTCATAGTT
TTTT
197





Human-Exon 46
32
 1
TGCCCATTAGGTTATTCATAGTTC
TTTC
198





Human-Exon 52
 1
-1
TAGAAAACAATTTAACAGGAAATA
TTTA
199





Human-Exon 52
 2
 1
CTGTTAAATTGTTTTCTATAAACC
TTTC
200





Human-Exon 52
 3
-1
GAAATAAAAAAGATGTTACTGTAT
TTTA
201





Human-Exon 52
 4
-1
AGAAATAAAAAAGATGTTACTGTA
TTTT
202





Human-Exon 52
 5
 1
CTATAAACCCTTATACAGTAACAT
TTTT
203





Human-Exon 52
 6
 1
TATAAACCCTTATACAGTAACATC
TTTC
204





Human-Exon 52
 7
 1
TTATTTCTAAAAGTGTTTTGGCTG
TTTT
205





Human-Exon 52
 8
 1
TATTTCTAAAAGTGTTTTGGCTGG
TTTT
206





Human-Exon 52
 9
 1
ATTTCTAAAAGTGTTTTGGCTGGT
TTTT
207





Human-Exon 52
10
 1
TTTCTAAAAGTGTTTTGGCTGGTC
TTTA
208





Human-Exon 52
11
 1
TAAAAGTGTTTTGGCTGGTCTCAC
TTTC
209





Human-Exon 52
12
-1
CATAATACAAAGTAAAGTACAATT
TTTA
210





Human-Exon 52
13
-1
ACATAATACAAAGTAAAGTACAAT
TTTT
211





Human-Exon 52
14
 1
GGCTGGTCTCACAATTGTACTTTA
TTTT
212





Human-Exon 52
15
 1
GCTGGTCTCACAATTGTACTTTAC
TTTG
213





Human-Exon 52
16
 1
CTTTGTATTATGTAAAAGGAATAC
TTTA
214





Human-Exon 52
17
 1
TATTATGTAAAAGGAATACACAAC
TTTG
215





Human-Exon 52
18
 1
TTCTTACAGGCAACAATGCAGGAT
TTTG
216





Human-Exon 52
19
 1
GAACAGAGGCGTCCCCAGTTGGAA
TTTG
217





Human-Exon 52
20
-1
GGCAGCGGTAATGAGTTCTTCCAA
TTTG
218





Human-Exon 52
21
-1
TCAAATTTTGGGCAGCGGTAATGA
TTTT
219





Human-Exon 52
22
 1
AAAAACAAGACCAGCAATCAAGAG
TTTG
220





Human-Exon 52
23
-1
TGTGTCCCATGCTTGTTAAAAAAC
TTTG
221





Human-Exon 52
24
 1
TTAACAAGCATGGGACACACAAAG
TTTT
222





Human-Exon 52
25
 1
TAACAAGCATGGGACACACAAAGC
TTTT
223





Human-Exon 52
26
 1
AACAAGCATGGGACACACAAAGCA
TTTT
224





Human-Exon 52
27
 1
ACAAGCATGGGACACACAAAGCAA
TTTA
225





Human-Exon 52
28
-1
TTGAAACTTGTCATGCATCTTGCT
TTTA
226





Human-Exon 52
29
-1
ATTGAAACTTGTCATGCATCTTGC
TTTT
227





Human-Exon 52
30
-1
TATTGAAACTTGTCATGCATCTTG
TTTT
228





Human-Exon 52
31
 1
AATAAAAACTTAAGTTCATATATC
TTTC
229





Human-Exon 50
 1
-1
GTGAATATATTATTGGATTTCTAT
TTTG
230





Human-Exon 50
 2
-1
AAGATAATTCATGAACATCTTAAT
TTTG
231





Human-Exon 50
 3
-1
ACAGAAAAGCATACACATTACTTA
TTTA
232





Human-Exon 50
 4
 1
CTGTTAAAGAGGAAGTTAGAAGAT
TTTT
233





Human-Exon 50
 5
 1
TGTTAAAGAGGAAGTTAGAAGATC
TTTC
234





Human-Exon 50
 6
-1
CCGCCTTCCACTCAGAGCTCAGAT
TTTA
235





Human-Exon 50
 7
-1
CCCTCAGCTCTTGAAGTAAACGGT
TTTG
236





Human-Exon 50
 8
 1
CTTCAAGAGCTGAGGGCAAAGCAG
TTTA
237





Human-Exon 50
 9
-1
AACAAATAGCTAGAGCCAAAGAGA
TTTG
238





Human-Exon 50
10
-1
GAACAAATAGCTAGAGCCAAAGAG
TTTT
239





Human-Exon 50
11
 1
GCTCTAGCTATTTGTTCAAAAGTG
TTTG
240





Human-Exon 50
12
 1
TTCAAAAGTGCAACTATGAAGTGA
TTTG
241





Human-Exon 50
13
-1
TCTCTCACCCAGTCATCACTTCAT
TTTC
242





Human-Exon 50
14
-1
CTCTCTCACCCAGTCATCACTTCA
TTTT
243





Human-Exon 43
 1
 1
tatatatatatatatTTTTCTCTT
TTTG
244





Human-Exon 43
 2
 1
TCTCTTTCTATAGACAGCTAATTC
tTTT
245





Human-Exon 43
 3
 1
CTCTTTCTATAGACAGCTAATTCA
TTTT
246





Human-Exon 43
 4
-1
AAACAGTAAAAAAATGAATTAGCT
TTTA
247





Human-Exon 43
 5
 1
TCTTTCTATAGACAGCTAATTCAT
TTTC
248





Human-Exon 43
 6
-1
AAAACAGTAAAAAAATGAATTAGC
TTTT
249





Human-Exon 43
 7
 1
TATAGACAGCTAATTCATTTTTTT
TTTC
250





Human-Exon 43
 8
-1
TATTCTGTAATATAAAAATTTTAA
TTTA
251





Human-Exon 43
 9
-1
ATATTCTGTAATATAAAAATTTTA
TTTT
252





Human-Exon 43
10
 1
TTTACTGTTTTAAAATTTTTATAT
TTTT
253





Human-Exon 43
11
 1
TTACTGTTTTAAAATTTTTATATT
TTTT
254





Human-Exon 43
12
 1
TACTGTTTTAAAATTTTTATATTA
TTTT
255





Human-Exon 43
13
 1
ACTGTTTTAAAATTTTTATATTAC
TTTT
256





Human-Exon 43
14
 1
CTGTTTTAAAATTTTTATATTACA
TTTA
257





Human-Exon 43
15
 1
AAAATTTTTATATTACAGAATATA
TTTT
258





Human-Exon 43
16
 1
AAATTTTTATATTACAGAATATAA
TTTA
259





Human-Exon 43
17
-1
TTGTAGACTATCTTTTATATTCTG
TTTG
260





Human-Exon 43
18
 1
TATATTACAGAATATAAAAGATAG
TTTT
261





Human-Exon 43
19
 1
ATATTACAGAATATAAAAGATAGT
TTTT
262





Human-Exon 43
20
 1
TATTACAGAATATAAAAGATAGTC
TTTA
263





Human-Exon 43
21
-1
CAATGCTGCTGTCTTCTTGCTATG
TTTG
264





Human-Exon 43
22
 1
CAATGGGAAAAAGTTAACAAAATG
TTTC
265





Human-Exon 43
23
-1
TGCAAGTATCAAGAAAAATATATG
TTTC
266





Human-Exon 43
24
 1
TCTTGATACTTGCAGAAATGATTT
TTTT
267





Human-Exon 43
25
 1
CTTGATACTTGCAGAAATGATTTG
TTTT
268





Human-Exon 43
26
 1
TTGATACTTGCAGAAATGATTTGT
TTTC
269





Human-Exon 43
27
 1
TTTTCAGGGAACTGTAGAATTTAT
TTTG
270





Human-Exon 43
28
-1
CATGGAGGGTACTGAAATAAATTC
TTTC
271





Human-Exon 43
29
-1
CCATGGAGGGTACTGAAATAAATT
TTTT
272





Human-Exon 43
30
 1
CAGGGAACTGTAGAATTTATTTCA
TTTT
273





Human-Exon 43
31
-1
TCCATGGAGGGTACTGAAATAAAT
TTTT
274





Human-Exon 43
32
 1
AGGGAACTGTAGAATTTATTTCAG
TTTC
275





Human-Exon 43
33
-1
TTCCATGGAGGGTACTGAAATAAA
TTTT
276





Human-Exon 43
34
-1
CCTGTCTTTTTTCCATGGAGGGTA
TTTC
277





Human-Exon 43
35
-1
CCCTGTCTTTTTTCCATGGAGGGT
TTTT
278





Human-Exon 43
36
-1
TCCCTGTCTTTTTTCCATGGAGGG
TTTT
279





Human-Exon 43
37
 1
TTTCAGTACCCTCCATGGAAAAAA
TTTA
280





Human-Exon 43
38
 1
AGTACCCTCCATGGAAAAAAGACA
TTTC
281





Human-Exon 6
 1
 1
AGTTTGCATGGTTCTTGCTCAAGG
TTTA
282





Human-Exon 6
 2
-1
ATAAGAAAATGCATTCCTTGAGCA
TTTC
283





Human-Exon 6
 3
-1
CATAAGAAAATGCATTCCTTGAGC
TTTT
284





Human-Exon 6
 4
 1
CATGGTTCTTGCTCAAGGAATGCA
TTTG
285





Human-Exon 6
 5
-1
ACCTACATGTGGAAATAAATTTTC
TTTG
286





Human-Exon 6
 6
-1
GACCTACATGTGGAAATAAATTTT
TTTT
287





Human-Exon 6
 7
-1
TGACCTACATGTGGAAATAAATTT
TTTT
288





Human-Exon 6
 8
 1
CTTATGAAAATTTATTTCCACATG
TTTT
289





Human-Exon 6
 9
 1
TTATGAAAATTTATTTCCACATGT
TTTC
290





Human-Exon 6
10
-1
ATTACATTTTTGACCTACATGTGG
TTTC
291





Human-Exon 6
11
-1
CATTACATTTTTGACCTACATGTG
TTTT
292





Human-Exon 6
12
-1
TCATTACATTTTTGACCTACATGT
TTTT
293





Human-Exon 6
13
 1
TTTCCACATGTAGGTCAAAAATGT
TTTA
294





Human-Exon 6
14
 1
CACATGTAGGTCAAAAATGTAATG
TTTC
295





Human-Exon 6
15
-1
TTGCAATCCAGCCATGATATTTTT
TTTG
296





Human-Exon 6
16
-1
ACTGTTGGTTTGTTGCAATCCAGC
TTTC
297





Human-Exon 6
17
-1
CACTGTTGGTTTGTTGCAATCCAG
TTTT
298





Human-Exon 6
18
 1
AATGCTCTCATCCATAGTCATAGG
TTTG
299





Human-Exon 6
19
-1
ATGTCTCAGTAATCTTCTTACCTA
TTTA
300





Human-Exon 6
20
-1
CAAGTTATTTAATGTCTCAGTAAT
TTTA
301





Human-Exon 6
21
-1
ACAAGTTATTTAATGTCTCAGTAA
TTTT
302





Human-Exon 6
22
 1
GACTCTGATGACATATTTTTCCCC
TTTA
303





Human-Exon 6
23
 1
TCCCCAGTATGGTTCCAGATCATG
TTTT
304





Human-Exon 6
24
 1
CCCCAGTATGGTTCCAGATCATGT
TTTT
305





Human-Exon 6
25
 1
CCCAGTATGGTTCCAGATCATGTC
TTTC
306





Human-Exon 7
 1
 1
TATTTGTCTTtgtgtatgtgtgta
TTTA
307





Human-Exon 7
 2
 1
TCTTtgtgtatgtgtgtatgtgta
TTTG
308





Human-Exon 7
 3
 1
tgtatgtgtgtatgtgtatgtgtt
TTtg
309





Human-Exon 7
 4
 1
AGGCCAGACCTATTTGACTGGAAT
ttTT
310





Human-Exon 7
 5
 1
GGCCAGACCTATTTGACTGGAATA
tTTA
311





Human-Exon 7
 6
 1
ACTGGAATAGTGTGGTTTGCCAGC
TTTG
312





Human-Exon 7
 7
 1
CCAGCAGTCAGCCACACAACGACT
TTTG
313





Human-Exon 7
 8
-1
TCTATGCCTAATTGATATCTGGCG
TTTC
314





Human-Exon 7
 9
-1
CCAACCTTCAGGATCGAGTAGTTT
TTTA
315





Human-Exon 7
10
 1
TGGACTACCACTGCTTTTAGTATG
TTTC
316





Human-Exon 7
11
 1
AGTATGGTAGAGTTTAATGTTTTC
TTTT
317





Human-Exon 7
12
 1
GTATGGTAGAGTTTAATGTTTTCA
TTTA
318





Human-Exon 8
 1
-1
AGACTCTAAAAGGATAATGAACAA
TTTG
319





Human-Exon 8
 2
 1
ACTTTGATTTGTTCATTATCCTTT
TTTA
320





Human-Exon 8
 3
-1
TATATTTGAGACTCTAAAAGGATA
TTTC
321





Human-Exon 8
 4
 1
ATTTGTTCATTATCCTTTTAGAGT
TTTG
322





Human-Exon 8
 5
-1
GTTTCTATATTTGAGACTCTAAAA
TTTG
323





Human-Exon 8
 6
-1
GGTTTCTATATTTGAGACTCTAAA
TTTT
324





Human-Exon 8
 7
-1
TGGTTTCTATATTTGAGACTCTAA
TTTT
325





Human-Exon 8
 8
 1
TTCATTATCCTTTTAGAGTCTCAA
TTTG
326





Human-Exon 8
 9
 1
AGAGTCTCAAATATAGAAACCAAA
TTTT
327





Human-Exon 8
10
 1
GAGTCTCAAATATAGAAACCAAAA
TTTA
328





Human-Exon 8
11
-1
CACTTCCTGGATGGCTTCAATGCT
TTTC
329





Human-Exon 8
12
 1
GCCTCAACAAGTGAGCATTGAAGC
TTTT
330





Human-Exon 8
13
 1
CCTCAACAAGTGAGCATTGAAGCC
TTTG
331





Human-Exon 8
14
-1
GGTGGCCTTGGCAACATTTCCACT
TTTA
332





Human-Exon 8
15
-1
GTCACTTTAGGTGGCCTTGGCAAC
TTTA
333





Human-Exon 8
16
-1
ATGATGTAACTGAAAATGTTCTTC
TTTG
334





Human-Exon 8
17
-1
CCTGTTGAGAATAGTGCATTTGAT
TTTA
335





Human-Exon 8
18
 1
CAGTTACATCATCAAATGCACTAT
TTTT
336





Human-Exon 8
19
 1
AGTTACATCATCAAATGCACTATT
TTTC
337





Human-Exon 8
20
-1
CACACTTTACCTGTTGAGAATAGT
TTTA
338





Human-Exon 8
21
 1
CTGTTTTATATGCATTTTTAGGTA
TTTT
339





Human-Exon 8
22
 1
TGTTTTATATGCATTTTTAGGTAT
TTTC
340





Human-Exon 8
23
 1
ATATGCATTTTTAGGTATTACGTG
TTTT
341





Human-Exon 8
24
 1
TATGCATTTTTAGGTATTACGTGC
TTTA
342





Human-Exon 8
25
 1
TAGGTATTACGTGCACatatatat
TTTT
343





Human-Exon 8
26
 1
AGGTATTACGTGCACatatatata
TTTT
344





Human-Exon 8
27
 1
GGTATTACGTGCACatatatatat
TTTA
345





Human-Exon 55
 1
-1
AGCAACAACTATAATATTGTGCAG
TTTA
346





Human-Exon 55
 2
 1
GTTCCTCCATCTTTCTCTTTTTAT
TTTA
347





Human-Exon 55
 3
 1
TCTTTTTATGGAGTTCACTAGGTG
TTTC
348





Human-Exon 55
 4
 1
TATGGAGTTCACTAGGTGCACCAT
TTTT
349





Human-Exon 55
 5
 1
ATGGAGTTCACTAGGTGCACCATT
TTTT
350





Human-Exon 55
 6
 1
TGGAGTTCACTAGGTGCACCATTC
TTTA
351





Human-Exon 55
 7
 1
ATAATTGCATCTGAACATTTGGTC
TTTA
352





Human-Exon 55
 8
 1
GTCCTTTGCAGGGTGAGTGAGCGA
TTTG
353





Human-Exon 55
 9
-1
TTCCAAAGCAGCCTCTCGCTCACT
TTTC
354





Human-Exon 55
10
 1
CAGGGTGAGTGAGCGAGAGGCTGC
TTTG
355





Human-Exon 55
11
 1
GAAGAAACTCATAGATTACTGCAA
TTTG
356





Human-Exon 55
12
-1
CAGGTCCAGGGGGAACTGTTGCAG
TTTC
357





Human-Exon 55
13
-1
CCAGGTCCAGGGGGAACTGTTGCA
TTTT
358





Human-Exon 55
14
-1
AGCTTCTGTAAGCCAGGCAAGAAA
TTTC
359





Human-Exon 55
15
 1
TTGCCTGGCTTACAGAAGCTGAAA
TTTC
360





Human-Exon 55
16
-1
CTTACGGGTAGCATCCTGTAGGAC
TTTC
361





Human-Exon 55
17
-1
CTCCCTTGGAGTCTTCTAGGAGCC
TTTA
362





Human-Exon 55
18
-1
ACTCCCTTGGAGTCTTCTAGGAGC
TTTT
363





Human-Exon 55
19
-1
ATCAGCTCTTTTACTCCCTTGGAG
TTTC
364





Human-Exon 55
20
 1
CGCTTTAGCACTCTTGTGGATCCA
TTTC
365





Human-Exon 55
21
 1
GCACTCTTGTGGATCCAATTGAAC
TTTA
366





Human-Exon 55
22
-1
TCCCTGGCTTGTCAGTTACAAGTA
TTTG
367





Human-Exon 55
23
-1
GTCCCTGGCTTGTCAGTTACAAGT
TTTT
368





Human-Exon 55
24
-1
TTTTGTCCCTGGCTTGTCAGTTAC
TTTG
369





Human-Exon 55
25
-1
GTTTTGTCCCTGGCTTGTCAGTTA
TTTT
370





Human-Exon 55
26
 1
TACTTGTAACTGACAAGCCAGGGA
TTTG
371





Human-G1-exon51

 1
gCTCCTACTCAGACTGTTACTCTG
TTTA
372





Human-G2-exon51

 1
taccatgtattgctaaacaaagta
TTTC
373





Human-G3-exon51

-1
attgaagagtaacaatttgagcca
TTTA
374





mouse-Exon23-G1

 1
aggctctgcaaagttctTTGAAAG
TTTG
375





mouse-Exon23-G2

 1
AAAGAGCAACAAAATGGCttcaac
TTTG
376





mouse-Exon23-G3

 1
AAAGAGCAATAAAATGGCttcaac
TTTG
377





mouse-Exon23-G4

-1
AAAGAACTTTGCAGAGCctcaaaa
TTTC
378





mouse-Exon23-G5

-1
ctgaatatctatgcattaataact
TTTA
379





mouse-Exon23-G6

-1
tattatattacagggcatattata
TTTC
380





mouse-Exon23-G7

 1
Aggtaagccgaggtttggccttta
TTTC
381





mouse-Exon23-G8

 1
cccagagtccttcaaagatattga
TTTA
382





*In this table, upper case letters represent nucleotides that align to the exon sequence of the gene. Lower case letters represent nucleotides that align to the intron sequence of the gene.













TABLE 7







gRNA sequences












Targeted




SEQ ID


gRNA Exon
Guide #
Strand
gRNA sequence*
PAM
NO.





Human-Exon 51
 4
 1
aaaaaggaaaaaagaagaaaaaga
tttt
383





Human-Exon 51
 5
 1
Caaaaaggaaaaaagaagaaaaag
tttt
384





Human-Exon 51
 6
 1
GCaaaaaggaaaaaagaagaaaaa
tttc
385





Human-Exon 51
 7
 1
UUUUGCaaaaaggaaaaaagaaga
tttt
386





Human-Exon 51
 8
 1
UUUUUGCaaaaaggaaaaaagaag
tttt
387





Human-Exon 51
 9
 1
GUUUUUGCaaaaaggaaaaaagaa
tttc
388





Human-Exon 51
10
 1
AUUUUGGGUUUUUGCaaaaaggaa
tttt
389





Human-Exon 51
11
 1
UAUUUUGGGUUUUUGCaaaaagga
tttt
390





Human-Exon 51
12
 1
AUAUUUUGGGUUUUUGCaaaaagg
tttt
391





Human-Exon 51
13
 1
AAUAUUUUGGGUUUUUGCaaaaag
tttc
392





Human-Exon 51
14
 1
GCUAAAAUAUUUUGGGUUUUUGCa
tttt
393





Human-Exon 51
15
 1
AGCUAAAAUAUUUUGGGUUUUUGC
tttt
394





Human-Exon 51
16
 1
GAGCUAAAAUAUUUUGGGUUUUUG
tttG
395





Human-Exon 51
17
 1
AGAGUAACAGUCUGAGUAGGAGCU
TTTT
396





Human-Exon 51
18
 1
CAGAGUAACAGUCUGAGUAGGAGC
TTTA
397





Human-Exon 51
19
-1
GUGACACAACCUGUGGUUACUAAG
TTTC
398





Human-Exon 51
20
-1
GGUUACUAAGGAAACUGCCAUCU
TTTG
399





Human-Exon 51
21
-1
AAGGAAACUGCCAUCUCCAAACUA
TTTC
400





Human-Exon 51
22
-1
AUCAUCAAGCAGAAGGUAUGAGAA
TTTT
401





Human-Exon 51
23
-1
AGCAGAAGGUAUGAGAAAAAAUGA
TTTA
402





Human-Exon 51
24
-1
GCAGAAGGUAUGAGAAAAAAUGAU
TTTT
403





Human-Exon 51
25
-1
UAAAAGUUGGCAGAAGUUUUUCUU
TTTA
404





Human-Exon 51
26
-1
AAAAGUUGGCAGAAGUUUUUCUUU
TTTT
405





Human-Exon 51
27
 1
GGUGGAAAAUCUUCAUUUUAAAGA
TTTT
406





Human-Exon 51
28
 1
UGGUGGAAAAUCUUCAUUUUAAAG
TTTT
407





Human-Exon 51
29
 1
UUGGUGGAAAAUCUUCAUUUUAAA
TTTC
408





Human-Exon 51
30
 1
GUGAUUGGUGGAAAAUCUUCAUUU
TTTA
409





Human-Exon 51
31
 1
CUAGGAGAGUAAAGUGAUUGGUGG
TTTT
410





Human-Exon 51
32
 1
UCUAGGAGAGUAAAGUGAUUGGUG
TTTC
411





Human-Exon 51
33
 1
CUGGUGGGAAAUGGUCUAGGAGA
TTTA
412





Human-Exon 45
 1
-1
guagcacacuguuuaaucuuuucu
tttg
413





Human-Exon 45
 2
-1
cacacuguuuaaucuuuucucaaa
TTTa
414





Human-Exon 45
 3
-1
acacuguuuaaucuuuucucaaau
TTTT
415





Human-Exon 45
 4
-1
cacuguuuaaucuuuucucaaauA
TTTT
416





Human-Exon 45
 5
 1
AUGUCUUUUUauuugagaaaagau
ttta
417





Human-Exon 45
 6
 1
AAGCCCCAUGUCUUUUUauuugag
tttt
418





Human-Exon 45
 7
 1
GAAGCCCCAUGUCUUUUUauuuga
tttc
419





Human-Exon 45
 8
 1
GUAAGAUACCAAAAAGGCAAAACA
TTTT
420





Human-Exon 45
 9
 1
UGUAAGAUACCAAAAAGGCAAAAC
TTTT
421





Human-Exon 45
10
 1
CUGUAAGAUACCAAAAAGGCAAAA
TTTG
422





Human-Exon 45
11
 1
GUUCCUGUAAGAUACCAAAAAGGC
TTTT
423





Human-Exon 45
12
 1
AGUUCCUGUAAGAUACCAAAAAGG
TTTG
424





Human-Exon 45
13
 1
UCCUGGAGUUCCUGUAAGAUACCA
TTTT
425





Human-Exon 45
14
 1
AUCCUGGAGUUCCUGUAAGAUACC
TTTT
426





Human-Exon 45
15
-1
GGGAAGAAAUAAUUCAGCAAUCCU
TTTG
427





Human-Exon 45
16
-1
GGAAGAAAUAAUUCAGCAAUCCUC
TTTT
428





Human-Exon 45
17
-1
GAAGAAAUAAUUCAGCAAUCCUCA
TTTT
429





Human-Exon 45
18
-1
AAAACAGAUGCCAGUAUUCUACAG
TTTC
430





Human-Exon 45
19
-1
AAACAGAUGCCAGUAUUCUACAGG
TTTT
431





Human-Exon 45
20
-1
AACAGAUGCCAGUAUUCUACAGGA
TTTT
432





Human-Exon 45
21
-1
GAAUCUGCGGUGGCAGGAGGUCUG
TTTG
433





Human-Exon 45
22
-1
AGGUCUGCAAACAGCUGUCAGACA
TTTC
434





Human-Exon 45
23
-1
GGUCUGCAAACAGCUGUCAGACAG
TTTT
435





Human-Exon 45
24
-1
GUCUGCAAACAGCUGUCAGACAGA
TTTT
436





Human-Exon 45
25
-1
UCUGCAAACAGCUGUCAGACAGAA
TTTT
437





Human-Exon 45
26
-1
UAGGGCGACAGAUCUAAUAGGAAU
TTTC
438





Human-Exon 45
27
-1
AGGGCGACAGAUCUAAUAGGAAUG
TTTT
439





Human-Exon 45
28
 1
UAAAGAAAGCUUAAAAAGUCUGCU
TTTT
440





Human-Exon 45
29
 1
CUAAAGAAAGCUUAAAAAGUCUGC
TTTA
441





Human-Exon 45
30
 1
AAAUAUUCUUCUAAAGAAAGCUUA
TTTT
442





Human-Exon 45
31
 1
GAAAUAUUCUUCUAAAGAAAGCUU
TTTT
443





Human-Exon 45
32
 1
UGAAAUAUUCUUCUAAAGAAAGCU
TTTA
444





Human-Exon 45
33
 1
UCUCUCAUGAAAUAUUCUUCUAAA
TTTC
445





Human-Exon 45
34
 1
AUAAUCUCUCAUGAAAUAUUCUUC
TTTA
446





Human-Exon 44
 1
 1
GCGUAUAUUUUUUGGUUAUACUGA
TTTG
447





Human-Exon 44
 2
 1
ucaagaaaaauagauggauuaugu
tttt
448





Human-Exon 44
 3
 1
aucaagaaaaauagauggauuaug
ttta
449





Human-Exon 44
 4
 1
CAGGUaaaagcauauggaucaaga
tttt
450





Human-Exon 44
 5
 1
GCAGGUaaaagcauauggaucaag
tttt
451





Human-Exon 44
 6
 1
UGCAGGUaaaagcauauggaucaa
tttc
452





Human-Exon 44
 7
-1
CAGGCGAUUUGACAGAUCUGUUGA
TTTC
453





Human-Exon 44
 8
 1
AGAUCUGUCAAAUCGCCUGCAGGU
tttt
454





Human-Exon 44
 9
 1
CAGAUCUGUCAAAUCGCCUGCAGG
tttA
455





Human-Exon 44
10
 1
GCCGCCAUUUCUCAACAGAUCUGU
TTTG
456





Human-Exon 44
11
-1
AAUGGCGGCGUUUUCAUUAUGAUA
TTTA
457





Human-Exon 44
12
 1
AUUAAAUAUCUUUAUAUCAUAAUG
TTTT
458





Human-Exon 44
13
-1
UGAGAAUUGGGAACAUGCUAAAUA
TTTG
459





Human-Exon 44
14
-1
GGUAAGUCUUUGAUUUGUUUUUUC
TTTC
460





Human-Exon 44
15
 1
AAAUACAAUUUCGAAAAAACAAAU
TTTG
461





Human-Exon 44
16
 1
AAGAUAAAUACAAUUUCGAAAAAA
TTTG
462





Human-Exon 44
17
 1
GCUGAAGAUAAAUACAAUUUCGAA
TTTT
463





Human-Exon 44
18
 1
UGCUGAAGAUAAAUACAAUUUCGA
TTTT
464





Human-Exon 44
19
 1
GUGCUGAAGAUAAAUACAAUUUCG
TTTT
465





Human-Exon 44
20
 1
UGUGCUGAAGAUAAAUACAAUUUC
TTTC
466





Human-Exon 44
21
-1
GCACAUCUGGACUCUUUAACUUCU
TTTA
467





Human-Exon 44
22
 1
UAAAGAGUCCAGAUGUGCUGAAGA
TTTA
468





Human-Exon 44
23
-1
AAGAUCAGGUUCUGAAGGGUGAUG
TTTC
469





Human-Exon 44
24
 1
UUCAGAACCUGAUCUUUAAGAAGU
TTTA
470





Human-Exon 44
25
 1
AAUAUAAUGAUGACAACAACAGUC
TTTT
471





Human-Exon 44
26
 1
UAAUAUAAUGAUGACAACAACAGU
TTTG
472





Human-Exon 53
 1
-1
UUUAUUUUUCCUUUUAUUCUAGUU
TTTC
473





Human-Exon 53
 2
 1
AAAGGAAAAAUAAAUAUAUAGUAG
TTTA
474





Human-Exon 53
 3
 1
UUUCAACUAGAAUAAAAGGAAAAA
TTTA
475





Human-Exon 53
 4
 1
AUUCUUUCAACUAGAAUAAAAGGA
TTTT
476





Human-Exon 53
 5
 1
AAUUCUUUCAACUAGAAUAAAAGG
TTTT
477





Human-Exon 53
 6
 1
GAAUUCUUUCAACUAGAAUAAAAG
TTTC
478





Human-Exon 53
 7
 1
AUUCUGAAUUCUUUCAACUAGAAU
TTTT
479





Human-Exon 53
 8
 1
GAUUCUGAAUUCUUUCAACUAGAA
TTTA
480





Human-Exon 53
 9
-1
CAGAACCGGAGGCAACAGUUGAAU
TTTC
481





Human-Exon 53
10
-1
GGAGGCAACAGUUGAAUGAAAUGU
TTTA
482





Human-Exon 53
11
-1
UAUACAGUAGAUGCAAUCCAAAAG
TTTT
483





Human-Exon 53
12
-1
GAUGCAAUCCAAAAGAAAAUCACA
TTTC
484





Human-Exon 53
13
-1
AAUCACAGAAACCAAGGUUAGUAU
TTTG
485





Human-Exon 53
14
-1
AGGUUAGUAUCAAAGAUACCUUU
TTTA
486





Human-Exon 53
15
-1
GGUUAGUAUCAAAGAUACCUUUUU
TTTT
487





Human-Exon 53
16
-1
AGUAUCAAAGAUACCUUUUUAAAA
TTTA
488





Human-Exon 53
17
-1
GUAUCAAAGAUACCUUUUUAAAAU
TTTT
489





Human-Exon 46
 1
-1
UGUUUGUGUCCCAGUUUGCAUUAA
TTTG
490





Human-Exon 46
 2
 1
CUGGGACACAAACAUGGCAAUUUA
TTTT
491





Human-Exon 46
 3
 1
ACUGGGACACAAACAUGGCAAUUU
TTTT
492





Human-Exon 46
 4
 1
AACUGGGACACAAACAUGGCAAUU
TTTA
493





Human-Exon 46
 5
 1
UAUUUGUUAAUGCAAACUGGGACA
TTTG
494





Human-Exon 46
 6
-1
ACAAAUAGUUUGAGAACUAUGUUG
tttC
495





Human-Exon 46
 7
-1
CAAAUAGUUUGAGAACUAUGUUGG
tttt
496





Human-Exon 46
 8
-1
AAAUAGUUUGAGAACUAUGUUGGa
tttt
497





Human-Exon 46
 9
-1
AUAGUUUGAGAACUAUGUUGGaaa
tilt
498





Human-Exon 46
10
-1
UAGUUUGAGAACUAUGUUGGaaaa
tttt
499





Human-Exon 46
11
-1
AGUUUGAGAACUAUGUUGGaaaaa
tttt
500





Human-Exon 46
12
 1
UAGUUCUCAAACUAUUUGUUAAUG
TTTG
501





Human-Exon 46
13
 1
UAuuuuuuuuuCCAACAUAGUUCU
TTTG
502





Human-Exon 46
14
-1
CUUCUUUCUCCAGGCUAGAAGAAC
TTTT
503





Human-Exon 46
15
 1
CUUCUAGCCUGGAGAAAGAAGAAU
TTTT
504





Human-Exon 46
16
 1
UCUUCUAGCCUGGAGAAAGAAGAA
TTTA
505





Human-Exon 46
17
 1
AUUCUUUUGUUCUUCUAGCCUGGA
TTTC
506





Human-Exon 46
18
-1
CAAAAGAAUAUCUUGUCAGAAUUU
TTTG
507





Human-Exon 46
19
-1
CUGGAAAAGAGCAGCAACUAAAAG
TTTT
508





Human-Exon 46
20
-1
CAAGUCAAGGUAAUUUUAUUUUCU
TTTG
509





Human-Exon 46
21
-1
CAAAUCCCCCAGGGCCUGCUUGCA
TTTA
510





Human-Exon 46
22
 1
AGGCCCUGGGGGAUUUGAGAAAAU
TTTT
511





Human-Exon 46
23
 1
CAGGCCCUGGGGGAUUUGAGAAAA
TTTA
512





Human-Exon 46
24
 1
CAAGCAGGCCCUGGGGGAUUUGAG
TTTT
513





Human-Exon 46
25
 1
GCAAGCAGGCCCUGGGGGAUUUGA
TTTC
514





Human-Exon 46
26
 1
GCAGAAAACCAAUGAUUGAAUUAA
TTTT
515





Human-Exon 46
27
 1
GGCAGAAAACCAAUGAUUGAAUUA
TTTT
516





Human-Exon 46
28
 1
GGGCAGAAAACCAAUGAUUGAAUU
TTTT
517





Human-Exon 46
29
 1
UGGGCAGAAAACCAAUGAUUGAAU
TTTA
518





Human-Exon 46
30
-1
AUUAGGUUAUUCAUAGUUCCUUGC
TTTA
519





Human-Exon 46
31
 1
AACUAUGAAUAACCUAAUGGGCAG
TTTT
520





Human-Exon 46
32
 1
GAACUAUGAAUAACCUAAUGGGCA
TTTC
521





Human-Exon 52
 1
-1
UAUUUCCUGUUAAAUUGUUUUCUA
TTTA
522





Human-Exon 52
 2
 1
GGUUUAUAGAAAACAAUUUAACAG
TTTC
523





Human-Exon 52
 3
-1
AUACAGUAACAUCUUUUUUAUUUC
TTTA
524





Human-Exon 52
 4
-1
UACAGUAACAUCUUUUUUAUUUCU
TTTT
525





Human-Exon 52
 5
 1
AUGUUACUGUAUAAGGGUUUAUAG
TTTT
526





Human-Exon 52
 6
 1
GAUGUUACUGUAUAAGGGUUUAUA
TTTC
527





Human-Exon 52
 7
 1
CAGCCAAAACACUUUUAGAAAUAA
TTTT
528





Human-Exon 52
 8
 1
CCAGCCAAAACACUUUUAGAAAUA
TTTT
529





Human-Exon 52
 9
 1
ACCAGCCAAAACACUUUUAGAAAU
TTTT
530





Human-Exon 52
10
 1
GACCAGCCAAAACACUUUUAGAAA
TTTA
531





Human-Exon 52
11
 1
GUGAGACCAGCCAAAACACUUUUA
TTTC
532





Human-Exon 52
12
-1
AAUUGUACUUUACUUUGUAUUAUG
TTTA
533





Human-Exon 52
13
-1
AUUGUACUUUACUUUGUAUUAUGU
TTTT
534





Human-Exon 52
14
 1
UAAAGUACAAUUGUGAGACCAGCC
TTTT
535





Human-Exon 52
15
 1
GUAAAGUACAAUUGUGAGACCAGC
TTTG
536





Human-Exon 52
16
 1
GUAUUCCUUUUACAUAAUACAAAG
TTTA
537





Human-Exon 52
17
 1
GUUGUGUAUUCCUUUUACAUAAUA
TTTG
538





Human-Exon 52
18
 1
AUCCUGCAUUGUUGCCUGUAAGAA
TTTG
539





Human-Exon 52
19
 1
UUCCAACUGGGGACGCCUCUGUUC
TTTG
540





Human-Exon 52
20
-1
UUGGAAGAACUCAUUACCGCUGCC
TTTG
541





Human-Exon 52
21
-1
UCAUUACCGCUGCCCAAAAUUUGA
TTTT
542





Human-Exon 52
22
 1
CUCUUGAUUGCUGGUCUUGUUUUU
TTTG
543





Human-Exon 52
23
-1
GUUUUUUAACAAGCAUGGGACACA
TTTG
544





Human-Exon 52
24
 1
CUUUGUGUGUCCCAUGCUUGUUAA
TTTT
545





Human-Exon 52
25
 1
GCUUUGUGUGUCCCAUGCUUGUUA
TTTT
546





Human-Exon 52
26
 1
UGCUUUGUGUGUCCCAUGCUUGUU
TTTT
547





Human-Exon 52
27
 1
UUGCUUUGUGUGUCCCAUGCUUGU
TTTA
548





Human-Exon 52
28
-1
AGCAAGAUGCAUGACAAGUUUCAA
TTTA
549





Human-Exon 52
29
-1
GCAAGAUGCAUGACAAGUUUCAAU
TTTT
550





Human-Exon 52
30
-1
CAAGAUGCAUGACAAGUUUCAAUA
TTTT
551





Human-Exon 52
31
 1
GAUAUAUGAACUUAAGUUUUUAUU
TTTC
552





Human-Exon 50
 1
-1
AUAGAAAUCCAAUAAUAUAUUCAC
TTTG
553





Human-Exon 50
 2
-1
AUUAAGAUGUUCAUGAAUUAUCUU
TTTG
554





Human-Exon 50
 3
-1
UAAGUAAUGUGUAUGCUUUUCUGU
TTTA
555





Human-Exon 50
 4
 1
AUCUUCUAACUUCCUCUUUAACAG
TTTT
556





Human-Exon 50
 5
 1
GAUCUUCUAACUUCCUCUUUAACA
TTTC
557





Human-Exon 50
 6
-1
AUCUGAGCUCUGAGUGGAAGGCGG
TTTA
558





Human-Exon 50
 7
-1
ACCGUUUACUUCAAGAGCUGAGGG
TTTG
559





Human-Exon 50
 8
 1
CUGCUUUGCCCUCAGCUCUUGAAG
TTTA
560





Human-Exon 50
 9
-1
UCUCUUUGGCUCUAGCUAUUUGUU
TTTG
561





Human-Exon 50
10
-1
CUCUUUGGCUCUAGCUAUUUGUUC
TTTT
562





Human-Exon 50
11
 1
CACUUUUGAACAAAUAGCUAGAGC
TTTG
563





Human-Exon 50
12
 1
UCACUUCAUAGUUGCACUUUUGAA
TTTG
564





Human-Exon 50
13
-1
AUGAAGUGAUGACUGGGUGAGAGA
TTTC
565





Human-Exon 50
14
-1
UGAAGUGAUGACUGGGUGAGAGAG
TTTT
566





Human-Exon 43
 1
 1
AAGAGAAAAauauauauauauaua
TTTG
567





Human-Exon 43
 2
 1
GAAUUAGCUGUCUAUAGAAAGAGA
tTTT
568





Human-Exon 43
 3
 1
UGAAUUAGCUGUCUAUAGAAAGAG
TTTT
569





Human-Exon 43
 4
-1
AGCUAAUUCAUUUUUUUACUGUUU
TTTA
570





Human-Exon 43
 5
 1
AUGAAUUAGCUGUCUAUAGAAAGA
TTTC
571





Human-Exon 43
 6
-1
GCUAAUUCAUUUUUUUACUGUUUU
TTTT
572





Human-Exon 43
 7
 1
AAAAAAAUGAAUUAGCUGUCUAUA
TTTC
573





Human-Exon 43
 8
-1
UUAAAAUUUUUAUAUUACAGAAUA
TTTA
574





Human-Exon 43
 9
-1
UAAAAUUUUUAUAUUACAGAAUAU
TTTT
575





Human-Exon 43
10
 1
AUAUAAAAAUUUUAAAACAGUAAA
TTTT
576





Human-Exon 43
11
 1
AAUAUAAAAAUUUUAAAACAGUAA
TTTT
577





Human-Exon 43
12
 1
UAAUAUAAAAAUUUUAAAACAGUA
TTTT
578





Human-Exon 43
13
 1
GUAAUAUAAAAAUUUUAAAACAGU
TTTT
579





Human-Exon 43
14
 1
UGUAAUAUAAAAAUUUUAAAACAG
TTTA
580





Human-Exon 43
15
 1
UAUAUUCUGUAAUAUAAAAAUUUU
TTTT
581





Human-Exon 43
16
 1
UUAUAUUCUGUAAUAUAAAAAUUU
TTTA
582





Human-Exon 43
17
-1
CAGAAUAUAAAAGAUAGUCUACAA
TTTG
583





Human-Exon 43
18
 1
CUAUCUUUUAUAUUCUGUAAUAUA
TTTT
584





Human-Exon 43
19
 1
ACUAUCUUUUAUAUUCUGUAAUAU
TTTT
585





Human-Exon 43
20
 1
GACUAUCUUUUAUAUUCUGUAAUA
TTTA
586





Human-Exon 43
21
-1
CAUAGCAAGAAGACAGCAGCAUUG
TTTG
587





Human-Exon 43
22
 1
CAUUUUGUUAACUUUUUCCCAUUG
TTTC
588





Human-Exon 43
23
-1
CAUAUAUUUUUCUUGAUACUUGCA
TTTC
589





Human-Exon 43
24
 1
AAAUCAUUUCUGCAAGUAUCAAGA
TTTT
590





Human-Exon 43
25
 1
CAAAUCAUUUCUGCAAGUAUCAAG
TTTT
591





Human-Exon 43
26
 1
ACAAAUCAUUUCUGCAAGUAUCAA
TTTC
592





Human-Exon 43
27
 1
AUAAAUUCUACAGUUCCCUGAAAA
TTTG
593





Human-Exon 43
28
-1
GAAUUUAUUUCAGUACCCUCCAUG
TTTC
594





Human-Exon 43
29
-1
AAUUUAUUUCAGUACCCUCCAUGG
TTTT
595





Human-Exon 43
30
 1
UGAAAUAAAUUCUACAGUUCCCUG
TTTT
596





Human-Exon 43
31
-1
AUUUAUUUCAGUACCCUCCAUGGA
TTTT
597





Human-Exon 43
32
 1
CUGAAAUAAAUUCUACAGUUCCCU
TTTC
598





Human-Exon 43
33
-1
UUUAUUUCAGUACCCUCCAUGGAA
TTTT
599





Human-Exon 43
34
-1
UACCCUCCAUGGAAAAAAGACAGG
TTTC
600





Human-Exon 43
35
-1
ACCCUCCAUGGAAAAAAGACAGGG
TTTT
601





Human-Exon 43
36
-1
CCCUCCAUGGAAAAAAGACAGGGA
TTTT
602





Human-Exon 43
37
 1
UUUUUUCCAUGGAGGGUACUGAAA
TTTA
603





Human-Exon 43
38
 1
UGUCUUUUUUCCAUGGAGGGUACU
TTTC
604





Human-Exon 6
 1
 1
CCUUGAGCAAGAACCAUGCAAACU
TTTA
605





Human-Exon 6
 2
-1
UGCUCAAGGAAUGCAUUUUCUUAU
TTTC
606





Human-Exon 6
 3
-1
GCUCAAGGAAUGCAUUUUCUUAUG
TTTT
607





Human-Exon 6
 4
 1
UGCAUUCCUUGAGCAAGAACCAUG
TTTG
608





Human-Exon 6
 5
-1
GAAAAUUUAUUUCCACAUGUAGGU
TTTG
609





Human-Exon 6
 6
-1
AAAAUUUAUUUCCACAUGUAGGUC
TTTT
610





Human-Exon 6
 7
-1
AAAUUUAUUUCCACAUGUAGGUCA
TTTT
611





Human-Exon 6
 8
 1
CAUGUGGAAAUAAAUUUUCAUAAG
TTTT
612





Human-Exon 6
 9
 1
ACAUGUGGAAAUAAAUUUUCAUAA
TTTC
613





Human-Exon 6
10
-1
CCACAUGUAGGUCAAAAAUGUAAU
TTTC
614





Human-Exon 6
11
-1
CACAUGUAGGUCAAAAAUGUAAUG
TTTT
615





Human-Exon 6
12
-1
ACAUGUAGGUCAAAAAUGUAAUGA
TTTT
616





Human-Exon 6
13
 1
ACAUUUUUGACCUACAUGUGGAAA
TTTA
617





Human-Exon 6
14
 1
CAUUACAUUUUUGACCUACAUGUG
TTTC
618





Human-Exon 6
15
-1
AAAAAUAUCAUGGCUGGAUUGCAA
TTTG
619





Human-Exon 6
16
-1
GCUGGAUUGCAACAAACCAACAGU
TTTC
620





Human-Exon 6
17
-1
CUGGAUUGCAACAAACCAACAGUG
TTTT
621





Human-Exon 6
18
 1
CCUAUGACUAUGGAUGAGAGCAUU
TTTG
622





Human-Exon 6
19
-1
UAGGUAAGAAGAUUACUGAGACAU
TTTA
623





Human-Exon 6
20
-1
AUUACUGAGACAUUAAAUAACUUG
TTTA
624





Human-Exon 6
21
-1
UUACUGAGACAUUAAAUAACUUGU
TTTT
625





Human-Exon 6
22
 1
GGGGAAAAAUAUGUCAUCAGAGUC
TTTA
626





Human-Exon 6
23
 1
CAUGAUCUGGAACCAUACUGGGGA
TTTT
627





Human-Exon 6
24
 1
ACAUGAUCUGGAACCAUACUGGGG
TTTT
628





Human-Exon 6
25
 1
GACAUGAUCUGGAACCAUACUGGG
TTTC
629





Human-Exon 7
 1
 1
uacacacauacacaAAGACAAAUA
TTTA
630





Human-Exon 7
 2
 1
uacacauacacacauacacaAAGA
TTTG
631





Human-Exon 7
 3
 1
aacacauacacauacacacauaca
TTtg
632





Human-Exon 7
 4
 1
AUUCCAGUCAAAUAGGUCUGGCCU
ttTT
633





Human-Exon 7
 5
 1
UAUUCCAGUCAAAUAGGUCUGGCC
tTTA
634





Human-Exon 7
 6
 1
GCUGGCAAACCACACUAUUCCAGU
TTTG
635





Human-Exon 7
 7
 1
AGUCGUUGUGUGGCUGACUGCUGG
TTTG
636





Human-Exon 7
 8
-1
CGCCAGAUAUCAAUUAGGCAUAGA
TTTC
637





Human-Exon 7
 9
-1
AAACUACUCGAUCCUGAAGGUUGG
TTTA
638





Human-Exon 7
10
 1
CAUACUAAAAGCAGUGGUAGUCCA
TTTC
639





Human-Exon 7
11
 1
GAAAACAUUAAACUCUACCAUACU
TTTT
640





Human-Exon 7
12
 1
UGAAAACAUUAAACUCUACCAUAC
TTTA
641





Human-Exon 8
 1
-1
UUGUUCAUUAUCCUUUUAGAGUCU
TTTG
642





Human-Exon 8
 2
 1
AAAGGAUAAUGAACAAAUCAAAGU
TTTA
643





Human-Exon 8
 3
-1
UAUCCUUUUAGAGUCUCAAAUAUA
TTTC
644





Human-Exon 8
 4
 1
ACUCUAAAAGGAUAAUGAACAAAU
TTTG
645





Human-Exon 8
 5
-1
UUUUAGAGUCUCAAAUAUAGAAAC
TTTG
646





Human-Exon 8
 6
-1
UUUAGAGUCUCAAAUAUAGAAACC
TTTT
647





Human-Exon 8
 7
-1
UUAGAGUCUCAAAUAUAGAAACCA
TTTT
648





Human-Exon 8
 8
 1
UUGAGACUCUAAAAGGAUAAUGAA
TTTG
649





Human-Exon 8
 9
 1
UUUGGUUUCUAUAUUUGAGACUCU
TTTT
650





Human-Exon 8
10
 1
UUUUGGUUUCUAUAUUUGAGACUC
TTTA
651





Human-Exon 8
11
-1
AGCAUUGAAGCCAUCCAGGAAGUG
TTTC
652





Human-Exon 8
12
 1
GCUUCAAUGCUCACUUGUUGAGGC
TTTT
653





Human-Exon 8
13
 1
GGCUUCAAUGCUCACUUGUUGAGG
TTTG
654





Human-Exon 8
14
-1
AGUGGAAAUGUUGCCAAGGCCACC
TTTA
655





Human-Exon 8
15
-1
GUUGCCAAGGCCACCUAAAGUGAC
TTTA
656





Human-Exon 8
16
-1
GAAGAACAUUUUCAGUUACAUCAU
TTTG
657





Human-Exon 8
17
-1
AUCAAAUGCACUAUUCUCAACAGG
TTTA
658





Human-Exon 8
18
 1
AUAGUGCAUUUGAUGAUGUAACUG
TTTT
659





Human-Exon 8
19
 1
AAUAGUGCAUUUGAUGAUGUAACU
TTTC
660





Human-Exon 8
20
-1
ACUAUUCUCAACAGGUAAAGUGUG
TTTA
661





Human-Exon 8
21
 1
UACCUAAAAAUGCAUAUAAAACAG
TTTT
662





Human-Exon 8
22
 1
AUACCUAAAAAUGCAUAUAAAACA
TTTC
663





Human-Exon 8
23
 1
CACGUAAUACCUAAAAAUGCAUAU
TTTT
664





Human-Exon 8
24
 1
GCACGUAAUACCUAAAAAUGCAUA
TTTA
665





Human-Exon 8
25
 1
auauauauGUGCACGUAAUACCUA
TTTT
666





Human-Exon 8
26
 1
uauauauauGUGCACGUAAUACCU
TTTT
667





Human-Exon 8
27
 1
auauauauauGUGCACGUAAUACC
TTTA
668





Human-Exon 55
 1
-1
CUGCACAAUAUUAUAGUUGUUGCU
TTTA
669





Human-Exon 55
 2
 1
AUAAAAAGAGAAAGAUGGAGGAAC
TTTA
670





Human-Exon 55
 3
 1
CACCUAGUGAACUCCAUAAAAAGA
TTTC
671





Human-Exon 55
 4
 1
AUGGUGCACCUAGUGAACUCCAUA
TTTT
672





Human-Exon 55
 5
 1
AAUGGUGCACCUAGUGAACUCCAU
TTTT
673





Human-Exon 55
 6
 1
GAAUGGUGCACCUAGUGAACUCCA
TTTA
674





Human-Exon 55
 7
 1
GACCAAAUGUUCAGAUGCAAUUAU
TTTA
675





Human-Exon 55
 8
 1
UCGCUCACUCACCCUGCAAAGGAC
TTTG
676





Human-Exon 55
 9
-1
AGUGAGCGAGAGGCUGCUUUGGAA
TTTC
677





Human-Exon 55
10
 1
GCAGCCUCUCGCUCACUCACCCUG
TTTG
678





Human-Exon 55
11
 1
UUGCAGUAAUCUAUGAGUUUCUUC
TTTG
679





Human-Exon 55
12
-1
CUGCAACAGUUCCCCCUGGACCUG
TTTC
680





Human-Exon 55
13
-1
UGCAACAGUUCCCCCUGGACCUGG
TTTT
681





Human-Exon 55
14
-1
UUUCUUGCCUGGCUUACAGAAGCU
TTTC
682





Human-Exon 55
15
 1
UUUCAGCUUCUGUAAGCCAGGCAA
TTTC
683





Human-Exon 55
16
-1
GUCCUACAGGAUGCUACCCGUAAG
TTTC
684





Human-Exon 55
17
-1
GGCUCCUAGAAGACUCCAAGGGAG
TTTA
685





Human-Exon 55
18
-1
GCUCCUAGAAGACUCCAAGGGAGU
TTTT
686





Human-Exon 55
19
-1
CUCCAAGGGAGUAAAAGAGCUGAU
TTTC
687





Human-Exon 55
20
 1
UGGAUCCACAAGAGUGCUAAAGCG
TTTC
688





Human-Exon 55
21
 1
GUUCAAUUGGAUCCACAAGAGUGC
TTTA
689





Human-Exon 55
22
-1
UACUUGUAACUGACAAGCCAGGGA
TTTG
690





Human-Exon 55
23
-1
ACUUGUAACUGACAAGCCAGGGAC
TTTT
691





Human-Exon 55
24
-1
GUAACUGACAAGCCAGGGACAAAA
TTTG
692





Human-Exon 55
25
-1
UAACUGACAAGCCAGGGACAAAAC
TTTT
693





Human-Exon 55
26
 1
UCCCUGGCUUGUCAGUUACAAGUA
TTTG
694





Human-G1-exon51

 1
CAGAGUAACAGUCUGAGUAGGAGc
TTTA
695





Human-G2-exon51

 1
uacuuuguuuagcaauacauggua
TTTC
696





Human-G3-exon51

-1
uggcucaaauuguuacucuucaau
TTTA
697





mouse-Exon23-G1

 1
CUUUCAAagaacuuugcagagccu
TTTG
698





mouse-Exon23-G2

 1
guugaaGCCAUUUUGUUGCUCUUU
TTTG
699





mouse-Exon23-G3

 1
guugaaGCCAUUUUAUUGCUCUUU
TTTG
700





mouse-Exon23-G4

-1
uuuugagGCUCUGCAAAGUUCUUU
TTTC
701





mouse-Exon23-G5

-1
aguuauuaaugcauagauauucag
TTTA
702





mouse-Exon23-G6

-1
uauaauaugcccuguaauauaaua
TTTC
703





mouse-Exon23-G7

 1
uaaaggccaaaccucggcuuaccU
TTTC
704





mouse-Exon23-G8

 1
ucaauaucuuugaaggacucuggg
TTTA
705





*In this table, upper case letters represent sgRNA nucleotides that align to the exon sequence of the gene. Lower case letters represent sgRNA nucleotides that align to the intron sequence of the gene.













TABLE 8







Genomic target sites for sgRNA in mouse Dmd


Exon 51














SEQ ID



ID sgRNA
Strand
Target site
NO:
PAM





Ex51-SA1
3′
AGAGTAACAGTCTGACTGG
706
CAG





Ex51-SD
5′
GAAATGATCATCAAACAGA
707
AGG





Ex51-SA-2
3′
CACTAGAGTAACAGTCTGAC
708
TGG
















TABLE 9







gRNA sequences targeting mouse Dmd Exon 51














SEQ ID



ID sgRNA
Strand
Target site
NO:
PAM





Ex51-SA1
3′
CCAGUCAGACUGUUACUCU
709
CAG





Ex51-SD
5′
UCUGUUUGAUGAUCAUUUC
710
AGG





Ex51-SA-2
3′
GUCAGACUGUUACUCUAGUG
711
TGG
















TABLE 10







Genomic target sequences for sgRNAs targeting


human Dmd Exon 51














SEQ ID



ID sgRNA
Strand
Target site
NO:
PAM





Ex51-SA
3
AGAGTAACAGTCTGAGTAG
712
GAG





Ex51-SD
5′
GAGATGATCATCAAGCAGA
713
AGG





Ex51-SA-2
3′
CACCAGAGTAACAGTCTGAG
714
TAG
















TABLE 11







sgRNA sequences targeting human Dmd Exon 51














SEQ ID



ID sgRNA
Strand
Target site
NO:
PAM





Ex51-SA
3′
CUACUCAGACUGUUACUCU
715
GAG





Ex51-SD
5′
UCUGCUUGAUGAUCAUCUC
716
AGG





Ex51-SA-2
3′
CUCAGACUGUUACUCUGGUG
717
TAG
















TABLE 12







Genomic target sequences for sgRNAs targeting 


sites in various human Dmd Exons














SEQ






ID



ID sgRNA
Strand
Target site
NO:
PAM





Exon51-#1
3′
CAGAGTAACAGTCTGAGTAG
947
GAG





Exon51-#2
3′
CACCAGAGTAACAGTCTGAG
718
TAG





Exon51-#3
3′
TATTTTGGGTTTTTGCAAAA
719
AGG





Exon51-#4
3′
AGTAGGAGCTAAAATATTTT
720
GGG





Exon51-#5
3′
GAGTAGGAGCTAAAATATTT
721
TGG





Exon51-#6
3′
ACCAGAGTAACAGTCTGAGT
722
AGG





Exon51-#7
5′
TCCTACTCAGACTGTTACTC
723
TGG





Exon51-#8
5′
TACTCTGGTGACACAACCTG
724
TGG





Exon51-#9
3′
GCAGTTTCCTTAGTAACCAC
725
AGG





Exon51-#10
5′
GACACAACCTGTGGTTACTA
726
AGG





Exon51-#11
3′
TGTCACCAGAGTAACAGTCT
727
GAG





Exon51-#12
3′
AGGTTGTGTCACCAGAGTAA
728
CAG





Exon51-#13
3′
AACCACAGGTTGTGTCACCA
729
GAG





Exon51-#14
3′
GTAACCACAGGTTGTGTCAC
730
CAG





Exon53-#1
5′
ATTTATTTTTCCTTTTATTC
731
TAG





Exon53-#2
5′
TTTCCTTTTATTCTAGTTGA
732
AAG





Exon53-#3
3′
TGATTCTGAATTCTTTCAAC
733
TAG





Exon53-#4
3′
AATTCTTTCAACTAGAATAA
734
AAG





Exon53-#6
5′
TTATTCTAGTTGAAAGAATT
735
CAG





Exon53-#7
5′
TAGTTGAAAGAATTCAGAAT
736
CAG





Exon53-#8
5′
AATTCAGAATCAGTGGGATG
737
AAG





Exon53-#9
3′
ATTCTTTCAACTAGAATAAA
738
AGG





Exon53-#10
5′
TTGAAAGAATTCAGAATCAG
739
TGG





Exon53-#11
5′
TGAAAGAATTCAGAATCAGT
740
GGG





Exon53-#12
3′
ACTGTTGCCTCCGGTTCTGA
741
AGG





Exon44-#1
3′
CAGATCTGTCAAATCGCCTG
742
CAG





Exon44-#2
3′
AAAACGCCGCCATTTCTCAA
743
CAG





Exon44-#3
3′
AGATCTGTCAAATCGCCTGC
744
AGG





Exon44-#4
3′
TATGGATCAAGAAAAATAGA
745
TGG





Exon44-#5
3′
CGCCTGCAGGTAAAAGCATA
746
TGG





Exon44-#6
5′
ATCCATATGCTTTTACCTGC
747
AGG





Exon44-#8
5′
TTGACAGATCTGTTGAGAAA
748
TGG





Exon44-#9
5′
ACAGATCTGTTGAGAAATGG
749
CGG





Exon44-#11
5′
GGCGATTTGACAGATCTGTT
750
GAG





Exon44-#13
5′
GGCGTTTTCATTATGATATA
751
AAG





Exon44-#14
5′
ATGATATAAAGATATTTAAT
752
CAG





Exon44-#15
5′
GATATTTAATCAGTGGCTAA
753
CAG





Exon44-#16
5′
ATTTAATCAGTGGCTAACAG
754
AAG





Exon44-#17
3′
AGAAACTGTTCAGCTTCTGT
755
TAG





Exon43-#1
5′
GTTTTAAAATTTTTATATTA
756
CAG





Exon43-#2
5′
TTTTATATTACAGAATATAA
757
AAG





Exon43-#3
5′
ATATTACAGAATATAAAAGA
758
TAG





Exon45-#1
3′
GTTCCTGTAAGATACCAAAA
759
AGG





Exon45-#2
5′
TTGCCTTTTTGGTATCTTAC
760
AGG





Exon45-#3
5′
TGGTATCTTACAGGAACTCC
761
AGG





Exon45-#4
5′
ATCTTACAGGAACTCCAGGA
762
TGG





Exon45-#5
3′
GCCGCTGCCCAATGCCATCC
763
TGG





Exon45-#6
5′
CAGGAACTCCAGGATGGCAT
764
TGG





Exon45-#7
5′
AGGAACTCCAGGATGGCATT
765
GGG





Exon45-#8
5′
TCCAGGATGGCATTGGGCAG
766
CGG





Exon45-#9
5′
GTCAGAACATTGAATGCAAC
767
TGG





Exon45-#10
3′
AGTTCCTGTAAGATACCAAA
768
AAG





Exon45-#11
3′
TGCCATCCTGGAGTTCCTGT
769
AAG





Exon45-#12
5′
TTGGTATCTTACAGGAACTC
770
CAG





Exon45-#13
3′
CGCTGCCCAATGCCATCCTG
771
GAG





Exon45-#14
5′
AACTCCAGGATGGCATTGGG
772
CAG





Exon45-#15
5′
GGGCAGCGGCAAACTGTTGT
773
CAG





Exon52-#1
3′
AGATCTGTCAAATCGCCTGC
774
AGG





Exon52-#2
3′
AATCCTGCATTGTTGCCTGT
775
AAG





Exon52-#3
5′
CGCTGAAGAACCCTGATACT
776
AAG





Exon52-#4
3′
GAACAAATATCCCTTAGTAT
777
CAG





Exon52-#5
3′
CTGTAAGAACAAATATCCCT
778
TAG





Exon52-#6
5′
CTAAGGGATATTTGTTCTTA
779
CAG





Exon52-#8
5′
TGTTCTTACAGGCAACAATG
780
CAG





Exon52-#9
5′
CAACAATGCAGGATTTGGAA
781
CAG





Exon52-#10
5′
ACAATGCAGGATTTGGAACA
782
GAG





Exon52-#11
5′
ATTTGGAACAGAGGCGTCCC
783
CAG





Exon52-#12
5′
ACAGAGGCGTCCCCAGTTGG
784
AAG





Exon2-#1
5′
TATTTTTTTATTTTGCATTT
785
TAG





Exon2-#2
5′
TTATTTTGCATTTTAGATGA
786
AAG





Exon2-#3
5′
ATTTTGCATTTTAGATGAAA
787
GAG





Exon2-#4
5′
TTGCATTTTAGATGAAAGAG
788
AAG





Exon2-#5
5′
ATGAAAGAGAAGATGTTCAA
789
AAG
















TABLE 13







gRNA sequences for targeting sites in various


human Dmd Exons














SEQ






ID



ID sgRNA
Strand
Target site
NO:
PAM





Exon51-#1
3′
CUACUCAGACUGUUACUCUG
790
GAG





Exon51-#2
3′
CUCAGACUGUUACUCUGGUG
791
TAG





Exon51-#3
3′
UUUUGCAAAAACCCAAAAUA
792
AGG





Exon51-#4
3′
AAAAUAUUUUAGCUCCUACU
793
GGG





Exon51-#5
3′
AAAUAUUUUAGCUCCUACUC
794
TGG





Exon51-#6
3′
ACUCAGACUGUUACUCUGGU
795
AGG





Exon51-#7
5′
GAGUAACAGUCUGAGUAGGA
796
TGG





Exon51-#8
5′
CAGGUUGUGUCACCAGAGUA
797
TGG





Exon51-#9
3′
GUGGUUACUAAGGAAACUGC
798
AGG





Exon51-#10
5′
UAGUAACCACAGGUUGUGUC
799
AGG





Exon51-#11
3′
AGACUGUUACUCUGGUGACA
800
GAG





Exon51-#12
3′
UUACUCUGGUGACACAACCU
801
CAG





Exon51-#13
3′
UGGUGACACAACCUGUGGUU
802
GAG





Exon51-#14
3′
GUGACACAACCUGUGGUUAC
803
CAG





Exon53-#1
5′
GAAUAAAAGGAAAAAUAAAU
804
TAG





Exon53-#2
5′
UCAACUAGAAUAAAAGGAAA
805
AAG





Exon53-#3
3′
GUUGAAAGAAUUCAGAAUCA
806
TAG





Exon53-#4
3′
UUAUUCUAGUUGAAAGAAUU
807
AAG





Exon53-#6
5′
AAUUCUUUCAACUAGAAUAA
808
CAG





Exon53-#7
5′
AUUCUGAAUUCUUUCAACUA
809
CAG





Exon53-#8
5′
CAUCCCACUGAUUCUGAAUU
810
AAG





Exon53-#9
3′
UUUAUUCUAGUUGAAAGAAU
811
AGG





Exon53-#10
5′
CUGAUUCUGAAUUCUUUCAA
812
TGG





Exon53-#11
5′
ACUGAUUCUGAAUUCUUUCA
813
GGG





Exon53-#12
3′
UCAGAACCGGAGGCAACAGU
814
AGG





Exon44-#1
3′
CAGGCGAUUUGACAGAUCUG
815
CAG





Exon44-#2
3′
UUGAGAAAUGGCGGCGUUUU
816
CAG





Exon44-#3
3′
GCAGGCGAUUUGACAGAUCU
817
AGG





Exon44-#4
3′
UCUAUUUUUCUUGAUCCAUA
818
TGG





Exon44-#5
3′
UAUGCUUUUACCUGCAGGCG
819
TGG





Exon44-#6
5′
GCAGGUAAAAGCAUAUGGAU
820
AGG





Exon44-#8
5′
UUUCUCAACAGAUCUGUCAA
821
TGG





Exon44-#9
5′
CCAUUUCUCAACAGAUCUGU
822
CGG





Exon44-#11
5′
AACAGAUCUGUCAAAUCGCC
823
GAG





Exon44-#13
5′
UAUAUCAUAAUGAAAACGCC
824
AAG





Exon44-#14
5′
AUUAAAUAUCUUUAUAUCAU
825
CAG





Exon44-#15
5′
UUAGCCACUGAUUAAAUAUC
826
CAG





Exon44-#16
5′
CUGUUAGCCACUGAUUAAAU
827
AAG





Exon44-#17
3′
ACAGAAGCUGAACAGUUUCU
828
TAG





Exon43-#1
5′
UAAUAUAAAAAUUUUAAAAC
829
CAG





Exon43-#2
5′
UUAUAUUCUGUAAUAUAAAA
830
AAG





Exon43-#3
5′
UCUUUUAUAUUCUGUAAUAU
831
TAG





Exon45-#1
3′
UUUUGGUAUCUUACAGGAAC
832
AGG





Exon45-#2
5′
GUAAGAUACCAAAAAGGCAA
833
AGG





Exon45-#3
5′
GGAGUUCCUGUAAGAUACCA
834
AGG





Exon45-#4
5′
UCCUGGAGUUCCUGUAAGAU
835
TGG





Exon45-#5
3′
GGAUGGCAUUGGGCAGCGGC
836
TGG





Exon45-#6
5′
AUGCCAUCCUGGAGUUCCUG
837
TGG





Exon45-#7
5′
AAUGCCAUCCUGGAGUUCCU
838
GGG





Exon45-#8
5′
CUGCCCAAUGCCAUCCUGGA
839
CGG





Exon45-#9
5′
GUUGCAUUCAAUGUUCUGAC
840
TGG





Exon45-#10
3′
UUUGGUAUCUUACAGGAACU
841
AAG





Exon45-#11
3′
ACAGGAACUCCAGGAUGGCA
842
AAG





Exon45-#12
5′
GAGUUCCUGUAAGAUACCAA
843
CAG





Exon45-#13
3′
CAGGAUGGCAUUGGGCAGCG
844
GAG





Exon45-#14
5′
CCCAAUGCCAUCCUGGAGUU
845
CAG





Exon45-#15
5′
ACAACAGUUUGCCGCUGCCC
846
CAG





Exon52-#1
3′
GCAGGCGAUUUGACAGAUCU
847
AGG





Exon52-#2
3′
ACAGGCAACAAUGCAGGAUU
848
AAG





Exon52-#3
5′
AGUAUCAGGGUUCUUCAGCG
849
AAG





Exon52-#4
3′
AUACUAAGGGAUAUUUGUUC
850
CAG





Exon52-#5
3′
AGGGAUAUUUGUUCUUACAG
851
TAG





Exon52-#6
5′
UAAGAACAAAUAUCCCUUAG
852
CAG





Exon52-#8
5′
CAUUGUUGCCUGUAAGAACA
853
CAG





Exon52-#9
5′
UUCCAAAUCCUGCAUUGUUG
854
CAG





Exon52-#10
5′
UGUUCCAAAUCCUGCAUUGU
855
GAG





Exon52-#11
5′
GGGACGCCUCUGUUCCAAAU
856
CAG





Exon52-#12
5′
CCAACUGGGGACGCCUCUGU
857
AAG





Exon2-#1
5′
ACAGAGGCGUCCCCAGUUGG
858
TAG





Exon2-#2
5′
UCAUCUAAAAUGCAAAAUAA
859
AAG





Exon2-#3
5′
UUUCAUCUAAAAUGCAAAAU
860
GAG





Exon2-#4
5′
CUCUUUCAUCUAAAAUGCAA
861
AAG





Exon2-#5
5′
UUGAACAUCUUCUCUUUCAU
862
AAG
















TABLE 14







Genomic targeting sequence for sgRNAs targeting


dog Dmd Exon 51














SEQ ID



ID sgRNA
Strand
Target site
NO:
PAM





Ex51-SA-2
3′
CACCAGAGTAACAGTCTGAC
863
TGG
















TABLE 15







gRNA sequence for targeting dog Dmd Exon 51














SEQ ID



ID sgRNA
Strand
Target site
NO:
PAM





Ex51-SA-2
3′
GUCAGACUGUUACUCUGGUG
864
TGG
















TABLE 16







Exon 43 & 45 gRNA sequences









sgRNA ID
Sequence (5′-3′)
SEQ ID NO.





Ex45-gRNA#3
CGCTGCCCAATGCCATCCTG
948





Ex45-gRNA#4
ATCTTACAGGAACTCCAGGA
949





Ex45-gRNA#5
AGGAACTCCAGGATGGCATT
950





Ex45-gRNA#6
CGCTGCCCAATGCCATCC
951





Ex43-gRNA#1
GTTTTAAAATTTTTATATTA
952





Ex43-gRNA#2
TTTTATATTACAGAATATAA
953





Ex43-gRNA#4
TATGTGTTACCTACCCTTGT
954





Ex43-gRNA#6
GTACAAGGACCGACAAGGGT
955
















TABLE 17







Exon 43 & 45 gRNA sequences









sgRNA ID
Sequence (5′-3′)
SEQ ID NO.





Ex45-gRNA#3
CAGGAUGGCAUUGGGCAGCG
956





Ex45-gRNA#4
UCCUGGAGUUCCUGUAAGAU
957





Ex45-gRNA#5
AAUGCCAUCCUGGAGUUCCU
958





Ex45-gRNA#6
GGAUGGCAUUGGGCAGCG
959





Ex43-gRNA#1
UAAUAUAAAAAUUUUAAAAC
960





Ex43-gRNA#2
UUAUAUUCUGUAAUAUAAAA
961





Ex43-gRNA#4
ACAAGGGUAGGUAACACAUA
962





Ex43-gRNA#6
ACCCUUGUCGGUCCUUGUAC
963





Ex45-gRNA#3′
CGCUGCCCAAUGCCAUCCUG
964





Ex45-gRNA#4′
AUCUUACAGGAACUCCAGGA
965





Ex45-gRNA#5′
AGGAACUCCAGGAUGGCAUU
966





Ex45-gRNA#6′
CGCUGCCCAAUGCCAUCC
967





Ex43-gRNA#1′
GUUUUAAAAUUUUUAUAUUA
968





Ex43-gRNA#2′
UUUUAUAUUACAGAAUAUAA
969





Ex43-gRNA#4′
UAUGUGUUACCUACCCUUGU
970





Ex43-gRNA#6′
GUACAAGGACCGACAAGGGU
971
















TABLE 18







gRNA sequences








Targeted















gRNA
Guide



SEQ ID

SEQ ID


Exon
#
Strand
PAM
DNA sequence*
NO.
RNA sequence*
NO.





Human-
 4
 1
tttt
tctttttcttcttttttccttttt
 972
ucuuuuucuucuuuuuuccuuuuu
1305


Exon 51












Human-
 5
 1
tttt
ctttttcttcttttttcctttttG
 973
cuuuuucuucuuuuuuccuuuuuG
1306


Exon 51












Human-
 6
 1
tttc
tttttcttcttttttcctttttGC
 974
uuuuucuucuuuuuuccuuuuuGC
1307


Exon 51












Human-
 7
 1
tttt
tcttcttttttcctttttGCAAAA
 975
ucuucuuuuuuccuuuuuGCAAAA
1308


Exon 51












Human-
 8
 1
tttt
cttcttttttcctttttGCAAAAA
 976
cuucuuuuuuccuuuuuGCAAAAA
1309


Exon 51












Human-
 9
 1
tttc
ttcttttttcctttttGCAAAAAC
 977
uucuuuuuuccuuuuuGCAAAAAC
1310


Exon 51












Human-
10
 1
tttt
ttcctttttGCAAAAACCCAAAAT
 978
uuccuuuuuGCAAAAACCCAAAAU
1311


Exon 51












Human-
11
 1
tttt
tcctttttGCAAAAACCCAAAATA
 979
uccuuuuuGCAAAAACCCAAAAUA
1312


Exon 51












Human-
12
 1
tttt
cctttttGCAAAAACCCAAAATAT
 980
ccuuuuuGCAAAAACCCAAAAUAU
1313


Exon 51












Human-
13
 1
tttc
ctttttGCAAAAACCCAAAATATT
 981
cuuuuuGCAAAAACCCAAAAUAUU
1314


Exon 51












Human-
14
 1
tttt
tGCAAAAACCCAAAATATTTTAGC
 982
uGCAAAAACCCAAAAUAUUUUAGC
1315


Exon 51












Human-
15
 1
tttt
GCAAAAACCCAAAATATTTTAGCT
 983
GCAAAAACCCAAAAUAUUUUAGCU
1316


Exon 51












Human-
16
 1
tttG
CAAAAACCCAAAATATTTTAGCTC
 984
CAAAAACCCAAAAUAUUUUAGCUC
1317


Exon 51












Human-
17
 1
TTTT
AGCTCCTACTCAGACTGTTACTCT
 985
AGCUCCUACUCAGACUGUUACUCU
1318


Exon 51












Human-
18
 1
TTTA
GCTCCTACTCAGACTGTTACTCTG
 986
GCUCCUACUCAGACUGUUACUCUG
1319


Exon 51












Human-
19
-1
TTTC
CTTAGTAACCACAGGTTGTGTCAC
 987
CUUAGUAACCACAGGUUGUGUCAC
1320


Exon 51












Human-
20
-1
TTTG
GAGATGGCAGTTTCCTTAGTAACC
 988
GAGAUGGCAGUUUCCUUAGUAACC
1321


Exon 51












Human-
21
-1
TTTC
TAGTTTGGAGATGGCAGTTTCCTT
 999
UAGUUUGGAGAUGGCAGUUUCCUU
1322


Exon 51












Human-
22
-1
TTTT
TTCTCATACCTTCTGCTTGATGAT
1000
UUCUCAUACCUUCUGCUUGAUGAU
1323


Exon 51












Human-
23
-1
TTTA
TCATTTTTTCTCATACCTTCTGCT
1001
UCAUUUUUUCUCAUACCUUCUGCU
1324


Exon 51












Human-
24
-1
TTTT
ATCATTTTTTCTCATACCTTCTGC
1002
AUCAUUUUUUCUCAUACCUUCUGC
1325


Exon 51












Human-
25
-1
TTTA
AAGAAAAACTTCTGCCAACTTTTA
1003
AAGAAAAACUUCUGCCAACUUUUA
1326


Exon 51












Human-
26
-1
TTTT
AAAGAAAAACTTCTGCCAACTTTT
1004
AAAGAAAAACUUCUGCCAACUUUU
1327


Exon 51












Human-
27
 1
TTTT
TCTTTAAAATGAAGATTTTCCACC
1005
UCUUUAAAAUGAAGAUUUUCCACC
1328


Exon 51












Human-
28
 1
TTTT
CTTTAAAATGAAGATTTTCCACCA
1006
CUUUAAAAUGAAGAUUUUCCACCA
1329


Exon 51












Human-
29
 1
TTTC
TTTAAAATGAAGATTTTCCACCAA
1007
UUUAAAAUGAAGAUUUUCCACCAA
1330


Exon 51












Human-
30
 1
TTTA
AAATGAAGATTTTCCACCAATCAC
1008
AAAUGAAGAUUUUCCACCAAUCAC
1331


Exon 51












Human-
31
 1
TTTT
CCACCAATCACTTTACTCTCCTAG
1009
CCACCAAUCACUUUACUCUCCUAG
1332


Exon 51












Human-
32
 1
TTTC
CACCAATCACTTTACTCTCCTAGA
1010
CACCAAUCACUUUACUCUCCUAGA
1333


Exon 51












Human-
33
 1
TTTA
CTCTCCTAGACCATTTCCCACCAG
1011
CUCUCCUAGACCAUUUCCCACCAG
1334


Exon 51












Human-
 1
-1
tttg
agaaaagattaaacagtgtgctac
1012
agaaaagauuaaacagugugcuac
1335


Exon 45












Human-
 2
-1
TTTa
tttgagaaaagattaaacagtgtg
1013
uuugagaaaagauuaaacagugug
1336


Exon 45












Human-
 3
-1
TTTT
atttgagaaaagattaaacagtgt
1014
auuugagaaaagauuaaacagugu
1337


Exon 45












Human-
 4
-1
TTTT
Tatttgagaaaagattaaacagtg
1015
Uauuugagaaaagauuaaacagug
1338


Exon 45












Human-
 5
 1
ttta
atcttttctcaaatAAAAAGACAT
1016
aucuuuucucaaauAAAAAGACAU
1339


Exon 45












Human-
 6
 1
tttt
ctcaaatAAAAAGACATGGGGCTT
1017
cucaaauAAAAAGACAUGGGGCUU
1340


Exon 45












Human-
 7
 1
tttc
tcaaatAAAAAGACATGGGGCTTC
1018
ucaaauAAAAAGACAUGGGGCUUC
1341


Exon 45












Human-
 8
 1
TTTT
TGTTTTGCCTTTTTGGTATCTTAC
1019
UGUUUUGCCUUUUUGGUAUCUUAC
1342


Exon 45












Human-
 9
 1
TTTT
GTTTTGCCTTTTTGGTATCTTACA
1020
GUUUUGCCUUUUUGGUAUCUUACA
1343


Exon 45












Human-
10
 1
TTTG
TTTTGCCTTTTTGGTATCTTACAG
1021
UUUUGCCUUUUUGGUAUCUUACAG
1344


Exon 45












Human-
11
 1
TTTT
GCCTTTTTGGTATCTTACAGGAAC
1022
GCCUUUUUGGUAUCUUACAGGAAC
1345


Exon 45












Human-
12
 1
TTTG
CCTTTTTGGTATCTTACAGGAACT
1023
CCUUUUUGGUAUCUUACAGGAACU
1346


Exon 45












Human-
13
 1
TTTT
TGGTATCTTACAGGAACTCCAGGA
1024
UGGUAUCUUACAGGAACUCCAGGA
1347


Exon 45












Human-
14
 1
TTTT
GGTATCTTACAGGAACTCCAGGAT
1025
GGUAUCUUACAGGAACUCCAGGAU
1348


Exon 45












Human-
15
-1
TTTG
AGGATTGCTGAATTATTTCTTCCC
1026
AGGAUUGCUGAAUUAUUUCUUCCC
1349


Exon 45












Human-
16
-1
TTTT
GAGGATTGCTGAATTATTTCTTCC
1027
GAGGAUUGCUGAAUUAUUUCUUCC
1350


Exon 45












Human-
17
-1
TTTT
TGAGGATTGCTGAATTATTTCTTC
1028
UGAGGAUUGCUGAAUUAUUUCUUC
1351


Exon 45












Human-
18
-1
TTTC
CTGTAGAATACTGGCATCTGTTTT
1029
CUGUAGAAUACUGGCAUCUGUUUU
1352


Exon 45












Human-
19
-1
TTTT
CCTGTAGAATACTGGCATCTGTTT
1030
CCUGUAGAAUACUGGCAUCUGUUU
1353


Exon 45












Human-
20
-1
TTTT
TCCTGTAGAATACTGGCATCTGTT
1031
UCCUGUAGAAUACUGGCAUCUGUU
1354


Exon 45












Human-
21
-1
TTTG
CAGACCTCCTGCCACCGCAGATTC
1032
CAGACCUCCUGCCACCGCAGAUUC
1355


Exon 45












Human-
22
-1
TTTC
TGTCTGACAGCTGTTTGCAGACCT
1033
UGUCUGACAGCUGUUUGCAGACCU
1356


Exon 45












Human-
23
-1
TTTT
CTGTCTGACAGCTGTTTGCAGACC
1034
CUGUCUGACAGCUGUUUGCAGACC
1357


Exon 45












Human-
24
-1
TTTT
TCTGTCTGACAGCTGTTTGCAGAC
1035
UCUGUCUGACAGCUGUUUGCAGAC
1358


Exon 45












Human-
25
-1
TTTT
TTCTGTCTGACAGCTGTTTGCAGA
1036
UUCUGUCUGACAGCUGUUUGCAGA
1359


Exon 45












Human-
26
-1
TTTC
ATTCCTATTAGATCTGTCGCCCTA
1037
AUUCCUAUUAGAUCUGUCGCCCUA
1360


Exon 45












Human-
27
-1
TTTT
CATTCCTATTAGATCTGTCGCCCT
1038
CAUUCCUAUUAGAUCUGUCGCCCU
1361


Exon 45












Human-
28
 1
TTTT
AGCAGACTTTTTAAGCTTTCTTTA
1039
AGCAGACUUUUUAAGCUUUCUUUA
1362


Exon 45












Human-
29
 1
TTTA
GCAGACTTTTTAAGCTTTCTTTAG
1040
GCAGACUUUUUAAGCUUUCUUUAG
1363


Exon 45












Human-
30
 1
TTTT
TAAGCTTTCTTTAGAAGAATATTT
1041
UAAGCUUUCUUUAGAAGAAUAUUU
1364


Exon 45












Human-
31
 1
TTTT
AAGCTTTCTTTAGAAGAATATTTC
1042
AAGCUUUCUUUAGAAGAAUAUUUC
1365


Exon 45












Human-
32
 1
TTTA
AGCTTTCTTTAGAAGAATATTTCA
1043
AGCUUUCUUUAGAAGAAUAUUUCA
1366


Exon 45












Human-
33
 1
TTTC
TTTAGAAGAATATTTCATGAGAGA
1044
UUUAGAAGAAUAUUUCAUGAGAGA
1367


Exon 45












Human-
34
 1
TTTA
GAAGAATATTTCATGAGAGATTAT
1045
GAAGAAUAUUUCAUGAGAGAUUAU
1368


Exon 45












Human-
 1
 1
TTTG
TCAGTATAACCAAAAAATATACGC
1046
UCAGUAUAACCAAAAAAUAUACGC
1369


Exon 44












Human-
 2
 1
tttt
acataatccatctatttttcttga
1047
acauaauccaucuauuuuucuuga
1370


Exon 44












Human-
 3
 1
ttta
cataatccatctatttttcttgat
1048
cauaauccaucuauuuuucuugau
1371


Exon 44












Human-
 4
 1
tttt
tcttgatccatatgcttttACCTG
1049
ucuugauccauaugcuuuuACCUG
1372


Exon 44












Human-
 5
 1
tttt
cttgatccatatgcttttACCTGC
1050
cuugauccauaugcuuuuACCUGC
1373


Exon 44












Human-
 6
 1
tttc
ttgatccatatgcttttACCTGCA
1051
uugauccauaugcuuuuACCUGCA
1374


Exon 44












Human-
 7
-1
TTTC
TCAACAGATCTGTCAAATCGCCTG
1052
UCAACAGAUCUGUCAAAUCGCCUG
1375


Exon 44












Human-
 8
 1
tttt
ACCTGCAGGCGATTTGACAGATCT
1053
ACCUGCAGGCGAUUUGACAGAUCU
1376


Exon 44












Human-
 9
 1
tttA
CCTGCAGGCGATTTGACAGATCTG
1054
CCUGCAGGCGAUUUGACAGAUCUG
1377


Exon 44












Human-
10
 1
TTTG
ACAGATCTGTTGAGAAATGGCGGC
1055
ACAGAUCUGUUGAGAAAUGGCGGC
1378


Exon 44












Human-
11
-1
TTTA
TATCATAATGAAAACGCCGCCATT
1056
UAUCAUAAUGAAAACGCCGCCAUU
1379


Exon 44












Human-
12
 1
TTTT
CATTATGATATAAAGATATTTAAT
1057
CAUUAUGAUAUAAAGAUAUUUAAU
1380


Exon 44












Human-
13
-1
TTTG
TATTTAGCATGTTCCCAATTCTCA
1058
UAUUUAGCAUGUUCCCAAUUCUCA
1381


Exon 44












Human-
14
-1
TTTC
GAAAAAACAAATCAAAGACTTACC
1059
GAAAAAACAAAUCAAAGACUUACC
1382


Exon 44












Human-
15
 1
TTTG
ATTTGTTTTTTCGAAATTGTATTT
1060
AUUUGUUUUUUCGAAAUUGUAUUU
1383


Exon 44












Human-
16
 1
TTTG
TTTTTTCGAAATTGTATTTATCTT
1061
UUUUUUCGAAAUUGUAUUUAUCUU
1384


Exon 44












Human-
17
 1
TTTT
TTCGAAATTGTATTTATCTTCAGC
1062
UUCGAAAUUGUAUUUAUCUUCAGC
1385


Exon 44












Human-
18
 1
TTTT
TCGAAATTGTATTTATCTTCAGCA
1063
UCGAAAUUGUAUUUAUCUUCAGCA
1386


Exon 44












Human-
19
 1
TTTT
CGAAATTGTATTTATCTTCAGCAC
1064
CGAAAUUGUAUUUAUCUUCAGCAC
1387


Exon 44












Human-
20
 1
TTTC
GAAATTGTATTTATCTTCAGCACA
1065
GAAAUUGUAUUUAUCUUCAGCACA
1388


Exon 44












Human-
21
-1
TTTA
AGAAGTTAAAGAGTCCAGATGTGC
1066
AGAAGUUAAAGAGUCCAGAUGUGC
1389


Exon 44












Human-
22
 1
TTTA
TCTTCAGCACATCTGGACTCTTTA
1067
UCUUCAGCACAUCUGGACUCUUUA
1390


Exon 44












Human-
23
-1
TTTC
CATCACCCTTCAGAACCTGATCTT
1068
CAUCACCCUUCAGAACCUGAUCUU
1391


Exon 44












Human-
24
 1
TTTA
ACTTCTTAAAGATCAGGTTCTGAA
1069
ACUUCUUAAAGAUCAGGUUCUGAA
1392


Exon 44












Human-
25
 1
TTTT
GACTGTTGTTGTCATCATTATATT
1070
GACUGUUGUUGUCAUCAUUAUAUU
1393


Exon 44












Human-
26
 1
TTTG
ACTGTTGTTGTCATCATTATATTA
1071
ACUGUUGUUGUCAUCAUUAUAUUA
1394


Exon 44












Human-
 1
-1
TTTC
AACTAGAATAAAAGGAAAAATAAA
1072
AACUAGAAUAAAAGGAAAAAUAAA
1395


Exon 53












Human-
 2
 1
TTTA
CTACTATATATTTATTTTTCCTTT
1073
CUACUAUAUAUUUAUUUUUCCUUU
1396


Exon 53












Human-
 3
 1
TTTA
TTTTTCCTTTTATTCTAGTTGAAA
1074
UUUUUCCUUUUAUUCUAGUUGAAA
1397


Exon 53












Human-
 4
 1
TTTT
TCCTTTTATTCTAGTTGAAAGAAT
1075
UCCUUUUAUUCUAGUUGAAAGAAU
1398


Exon 53












Human-
 5
 1
TTTT
CCTTTTATTCTAGTTGAAAGAATT
1076
CCUUUUAUUCUAGUUGAAAGAAUU
1399


Exon 53












Human-
 6
 1
TTTC
CTTTTATTCTAGTTGAAAGAATTC
1077
CUUUUAUUCUAGUUGAAAGAAUUC
1400


Exon 53












Human-
 7
 1
TTTT
ATTCTAGTTGAAAGAATTCAGAAT
1078
AUUCUAGUUGAAAGAAUUCAGAAU
1401


Exon 53












Human-
 8
 1
TTTA
TTCTAGTTGAAAGAATTCAGAATC
1079
UUCUAGUUGAAAGAAUUCAGAAUC
1402


Exon 53












Human-
 9
-1
TTTC
ATTCAACTGTTGCCTCCGGTTCTG
1080
AUUCAACUGUUGCCUCCGGUUCUG
1403


Exon 53












Human-
10
-1
TTTA
ACATTTCATTCAACTGTTGCCTCC
1081
ACAUUUCAUUCAACUGUUGCCUCC
1404


Exon 53












Human-
11
-1
TTTT
CTTTTGGATTGCATCTACTGTATA
1082
CUUUUGGAUUGCAUCUACUGUAUA
1405


Exon 53












Human-
12
-1
TTTC
TGTGATTTTCTTTTGGATTGCATC
1083
UGUGAUUUUCUUUUGGAUUGCAUC
1406


Exon 53












Human-
13
-1
TTTG
ATACTAACCTTGGTTTCTGTGATT
1084
AUACUAACCUUGGUUUCUGUGAUU
1407


Exon 53












Human-
14
-1
TTTA
AAAAGGTATCTTTGATACTAACCT
1085
AAAAGGUAUCUUUGAUACUAACCU
1408


Exon 53












Human-
15
-1
TTTT
AAAAAGGTATCTTTGATACTAACC
1086
AAAAAGGUAUCUUUGAUACUAACC
1409


Exon 53












Human-
16
-1
TTTA
TTTTAAAAAGGTATCTTTGATACT
1087
UUUUAAAAAGGUAUCUUUGAUACU
1410


Exon 53












Human-
17
-1
TTTT
ATTTTAAAAAGGTATCTTTGATAC
1088
AUUUUAAAAAGGUAUCUUUGAUAC
1411


Exon 53












Human-
 1
-1
TTTG
TTAATGCAAACTGGGACACAAACA
1089
UUAAUGCAAACUGGGACACAAACA
1412


Exon 46












Human-
 2
 1
TTTT
TAAATTGCCATGTTTGTGTCCCAG
1090
UAAAUUGCCAUGUUUGUGUCCCAG
1413


Exon 46












Human-
 3
 1
TTTT
AAATTGCCATGTTTGTGTCCCAGT
1091
AAAUUGCCAUGUUUGUGUCCCAGU
1414


Exon 46












Human-
 4
 1
TTTA
AATTGCCATGTTTGTGTCCCAGTT
1092
AAUUGCCAUGUUUGUGUCCCAGUU
1415


Exon 46












Human-
 5
 1
TTTG
TGTCCCAGTTTGCATTAACAAATA
1093
UGUCCCAGUUUGCAUUAACAAAUA
1416


Exon 46












Human-
 6
-1
tttC
CAACATAGTTCTCAAACTATTTGT
1094
CAACAUAGUUCUCAAACUAUUUGU
1417


Exon 46












Human-
 7
-1
tttt
CCAACATAGTTCTCAAACTATTTG
1095
CCAACAUAGUUCUCAAACUAUUUG
1418


Exon 46












Human-
 8
-1
tttt
tCCAACATAGTTCTCAAACTATTT
1096
uCCAACAUAGUUCUCAAACUAUUU
1419


Exon 46












Human-
 9
-1
tttt
tttCCAACATAGTTCTCAAACTAT
1097
uuuCCAACAUAGUUCUCAAACUAU
1420


Exon 46












Human-
10
-1
tttt
ttttCCAACATAGTTCTCAAACTA
1098
uuuuCCAACAUAGUUCUCAAACUA
1421


Exon 46












Human-
11
-1
tttt
tttttCCAACATAGTTCTCAAACT
1099
uuuuuCCAACAUAGUUCUCAAACU
1422


Exon 46












Human-
12
 1
TTTG
CATTAACAAATAGTTTGAGAACTA
1100
CAUUAACAAAUAGUUUGAGAACUA
1423


Exon 46












Human-
13
 1
TTTG
AGAACTATGTTGGaaaaaaaaaTA
1101
AGAACUAUGUUGGaaaaaaaaaUA
1424


Exon 46












Human-
14
-1
TTTT
GTTCTTCTAGCCTGGAGAAAGAAG
1102
GUUCUUCUAGCCUGGAGAAAGAAG
1425


Exon 46












Human-
15
 1
TTTT
ATTCTTCTTTCTCCAGGCTAGAAG
1103
AUUCUUCUUUCUCCAGGCUAGAAG
1426


Exon 46












Human-
16
 1
TTTA
TTCTTCTTTCTCCAGGCTAGAAGA
1104
UUCUUCUUUCUCCAGGCUAGAAGA
1427


Exon 46












Human-
17
 1
TTTC
TCCAGGCTAGAAGAACAAAAGAAT
1105
UCCAGGCUAGAAGAACAAAAGAAU
1428


Exon 46












Human-
18
-1
TTTG
AAATTCTGACAAGATATTCTTTTG
1106
AAAUUCUGACAAGAUAUUCUUUUG
1429


Exon 46












Human-
19
-1
TTTT
CTTTTAGTTGCTGCTCTTTTCCAG
1107
CUUUUAGUUGCUGCUCUUUUCCAG
1430


Exon 46












Human-
20
-1
TTTG
AGAAAATAAAATTACCTTGACTTG
1108
AGAAAAUAAAAUUACCUUGACUUG
1431


Exon 46












Human-
21
-1
TTTA
TGCAAGCAGGCCCTGGGGGATTTG
1109
UGCAAGCAGGCCCUGGGGGAUUUG
1432


Exon 46












Human-
22
 1
TTTT
ATTTTCTCAAATCCCCCAGGGCCT
1110
AUUUUCUCAAAUCCCCCAGGGCCU
1433


Exon 46












Human-
23
 1
TTTA
TTTTCTCAAATCCCCCAGGGCCTG
1111
UUUUCUCAAAUCCCCCAGGGCCUG
1434


Exon 46












Human-
24
 1
TTTT
CTCAAATCCCCCAGGGCCTGCTTG
1112
CUCAAAUCCCCCAGGGCCUGCUUG
1435


Exon 46












Human-
25
 1
TTTC
TCAAATCCCCCAGGGCCTGCTTGC
1113
UCAAAUCCCCCAGGGCCUGCUUGC
1436


Exon 46












Human-
26
 1
TTTT
TTAATTCAATCATTGGTTTTCTGC
1114
UUAAUUCAAUCAUUGGUUUUCUGC
1437


Exon 46












Human-
27
 1
TTTT
TAATTCAATCATTGGTTTTCTGCC
1115
UAAUUCAAUCAUUGGUUUUCUGCC
1438


Exon 46












Human-
28
 1
TTTT
AATTCAATCATTGGTTTTCTGCCC
1116
AAUUCAAUCAUUGGUUUUCUGCCC
1439


Exon 46












Human-
29
 1
TTTA
ATTCAATCATTGGTTTTCTGCCCA
1117
AUUCAAUCAUUGGUUUUCUGCCCA
1440


Exon 46












Human-
30
-1
TTTA
GCAAGGAACTATGAATAACCTAAT
1118
GCAAGGAACUAUGAAUAACCUAAU
1441


Exon 46












Human-
31
 1
TTTT
CTGCCCATTAGGTTATTCATAGTT
1119
CUGCCCAUUAGGUUAUUCAUAGUU
1442


Exon 46












Human-
32
 1
TTTC
TGCCCATTAGGTTATTCATAGTTC
1120
UGCCCAUUAGGUUAUUCAUAGUUC
1443


Exon 46












Human-
 1
-1
TTTA
TAGAAAACAATTTAACAGGAAATA
1121
UAGAAAACAAUUUAACAGGAAAUA
1444


Exon 52












Human-
 2
 1
TTTC
CTGTTAAATTGTTTTCTATAAACC
1122
CUGUUAAAUUGUUUUCUAUAAACC
1445


Exon 52












Human-
 3
-1
TTTA
GAAATAAAAAAGATGTTACTGTAT
1123
GAAAUAAAAAAGAUGUUACUGUAU
1446


Exon 52












Human-
 4
-1
TTTT
AGAAATAAAAAAGATGTTACTGTA
1124
AGAAAUAAAAAAGAUGUUACUGUA
1447


Exon 52












Human-
 5
 1
TTTT
CTATAAACCCTTATACAGTAACAT
1125
CUAUAAACCCUUAUACAGUAACAU
1448


Exon 52












Human-
 6
 1
TTTC
TATAAACCCTTATACAGTAACATC
1126
UAUAAACCCUUAUACAGUAACAUC
1449


Exon 52












Human-
 7
 1
TTTT
TTATTTCTAAAAGTGTTTTGGCTG
1127
UUAUUUCUAAAAGUGUUUUGGCUG
1450


Exon 52












Human-
 8
 1
TTTT
TATTTCTAAAAGTGTTTTGGCTGG
1128
UAUUUCUAAAAGUGUUUUGGCUGG
1451


Exon 52












Human-
 9
 1
TTTT
ATTTCTAAAAGTGTTTTGGCTGGT
1129
AUUUCUAAAAGUGUUUUGGCUGGU
1452


Exon 52












Human-
10
 1
TTTA
TTTCTAAAAGTGTTTTGGCTGGTC
1130
UUUCUAAAAGUGUUUUGGCUGGUC
1453


Exon 52












Human-
11
 1
TTTC
TAAAAGTGTTTTGGCTGGTCTCAC
1131
UAAAAGUGUUUUGGCUGGUCUCAC
1454


Exon 52












Human-
12
-1
TTTA
CATAATACAAAGTAAAGTACAATT
1132
CAUAAUACAAAGUAAAGUACAAUU
1455


Exon 52












Human-
13
-1
TTTT
ACATAATACAAAGTAAAGTACAAT
1133
ACAUAAUACAAAGUAAAGUACAAU
1456


Exon 52












Human-
14
 1
TTTT
GGCTGGTCTCACAATTGTACTTTA
1134
GGCUGGUCUCACAAUUGUACUUUA
1457


Exon 52












Human-
15
 1
TTTG
GCTGGTCTCACAATTGTACTTTAC
1135
GCUGGUCUCACAAUUGUACUUUAC
1458


Exon 52












Human-
16
 1
TTTA
CTTTGTATTATGTAAAAGGAATAC
1136
CUUUGUAUUAUGUAAAAGGAAUAC
1459


Exon 52












Human-
17
 1
TTTG
TATTATGTAAAAGGAATACACAAC
1137
UAUUAUGUAAAAGGAAUACACAAC
1460


Exon 52












Human-
18
 1
TTTG
TTCTTACAGGCAACAATGCAGGAT
1138
UUCUUACAGGCAACAAUGCAGGAU
1461


Exon 52












Human-
19
 1
TTTG
GAACAGAGGCGTCCCCAGTTGGAA
1139
GAACAGAGGCGUCCCCAGUUGGAA
1462


Exon 52












Human-
20
-1
TTTG
GGCAGCGGTAATGAGTTCTTCCAA
1140
GGCAGCGGUAAUGAGUUCUUCCAA
1463


Exon 52












Human-
21
-1
TTTT
TCAAATTTTGGGCAGCGGTAATGA
1141
UCAAAUUUUGGGCAGCGGUAAUGA
1464


Exon 52












Human-
22
 1
TTTG
AAAAACAAGACCAGCAATCAAGAG
1142
AAAAACAAGACCAGCAAUCAAGAG
1465


Exon 52












Human-
23
-1
TTTG
TGTGTCCCATGCTTGTTAAAAAAC
1143
UGUGUCCCAUGCUUGUUAAAAAAC
1466


Exon 52












Human-
24
 1
TTTT
TTAACAAGCATGGGACACACAAAG
1144
UUAACAAGCAUGGGACACACAAAG
1467


Exon 52












Human-
25
 1
TTTT
TAACAAGCATGGGACACACAAAGC
1145
UAACAAGCAUGGGACACACAAAGC
1468


Exon 52












Human-
26
 1
TTTT
AACAAGCATGGGACACACAAAGCA
1146
AACAAGCAUGGGACACACAAAGCA
1469


Exon 52












Human-
27
 1
TTTA
ACAAGCATGGGACACACAAAGCAA
1147
ACAAGCAUGGGACACACAAAGCAA
1470


Exon 52












Human-
28
-1
TTTA
TTGAAACTTGTCATGCATCTTGCT
1148
UUGAAACUUGUCAUGCAUCUUGCU
1471


Exon 52












Human-
29
-1
TTTT
ATTGAAACTTGTCATGCATCTTGC
1149
AUUGAAACUUGUCAUGCAUCUUGC
1472


Exon 52












Human-
30
-1
TTTT
TATTGAAACTTGTCATGCATCTTG
1150
UAUUGAAACUUGUCAUGCAUCUUG
1473


Exon 52












Human-
31
 1
TTTC
AATAAAAACTTAAGTTCATATATC
1151
AAUAAAAACUUAAGUUCAUAUAUC
1474


Exon 52












Human-
 1
-1
TTTG
GTGAATATATTATTGGATTTCTAT
1152
GUGAAUAUAUUAUUGGAUUUCUAU
1475


Exon 50












Human-
 2
-1
TTTG
AAGATAATTCATGAACATCTTAAT
1153
AAGAUAAUUCAUGAACAUCUUAAU
1476


Exon 50












Human-
 3
-1
TTTA
ACAGAAAAGCATACACATTACTTA
1154
ACAGAAAAGCAUACACAUUACUUA
1477


Exon 50












Human-
 4
 1
TTTT
CTGTTAAAGAGGAAGTTAGAAGAT
1155
CUGUUAAAGAGGAAGUUAGAAGAU
1478


Exon 50












Human-
 5
 1
TTTC
TGTTAAAGAGGAAGTTAGAAGATC
1156
UGUUAAAGAGGAAGUUAGAAGAUC
1479


Exon 50












Human-
 6
-1
TTTA
CCGCCTTCCACTCAGAGCTCAGAT
1157
CCGCCUUCCACUCAGAGCUCAGAU
1480


Exon 50












Human-
 7
-1
TTTG
CCCTCAGCTCTTGAAGTAAACGGT
1158
CCCUCAGCUCUUGAAGUAAACGGU
1481


Exon 50












Human-
 8
 1
TTTA
CTTCAAGAGCTGAGGGCAAAGCAG
1159
CUUCAAGAGCUGAGGGCAAAGCAG
1482


Exon 50












Human-
 9
-1
TTTG
AACAAATAGCTAGAGCCAAAGAGA
1160
AACAAAUAGCUAGAGCCAAAGAGA
1483


Exon 50












Human-
10
-1
TTTT
GAACAAATAGCTAGAGCCAAAGAG
1161
GAACAAAUAGCUAGAGCCAAAGAG
1484


Exon 50












Human-
11
 1
TTTG
GCTCTAGCTATTTGTTCAAAAGTG
1162
GCUCUAGCUAUUUGUUCAAAAGUG
1485


Exon 50












Human-
12
 1
TTTG
TTCAAAAGTGCAACTATGAAGTGA
1163
UUCAAAAGUGCAACUAUGAAGUGA
1486


Exon 50












Human-
13
-1
TTTC
TCTCTCACCCAGTCATCACTTCAT
1164
UCUCUCACCCAGUCAUCACUUCAU
1487


Exon 50












Human-
14
-1
TTTT
CTCTCTCACCCAGTCATCACTTCA
1165
CUCUCUCACCCAGUCAUCACUUCA
1488


Exon 50












Human-
 1
 1
TTTG
tatatatatatatatTTTTCTCTT
1166
uauauauauauauauUUUUCUCUU
1489


Exon 43












Human-
 2
 1
tTTT
TCTCTTTCTATAGACAGCTAATTC
1167
UCUCUUUCUAUAGACAGCUAAUUC
1490


Exon 43












Human-
 3
 1
TTTT
CTCTTTCTATAGACAGCTAATTCA
1168
CUCUUUCUAUAGACAGCUAAUUCA
1491


Exon 43












Human-
 4
-1
TTTA
AAACAGTAAAAAAATGAATTAGCT
1169
AAACAGUAAAAAAAUGAAUUAGCU
1492


Exon 43












Human-
 5
 1
TTTC
TCTTTCTATAGACAGCTAATTCAT
1170
UCUUUCUAUAGACAGCUAAUUCAU
1493


Exon 43












Human-
 6
-1
TTTT
AAAACAGTAAAAAAATGAATTAGC
1171
AAAACAGUAAAAAAAUGAAUUAGC
1494


Exon 43












Human-
 7
 1
TTTC
TATAGACAGCTAATTCATTTTTTT
1172
UAUAGACAGCUAAUUCAUUUUUUU
1495


Exon 43












Human-
 8
-1
TTTA
TATTCTGTAATATAAAAATTTTAA
1173
UAUUCUGUAAUAUAAAAAUUUUAA
1496


Exon 43












Human-
 9
-1
TTTT
ATATTCTGTAATATAAAAATTTTA
1174
AUAUUCUGUAAUAUAAAAAUUUUA
1497


Exon 43












Human-
10
 1
TTTT
TTTACTGTTTTAAAATTTTTATAT
1175
UUUACUGUUUUAAAAUUUUUAUAU
1498


Exon 43












Human-
11
 1
TTTT
TTACTGTTTTAAAATTTTTATATT
1176
UUACUGUUUUAAAAUUUUUAUAUU
1499


Exon 43












Human-
12
 1
TTTT
TACTGTTTTAAAATTTTTATATTA
1177
UACUGUUUUAAAAUUUUUAUAUUA
1500


Exon 43












Human-
13
 1
TTTT
ACTGTTTTAAAATTTTTATATTAC
1178
ACUGUUUUAAAAUUUUUAUAUUAC
1501


Exon 43












Human-
14
 1
TTTA
CTGTTTTAAAATTTTTATATTACA
1179
CUGUUUUAAAAUUUUUAUAUUACA
1502


Exon 43












Human-
15
 1
TTTT
AAAATTTTTATATTACAGAATATA
1180
AAAAUUUUUAUAUUACAGAAUAUA
1503


Exon 43












Human-
16
 1
TTTA
AAATTTTTATATTACAGAATATAA
1181
AAAUUUUUAUAUUACAGAAUAUAA
1504


Exon 43












Human-
17
-1
TTTG
TTGTAGACTATCTTTTATATTCTG
1182
UUGUAGACUAUCUUUUAUAUUCUG
1505


Exon 43












Human-
18
 1
TTTT
TATATTACAGAATATAAAAGATAG
1183
UAUAUUACAGAAUAUAAAAGAUAG
1506


Exon 43












Human-
19
 1
TTTT
ATATTACAGAATATAAAAGATAGT
1184
AUAUUACAGAAUAUAAAAGAUAGU
1507


Exon 43












Human-
20
 1
TTTA
TATTACAGAATATAAAAGATAGTC
1185
UAUUACAGAAUAUAAAAGAUAGUC
1508


Exon 43












Human-
21
-1
TTTG
CAATGCTGCTGTCTTCTTGCTATG
1186
CAAUGCUGCUGUCUUCUUGCUAUG
1509


Exon 43












Human-
22
 1
TTTC
CAATGGGAAAAAGTTAACAAAATG
1187
CAAUGGGAAAAAGUUAACAAAAUG
1510


Exon 43












Human-
23
-1
TTTC
TGCAAGTATCAAGAAAAATATATG
1188
UGCAAGUAUCAAGAAAAAUAUAUG
1511


Exon 43












Human-
24
 1
TTTT
TCTTGATACTTGCAGAAATGATTT
1189
UCUUGAUACUUGCAGAAAUGAUUU
1512


Exon 43












Human-
25
 1
TTTT
CTTGATACTTGCAGAAATGATTTG
1190
CUUGAUACUUGCAGAAAUGAUUUG
1513


Exon 43












Human-
26
 1
TTTC
TTGATACTTGCAGAAATGATTTGT
1191
UUGAUACUUGCAGAAAUGAUUUGU
1514


Exon 43












Human-
27
 1
TTTG
TTTTCAGGGAACTGTAGAATTTAT
1192
UUUUCAGGGAACUGUAGAAUUUAU
1515


Exon 43












Human-
28
-1
TTTC
CATGGAGGGTACTGAAATAAATTC
1193
CAUGGAGGGUACUGAAAUAAAUUC
1516


Exon 43












Human-
29
-1
TTTT
CCATGGAGGGTACTGAAATAAATT
1194
CCAUGGAGGGUACUGAAAUAAAUU
1517


Exon 43












Human-
30
 1
TTTT
CAGGGAACTGTAGAATTTATTTCA
1195
CAGGGAACUGUAGAAUUUAUUUCA
1518


Exon 43












Human-
31
-1
TTTT
TCCATGGAGGGTACTGAAATAAAT
1196
UCCAUGGAGGGUACUGAAAUAAAU
1519


Exon 43












Human-
32
 1
TTTC
AGGGAACTGTAGAATTTATTTCAG
1197
AGGGAACUGUAGAAUUUAUUUCAG
1520


Exon 43












Human-
33
-1
TTTT
TTCCATGGAGGGTACTGAAATAAA
1198
UUCCAUGGAGGGUACUGAAAUAAA
1521


Exon 43












Human-
34
-1
TTTC
CCTGTCTTTTTTCCATGGAGGGTA
1199
CCUGUCUUUUUUCCAUGGAGGGUA
1522


Exon 43












Human-
35
-1
TTTT
CCCTGTCTTTTTTCCATGGAGGGT
1200
CCCUGUCUUUUUUCCAUGGAGGGU
1523


Exon 43












Human-
36
-1
TTTT
TCCCTGTCTTTTTTCCATGGAGGG
1201
UCCCUGUCUUUUUUCCAUGGAGGG
1524


Exon 43












Human-
37
 1
TTTA
TTTCAGTACCCTCCATGGAAAAAA
1202
UUUCAGUACCCUCCAUGGAAAAAA
1525


Exon 43












Human-
38
 1
TTTC
AGTACCCTCCATGGAAAAAAGACA
1203
AGUACCCUCCAUGGAAAAAAGACA
1526


Exon 43












Human-
 1
 1
TTTA
AGTTTGCATGGTTCTTGCTCAAGG
1204
AGUUUGCAUGGUUCUUGCUCAAGG
1527


Exon 6












Human-
 2
-1
TTTC
ATAAGAAAATGCATTCCTTGAGCA
1205
AUAAGAAAAUGCAUUCCUUGAGCA
1528


Exon 6












Human-
 3
-1
TTTT
CATAAGAAAATGCATTCCTTGAGC
1206
CAUAAGAAAAUGCAUUCCUUGAGC
1529


Exon 6












Human-
 4
 1
TTTG
CATGGTTCTTGCTCAAGGAATGCA
1207
CAUGGUUCUUGCUCAAGGAAUGCA
1530


Exon 6












Human-
 5
-1
TTTG
ACCTACATGTGGAAATAAATTTTC
1208
ACCUACAUGUGGAAAUAAAUUUUC
1531


Exon 6












Human-
 6
-1
TTTT
GACCTACATGTGGAAATAAATTTT
1209
GACCUACAUGUGGAAAUAAAUUUU
1532


Exon 6












Human-
 7
-1
TTTT
TGACCTACATGTGGAAATAAATTT
1210
UGACCUACAUGUGGAAAUAAAUUU
1533


Exon 6












Human-
 8
 1
TTTT
CTTATGAAAATTTATTTCCACATG
1211
CUUAUGAAAAUUUAUUUCCACAUG
1534


Exon 6












Human-
 9
 1
TTTC
TTATGAAAATTTATTTCCACATGT
1212
UUAUGAAAAUUUAUUUCCACAUGU
1535


Exon 6












Human-
10
-1
TTTC
ATTACATTTTTGACCTACATGTGG
1213
AUUACAUUUUUGACCUACAUGUGG
1536


Exon 6












Human-
11
-1
TTTT
CATTACATTTTTGACCTACATGTG
1214
CAUUACAUUUUUGACCUACAUGUG
1537


Exon 6












Human-
12
-1
TTTT
TCATTACATTTTTGACCTACATGT
1215
UCAUUACAUUUUUGACCUACAUGU
1538


Exon 6












Human-
13
 1
TTTA
TTTCCACATGTAGGTCAAAAATGT
1216
UUUCCACAUGUAGGUCAAAAAUGU
1539


Exon 6












Human-
14
 1
TTTC
CACATGTAGGTCAAAAATGTAATG
1217
CACAUGUAGGUCAAAAAUGUAAUG
1540


Exon 6












Human-
15
-1
TTTG
TTGCAATCCAGCCATGATATTTTT
1218
UUGCAAUCCAGCCAUGAUAUUUUU
1541


Exon 6












Human-
16
-1
TTTC
ACTGTTGGTTTGTTGCAATCCAGC
1219
ACUGUUGGUUUGUUGCAAUCCAGC
1542


Exon 6












Human-
17
-1
TTTT
CACTGTTGGTTTGTTGCAATCCAG
1220
CACUGUUGGUUUGUUGCAAUCCAG
1543


Exon 6












Human-
18
 1
TTTG
AATGCTCTCATCCATAGTCATAGG
1221
AAUGCUCUCAUCCAUAGUCAUAGG
1544


Exon 6












Human-
19
-1
TTTA
ATGTCTCAGTAATCTTCTTACCTA
1222
AUGUCUCAGUAAUCUUCUUACCUA
1545


Exon 6












Human-
20
-1
TTTA
CAAGTTATTTAATGTCTCAGTAAT
1223
CAAGUUAUUUAAUGUCUCAGUAAU
1546


Exon 6












Human-
21
-1
TTTT
ACAAGTTATTTAATGTCTCAGTAA
1224
ACAAGUUAUUUAAUGUCUCAGUAA
1547


Exon 6












Human-
22
 1
TTTA
GACTCTGATGACATATTTTTCCCC
1225
GACUCUGAUGACAUAUUUUUCCCC
1548


Exon 6












Human-
23
 1
TTTT
TCCCCAGTATGGTTCCAGATCATG
1226
UCCCCAGUAUGGUUCCAGAUCAUG
1549


Exon 6












Human-
24
 1
TTTT
CCCCAGTATGGTTCCAGATCATGT
1227
CCCCAGUAUGGUUCCAGAUCAUGU
1550


Exon 6












Human-
25
 1
TTTC
CCCAGTATGGTTCCAGATCATGTC
1228
CCCAGUAUGGUUCCAGAUCAUGUC
1551


Exon 6












Human-
 1
 1
TTTA
TATTTGTCTTtgtgtatgtgtgta
1229
UAUUUGUCUUuguguaugugugua
1552


Exon 7












Human-
 2
 1
TTTG
TCTTtgtgtatgtgtgtatgtgta
1230
UCUUuguguauguguguaugugua
1553


Exon 7












Human-
 3
 1
TTtg
tgtatgtgtgtatgtgtatgtgtt
1231
uguauguguguauguguauguguu
1554


Exon 7












Human-
 4
 1
ttTT
AGGCCAGACCTATTTGACTGGAAT
1232
AGGCCAGACCUAUUUGACUGGAAU
1555


Exon 7












Human-
 5
 1
tTTA
GGCCAGACCTATTTGACTGGAATA
1233
GGCCAGACCUAUUUGACUGGAAUA
1556


Exon 7












Human-
 6
 1
TTTG
ACTGGAATAGTGTGGTTTGCCAGC
1234
ACUGGAAUAGUGUGGUUUGCCAGC
1557


Exon 7












Human-
 7
 1
TTTG
CCAGCAGTCAGCCACACAACGACT
1235
CCAGCAGUCAGCCACACAACGACU
1558


Exon 7












Human-
 8
-1
TTTC
TCTATGCCTAATTGATATCTGGCG
1236
UCUAUGCCUAAUUGAUAUCUGGCG
1559


Exon 7












Human-
 9
-1
TTTA
CCAACCTTCAGGATCGAGTAGTTT
1237
CCAACCUUCAGGAUCGAGUAGUUU
1560


Exon 7












Human-
10
 1
TTTC
TGGACTACCACTGCTTTTAGTATG
1238
UGGACUACCACUGCUUUUAGUAUG
1561


Exon 7












Human-
11
 1
TTTT
AGTATGGTAGAGTTTAATGTTTTC
1239
AGUAUGGUAGAGUUUAAUGUUUUC
1562


Exon 7












Human-
12
 1
TTTA
GTATGGTAGAGTTTAATGTTTTCA
1240
GUAUGGUAGAGUUUAAUGUUUUCA
1563


Exon 7












Human-
 1
-1
TTTG
AGACTCTAAAAGGATAATGAACAA
1241
AGACUCUAAAAGGAUAAUGAACAA
1564


Exon 8












Human-
 2
 1
TTTA
ACTTTGATTTGTTCATTATCCTTT
1242
ACUUUGAUUUGUUCAUUAUCCUUU
1565


Exon 8












Human-
 3
-1
TTTC
TATATTTGAGACTCTAAAAGGATA
1243
UAUAUUUGAGACUCUAAAAGGAUA
1566


Exon 8












Human-
 4
 1
TTTG
ATTTGTTCATTATCCTTTTAGAGT
1244
AUUUGUUCAUUAUCCUUUUAGAGU
1567


Exon 8












Human-
 5
-1
TTTG
GTTTCTATATTTGAGACTCTAAAA
1245
GUUUCUAUAUUUGAGACUCUAAAA
1568


Exon 8












Human-
 6
-1
TTTT
GGTTTCTATATTTGAGACTCTAAA
1246
GGUUUCUAUAUUUGAGACUCUAAA
1569


Exon 8












Human-
 7
-1
TTTT
TGGTTTCTATATTTGAGACTCTAA
1247
UGGUUUCUAUAUUUGAGACUCUAA
1570


Exon 8












Human-
 8
 1
TTTG
TTCATTATCCTTTTAGAGTCTCAA
1248
UUCAUUAUCCUUUUAGAGUCUCAA
1571


Exon 8












Human-
 9
 1
TTTT
AGAGTCTCAAATATAGAAACCAAA
1249
AGAGUCUCAAAUAUAGAAACCAAA
1572


Exon 8












Human-
10
 1
TTTA
GAGTCTCAAATATAGAAACCAAAA
1250
GAGUCUCAAAUAUAGAAACCAAAA
1573


Exon 8












Human-
11
-1
TTTC
CACTTCCTGGATGGCTTCAATGCT
1251
CACUUCCUGGAUGGCUUCAAUGCU
1574


Exon 8












Human-
12
 1
TTTT
GCCTCAACAAGTGAGCATTGAAGC
1252
GCCUCAACAAGUGAGCAUUGAAGC
1575


Exon 8












Human-
13
 1
TTTG
CCTCAACAAGTGAGCATTGAAGCC
1253
CCUCAACAAGUGAGCAUUGAAGCC
1576


Exon 8












Human-
14
-1
TTTA
GGTGGCCTTGGCAACATTTCCACT
1254
GGUGGCCUUGGCAACAUUUCCACU
1577


Exon 8












Human-
15
-1
TTTA
GTCACTTTAGGTGGCCTTGGCAAC
1255
GUCACUUUAGGUGGCCUUGGCAAC
1578


Exon 8












Human-
16
-1
TTTG
ATGATGTAACTGAAAATGTTCTTC
1256
AUGAUGUAACUGAAAAUGUUCUUC
1579


Exon 8












Human-
17
-1
TTTA
CCTGTTGAGAATAGTGCATTTGAT
1257
CCUGUUGAGAAUAGUGCAUUUGAU
1580


Exon 8












Human-
18
 1
TTTT
CAGTTACATCATCAAATGCACTAT
1258
CAGUUACAUCAUCAAAUGCACUAU
1581


Exon 8












Human-
19
 1
TTTC
AGTTACATCATCAAATGCACTATT
1259
AGUUACAUCAUCAAAUGCACUAUU
1582


Exon 8












Human-
20
-1
TTTA
CACACTTTACCTGTTGAGAATAGT
1260
CACACUUUACCUGUUGAGAAUAGU
1583


Exon 8












Human-
21
 1
TTTT
CTGTTTTATATGCATTTTTAGGTA
1261
CUGUUUUAUAUGCAUUUUUAGGUA
1584


Exon 8












Human-
22
 1
TTTC
TGTTTTATATGCATTTTTAGGTAT
1262
UGUUUUAUAUGCAUUUUUAGGUAU
1585


Exon 8












Human-
23
 1
TTTT
ATATGCATTTTTAGGTATTACGTG
1263
AUAUGCAUUUUUAGGUAUUACGUG
1586


Exon 8












Human-
24
 1
TTTA
TATGCATTTTTAGGTATTACGTGC
1264
UAUGCAUUUUUAGGUAUUACGUGC
1587


Exon 8












Human-
25
 1
TTTT
TAGGTATTACGTGCACatatatat
1265
UAGGUAUUACGUGCACauauauau
1588


Exon 8












Human-
26
 1
TTTT
AGGTATTACGTGCACatatatata
1266
AGGUAUUACGUGCACauauauaua
1589


Exon 8












Human-
27
 1
TTTA
GGTATTACGTGCACatatatatat
1267
GGUAUUACGUGCACauauauauau
1590


Exon 8












Human-
 1
-1
TTTA
AGCAACAACTATAATATTGTGCAG
1268
AGCAACAACUAUAAUAUUGUGCAG
1591


Exon 55












Human-
 2
 1
TTTA
GTTCCTCCATCTTTCTCTTTTTAT
1269
GUUCCUCCAUCUUUCUCUUUUUAU
1592


Exon 55












Human-
 3
 1
TTTC
TCTTTTTATGGAGTTCACTAGGTG
1270
UCUUUUUAUGGAGUUCACUAGGUG
1593


Exon 55












Human-
 4
 1
TTTT
TATGGAGTTCACTAGGTGCACCAT
1271
UAUGGAGUUCACUAGGUGCACCAU
1594


Exon 55












Human-
 5
 1
TTTT
ATGGAGTTCACTAGGTGCACCATT
1272
AUGGAGUUCACUAGGUGCACCAUU
1595


Exon 55












Human-
 6
 1
TTTA
TGGAGTTCACTAGGTGCACCATTC
1273
UGGAGUUCACUAGGUGCACCAUUC
1596


Exon 55












Human-
 7
 1
TTTA
ATAATTGCATCTGAACATTTGGTC
1274
AUAAUUGCAUCUGAACAUUUGGUC
1597


Exon 55












Human-
 8
 1
TTTG
GTCCTTTGCAGGGTGAGTGAGCGA
1275
GUCCUUUGCAGGGUGAGUGAGCGA
1598


Exon 55












Human-
 9
-1
TTTC
TTCCAAAGCAGCCTCTCGCTCACT
1276
UUCCAAAGCAGCCUCUCGCUCACU
1599


Exon 55












Human-
10
 1
TTTG
CAGGGTGAGTGAGCGAGAGGCTGC
1277
CAGGGUGAGUGAGCGAGAGGCUGC
1600


Exon 55












Human-
11
 1
TTTG
GAAGAAACTCATAGATTACTGCAA
1278
GAAGAAACUCAUAGAUUACUGCAA
1601


Exon 55












Human-
12
-1
TTTC
CAGGTCCAGGGGGAACTGTTGCAG
1279
CAGGUCCAGGGGGAACUGUUGCAG
1602


Exon 55












Human-
13
-1
TTTT
CCAGGTCCAGGGGGAACTGTTGCA
1280
CCAGGUCCAGGGGGAACUGUUGCA
1603


Exon 55












Human-
14
-1
TTTC
AGCTTCTGTAAGCCAGGCAAGAAA
1281
AGCUUCUGUAAGCCAGGCAAGAAA
1604


Exon 55












Human-
15
 1
TTTC
TTGCCTGGCTTACAGAAGCTGAAA
1282
UUGCCUGGCUUACAGAAGCUGAAA
1605


Exon 55












Human-
16
-1
TTTC
CTTACGGGTAGCATCCTGTAGGAC
1283
CUUACGGGUAGCAUCCUGUAGGAC
1606


Exon 55












Human-
17
-1
TTTA
CTCCCTTGGAGTCTTCTAGGAGCC
1284
CUCCCUUGGAGUCUUCUAGGAGCC
1607


Exon 55












Human-
18
-1
TTTT
ACTCCCTTGGAGTCTTCTAGGAGC
1285
ACUCCCUUGGAGUCUUCUAGGAGC
1608


Exon 55












Human-
19
-1
TTTC
ATCAGCTCTTTTACTCCCTTGGAG
1286
AUCAGCUCUUUUACUCCCUUGGAG
1609


Exon 55












Human-
20
 1
TTTC
CGCTTTAGCACTCTTGTGGATCCA
1287
CGCUUUAGCACUCUUGUGGAUCCA
1610


Exon 55












Human-
21
 1
TTTA
GCACTCTTGTGGATCCAATTGAAC
1288
GCACUCUUGUGGAUCCAAUUGAAC
1611


Exon 55












Human-
22
-1
TTTG
TCCCTGGCTTGTCAGTTACAAGTA
1289
UCCCUGGCUUGUCAGUUACAAGUA
1612


Exon 55












Human-
23
-1
TTTT
GTCCCTGGCTTGTCAGTTACAAGT
1290
GUCCCUGGCUUGUCAGUUACAAGU
1613


Exon 55












Human-
24
-1
TTTG
TTTTGTCCCTGGCTTGTCAGTTAC
1291
UUUUGUCCCUGGCUUGUCAGUUAC
1614


Exon 55












Human-
25
-1
TTTT
GTTTTGTCCCTGGCTTGTCAGTTA
1292
GUUUUGUCCCUGGCUUGUCAGUUA
1615


Exon 55












Human-
26
 1
TTTG
TACTTGTAACTGACAAGCCAGGGA
1293
UACUUGUAACUGACAAGCCAGGGA
1616


Exon 55












Human-

 1
TTTA
gCTCCTACTCAGACTGTTACTCTG
1294
gCUCCUACUCAGACUGUUACUCUG
1617


G1-exon51












Human-

 1
TTTC
taccatgtattgctaaacaaagta
1295
uaccauguauugcuaaacaaagua
1618


G2-exon51












Human-

-1
TTTA
attgaagagtaacaatttgagcca
1296
auugaagaguaacaauuugagcca
1619


G3-exon51












mouse-

 1
TTTG
aggctctgcaaagttctTTGAAAG
1297
aggcucugcaaaguucuUUGAAAG
1620


Exon23-









G1












mouse-

 1
TTTG
AAAGAGCAACAAAATGGCttcaac
1298
AAAGAGCAACAAAAUGGCuucaac
1621


Exon23-









G2












mouse-

 1
TTTG
AAAGAGCAATAAAATGGCttcaac
1299
AAAGAGCAAUAAAAUGGCuucaac
1622


Exon23-









G3












mouse-

-1
TTTC
AAAGAACTTTGCAGAGCctcaaaa
1300
AAAGAACUUUGCAGAGCcucaaaa
1623


Exon23-









G4












mouse-

-1
TTTA
ctgaatatctatgcattaataact
1301
cugaauaucuaugcauuaauaacu
1624


Exon23-









G5












mouse-

-1
TTTC
tattatattacagggcatattata
1302
ummaummacagggcaummaua
1625


Exon23-









G6












mouse-

 1
TTTC
Aggtaagccgaggtttggccttta
1303
Agguaagccgagguuuggccuuua
1626


Exon23-









G7












mouse-

 1
TTTA
cccagagtccttcaaagatattga
1304
cccagaguccuucaaagauauuga
1627


Exon23-









G8





*In this table, upper case letters represent sgRNA nucleotides that align to the exon sequence of the gene. Lower case letters represent sgRNA nucleotides that align to the intron sequence of the gene













TABLE 19







Additional gRNA targeting sequences




















SEQ



Name
Species
Gene
Target
Strand
Sequence
ID NO
PAM





DCR1
Human
DMD
Intron
+
attggctttgatttcccta
1628
GGG





50









DCR2
Human
DMD
Intron
-
tgtagagtaagtcagccta
1629
TGG





50









DCR3
Human
DMD
Exon
+
cctactcagactgttactc
1630
TGG





51-55′









DCR4
Human
DMD
Exon
+
ttggacagaacttaccgac
1631
TGG





51-53′









DCR5
Human
DMD
Intron
-
cagttgcctaagaactggt
1632
GGG





51









DCR6
Human
DMD
Intron
-
GGGCTCCACCCTCACGAGT
1633
GGG





44









DCR7
Human
DMD
Intron
+
TTTGCTTCGCTATAAAACG
1634
AGG





55









DCR8
Human
DMD
Exon 41
+
TCTGAGGATGGGGCCGCAA
1635
TGG





DCR9
Human
DMD
Exon 44
-
GATCTGTCAAATCGCCTGC
1636
AGG





DCR10
Human
DMD
Exon 45
+
CCAGGATGGCATTGGGCAG
1637
CGG





DCR11
Human
DMD
Exon 45
+
CTGAATCTGCGGTGGCAGG
1638
AGG





DCR12
Human
DMD
Exon 46
-
TTCTTTTGTTCTTCTAGCc
1639
TGG





DCR13
Human
DMD
Exon 46
+
GAAAAGCTTGAGCAAGTCA
1640
AGG





DCR14
Human
DMD
Exon 47
+
GAAGAGTTGCCCCTGCGCC
1641
AGG





DCR15
Human
DMD
Exon 47
+
ACAAATCTCCAGTGGATAA
1642
AGG





DCR16
Human
DMD
Exon 48
-
TGTTTCTCAGGTAAAGCTC
1643
TGG





DCR17
Human
DMD
Exon 48
+
GAAGGACCATTTGACGTTa
1644
AGG





DCR18
Human
DMD
Exon 49
-
AACTGCTATTTCAGTTTCc
1645
TGG





DCR19
Human
DMD
Exon 49
+
CCAGCCACTCAGCCAGTGA
1646
AGG





DCR20
Human
DMD
Exon 50
+
gtatgcttttctgttaaag
1647
AGG





DCR21
Human
DMD
Exon 50
+
CTCCTGGACTGACCACTAT
1648
TGG





DCR22
Human
DMD
Exon 52
+
GAACAGAGGCGTCCCCAGT
1649
TGG





DCR23
Human
DMD
Exon 52
+
GAGGCTAGAACAATCATTA
1650
CGG





DCR24
Human
DMD
Exon 53
+
ACAAGAACACCTTCAGAAC
1651
CGG





DCR25
Human
DMD
Exon 53
-
GGTTTCTGTGATTTTCTTT
1652
TGG





DCR26
Human
DMD
Exon 54
+
GGCCAAAGACCTCCGCCAG
1653
TGG





DCR27
Human
DMD
Exon 54
+
TTGGAGAAGCATTCATAAA
1654
AGG





DCR28
Human
DMD
Exon 55
-
TCGCTCACTCACCctgcaa
1655
AGG





DCR29
Human
DMD
Exon 55
+
AAAAGAGCTGATGAAACAA
1656
TGG





DCR30
Human
DMD
5′UTR/
+
TAcACTTTTCaAAATGCTT
1657
TGG





Exon 1









DCR31
Human
DMD
Exon 51
+
gagatgatcatcaagcaga
1658
AGG





DCR32
Mouse
DMD
mdx
+
ctttgaaagagcaaTaaaa
1659
TGG





DCR33
Human
DMD
Intron
-
CACAAAAGTCAAATCGGAA
1660
TGG





44









DCR34
Human
DMD
Intron
-
ATTTCAATATAAGATTCGG
1661
AGG





44









DCR35
Human
DMD
Intron
-
CTTAAGCAATCCCGAACTC
1662
TGG





55









DCR36
Human
DMD
Intron
-
CCTTCTTTATCCCCTATCG
1663
AGG





55









DCR40
Mouse
DMD
Exon 23
-
aggccaaacctcggcttac
1664
NNGRR





DCR41
Mouse
DMD
Exon 23
+
TTCGAAAATTTCAGgtaag
1665
NNGRR





DCR42
Mouse
DMD
Exon 23
+
gcagaacaggagataacag
1666
NNGRRT





DCR43
Mouse
ACV
Exon 1
+
gcggccctcgcccttctct
1667
ggggat




R2B










DCR48
Human
DMD
Intron
-
TAGTGATCGTGGATACGAG
1668
AGG





45









DCR49
Human
DMD
Intron
-
TACAGCCCTCGGTGTATAT
1669
TGG





45









DCR50
Human
DMD
Intron
-
GGAAGGAATTAAGCCCGAA
1670
TGG





52









DCR51
Human
DMD
Intron
-
GGAACAGCTTTCGTAGTTG
1671
AGG





53









DCR52
Human
DMD
Intron
+
ATAAAGTCCAGTGTCGATC
1672
AGG





54









DCR53


Intron
+
AAAACCAGAGCTTCGGTCA
1673
AGG





54









DCR54
Mouse
Rosa26
ZFN
+
GAGTCTTCTGGGCAGGCTTAA
1674
TGG





region









DCR55
Mouse
Rosa26
mRNA
-
TCGGGTGAGCATGTCTTTAAT
1675
TGG





DCR49
Human
DMD
Ex 51
-
gtgtcaccagagtaacagt
1676
ctgagt





DCR50
Human
DMD
Ex 51
+
tgatcatcaagcagaaggt
1677
atgag





DCR60
Mouse
DMD
Exon 23
+
AACTTCGAAAATTTCAGgta
1678
agccgagg





DCR61
Mouse
DMD
Intron
+
gaaactcatcaaatatgcgt
1679
gttagtgt





22









DCR62
Mouse
DMD
Intron
-
tcatttacactaacacgcat
1680
atttgatg





22









DCR63
Mouse
DMD
Intron
+
gaatgaaactcatcaaatat
1681
gcgtgtta





22









DCR64
Mouse
DMD
Intron
-
tcatcaatatctttgaagga
1682
ctctgggt





23









DCR65
Mouse
DMD
Intron
-
tgttttcataggaaaaatag
1683
gcaagttg





23









DCR66
Mouse
DMD
Intron
+
aattggaaaatgtgatggga
1684
aacagata





23









DCR67
Human
DMD
Exon 51
+
atgatcatcaagcagaaggt
1685
atgagaaa





DCR68
Human
DMD
Exon 51
+
agatgatcatcaagcagaag
1686
gtatgaga





DCR69
Human
DMD
Exon 51
-
cattttttctcataccttct
1687
gcttgatg





DCR70
Human
DMD
Exon 51
+
tcctactcagactgttactc
1688
tggtgaca





DCR71
Human
DMD
Exon 51
-
acaggttgtgtcaccagagt
1689
aacagtct





DCR72
Human
DMD
Exon 51
-
ttatcattttttctcatacc
1690
ttctgctt





DCR73
Human
DMD
Intron
-
ttgcctaagaactggtggga
1691
aatggtct





51









DCR74
Human
DMD
Intron
-
aaacagttgcctaagaactg
1692
gtgggaaa





51









DCR75
Human
DMD
Intron
+
tttcccaccagttcttaggc
1693
aactgttt





51









DCR76
Human
DMD
Intron
+
tggctttgatttccctaggg
1694
tccagctt





50









DCR77
Human
DMD
Intron
-
tagggaaatcaaagccaatg
1695
aaacgttc





50









DCR78
Human
DMD
Intron
-
gaccctagggaaatcaaagc
1696
caatgaaa





50









DCR79
Human
DMD
Intron
-
TGAGGGCTCCACCCTCACGA
1697
GTGGGT





44



TT





DCR80
Human
DMD
Intron
-
AAGGATTGAGGGCTCCACCC
1698
TCACGA





44



GT





DCR81
Human
DMD
Intron
-
GCTCCACCCTCACGAGTGGG
1699
TTTGGT





44



TC





DCR82
Human
DMD
Intron
-
TATCCCCTATCGAGGAAACC
1700
ACGAGT





55



TT





DCR83
Human
DMD
Intron
+
GATAAAGAAGGCCTATTTCA
1701
TAGAGT





55



TG





DCR84
Human
DMD
Intron
-
AGGCCTTCTTTATCCCCTAT
1702
CGAGG





55



AAA





DCR85
Human
DMD
Intron
-
TGAGGGCTCCACCCTCACGA
1703
GTGGGT





44









DCR86
Human
DMD
Intron
+
GATAAAGAAGGCCTATTTCA
1704
TAGAGT





55









DCR1
Human
DMD
Intron
+
attggctttgatttcccta
1705
GGG





50









DCR2
Human
DMD
Intron
-
tgtagagtaagtcagccta
1706
TGG





50









DCR3
Human
DMD
Exon
+
cctactcagactgttactc
1707
TGG





51-5′









DCR4
Human
DMD
Exon
+
ttggacagaacttaccgac
1708
TGG





51-3′









DCR5
Human
DMD
Intron
-
cagttgcctaagaactggt
1709
GGG





51









DCR6
Human
DMD
Intron
-
GGGCTCCACCCTCACGAGT
1710
GGG





44









DCR7
Human
DMD
Intron
+
TTTGCTTCGCTATAAAACG
1711
AGG





55









DCR8
Human
DMD
Exon 41
+
TCTGAGGATGGGGCCGCAA
1712
TGG





DCR9
Human
DMD
Exon 44
-
GATCTGTCAAATCGCCTGC
1713
AGG





DCR10
Human
DMD
Exon 45
+
CCAGGATGGCATTGGGCAG
1714
CGG





DCR11
Human
DMD
Exon 45
+
CTGAATCTGCGGTGGCAGG
1715
AGG





DCR12
Human
DMD
Exon 46
-
TTCTTTTGTTCTTCTAGCc
1716
TGG





DCR13
Human
DMD
Exon 46
+
GAAAAGCTTGAGCAAGTCA
1717
AGG





DCR14
Human
DMD
Exon 47
+
GAAGAGTTGCCCCTGCGCC
1718
AGG





DCR15
Human
DMD
Exon 47
+
ACAAATCTCCAGTGGATAA
1719
AGG





DCR16
Human
DMD
Exon 48
-
TGTTTCTCAGGTAAAGCTC
1720
TGG





DCR17
Human
DMD
Exon 48
+
GAAGGACCATTTGACGTTa
1721
AGG





DCR18
Human
DMD
Exon 49
-
AACTGCTATTTCAGTTTCc
1722
TGG





DCR19
Human
DMD
Exon 49
+
CCAGCCACTCAGCCAGTGA
1723
AGG





DCR20
Human
DMD
Exon 50
+
gtatgcttttctgttaaag
1724
AGG





DCR21
Human
DMD
Exon 50
+
CTCCTGGACTGACCACTAT
1725
TGG





DCR22
Human
DMD
Exon 52
+
GAACAGAGGCGTCCCCAGT
1726
TGG





DCR23
Human
DMD
Exon 52
+
GAGGCTAGAACAATCATTA
1727
CGG





DCR24
Human
DMD
Exon 53
+
ACAAGAACACCTTCAGAAC
1728
CGG





DCR25
Human
DMD
Exon 53
-
GGTTTCTGTGATTTTCTTT
1729
TGG





DCR26
Human
DMD
Exon 54
+
GGCCAAAGACCTCCGCCAG
1730
TGG





DCR27
Human
DMD
Exon 54
+
TTGGAGAAGCATTCATAAA
1731
AGG





DCR28
Human
DMD
Exon 55
-
TCGCTCACTCACCctgcaa
1732
AGG





DCR29
Human
DMD
Exon 55
+
AAAAGAGCTGATGAAACAA
1733
TGG





DCR30
Human
DMD
5′UTR/
+
TAcACTTTTCaAAATGCTT
1734
TGG





Exon 1









DCR31
Human
DMD
Exon 51
+
gagatgatcatcaagcaga
1735
AGG





DCR32
Mouse
DMD
mdx
+
ctttgaaagagcaaTaaaa
1736
TGG





DCR33
Human
DMD
Intron
-
CACAAAAGTCAAATCGGAA
1737
TGG





44









DCR34
Human
DMD
Intron
-
ATTTCAATATAAGATTCGG
1738
AGG





44









DCR35
Human
DMD
Intron
-
CTTAAGCAATCCCGAACTC
1739
TGG





55









DCR36
Human
DMD
Intron
-
CCTTCTTTATCCCCTATCG
1740
AGG





55









DCR40
Mouse
DMD
Exon 23
-
aggccaaacctcggcttac
1741
NNGRR





DCR41
Mouse
DMD
Exon 23
+
TTCGAAAATTTCAGgtaag
1742
NNGRR





DCR42
Mouse
DMD
Exon 23
+
gcagaacaggagataacag
1743
NNGRRT





DCR43
Mouse
ACV
Exon 1
+
gcggccctcgcccttctct
1744
ggggat




R2B










DCR48
Human
DMD
Intron
-
TAGTGATCGTGGATACGAG
1745
AGG





45









DCR49
Human
DMD
Intron
-
TACAGCCCTCGGTGTATAT
1746
TGG





45









DCR50
Human
DMD
Intron
-
GGAAGGAATTAAGCCCGAA
1747
TGG





52









DCR51
Human
DMD
Intron
-
GGAACAGCTTTCGTAGTTG
1748
AGG





53









DCR52
Human
DMD
Intron
+
ATAAAGTCCAGTGTCGATC
1749
AGG





54









DCR53


Intron
+
AAAACCAGAGCTTCGGTCA
1750
AGG





54









DCR54
Mouse
Rosa26
ZFN
+
GAGTCTTCTGGGCAGGCTTAA
1751
TGG





region









DCR55
Mouse
Rosa26
mRNA
-
TCGGGTGAGCATGTCTTTAAT
1752
TGG





DCR49
Human
DMD
Ex 51
-
gtgtcaccagagtaacagt
1753
ctgagt





DCR50
Human
DMD
Ex 51
+
tgatcatcaagcagaaggt
1754
atgag





DCR60
Mouse
DMD
Exon 23
+
AACTTCGAAAATTTCAGgta
1755
agccgagg





DCR61
Mouse
DMD
Intron
+
gaaactcatcaaatatgcgt
1756
gttagtgt





22









DCR62
Mouse
DMD
Intron
-
tcatttacactaacacgcat
1757
atttgatg





22









DCR63
Mouse
DMD
Intron
+
gaatgaaactcatcaaatat
1758
gcgtgtta





22









DCR64
Mouse
DMD
Intron
-
tcatcaatatctttgaagga
1759
ctctgggt





23









DCR65
Mouse
DMD
Intron
-
tgttttcataggaaaaatag
1760
gcaagttg





23









DCR66
Mouse
DMD
Intron
+
aattggaaaatgtgatggga
1761
aacagata




23










DCR67
Human
DMD
Exon 51
+
atgatcatcaagcagaaggt
1762
atgagaaa





DCR68
Human
DMD
Exon 51
+
agatgatcatcaagcagaag
1763
gtatgaga





DCR69
Human
DMD
Exon 51
-
cattttttctcataccttct
1764
gcttgatg





DCR70
Human
DMD
Exon 51
+
tcctactcagactgttactc
1765
tggtgaca





DCR71
Human
DMD
Exon 51
-
acaggttgtgtcaccagagt
1766
aacagtct





DCR72
Human
DMD
Exon 51
-
ttatcattttttctcatacc
1767
ttctgctt





DCR73
Human
DMD
Intron
-
ttgcctaagaactggtggga
1768
aatggtct





51









DCR74
Human
DMD
Intron
-
aaacagttgcctaagaactg
1769
gtgggaaa





51









DCR75
Human
DMD
Intron
+
tttcccaccagttcttaggc
1770
aactgttt





51









DCR76
Human
DMD
Intron
+
tggctttgatttccctaggg
1771
tccagctt





50









DCR77
Human
DMD
Intron
-
tagggaaatcaaagccaatg
1772
aaacgttc





50









DCR78
Human
DMD
Intron
-
gaccctagggaaatcaaagc
1773
caatgaaa





50









DCR79
Human
DMD
Intron
-
TGAGGGCTCCACCCTCACGA
1774
GTGGGT





44



TT





DCR80
Human
DMD
Intron
-
AAGGATTGAGGGCTCCACCC
1775
TCACGA





44



GT





DCR81
Human
DMD
Intron
-
GCTCCACCCTCACGAGTGGG
1776
TTTGGT





44



TC





DCR82
Human
DMD
Intron
-
TATCCCCTATCGAGGAAACC
1777
ACGAGT





55



TT





DCR83
Human
DMD
Intron
+
GATAAAGAAGGCCTATTTCA
1778
TAGAGT





55



TG





DCR84
Human
DMD
Intron
-
AGGCCTTCTTTATCCCCTAT
1779
CGAGG





55



AAA





DCR85
Human
DMD
Intron
-
TGAGGGCTCCACCCTCACGA
1780
GTGGGT





44









DCR86
Human
DMD
Intron
+
GATAAAGAAGGCCTATTTCA
1781
TAGAGT





55











DMD


UAGAAGAUCUGAGCUCUGAG
1782








DMD


AGAUCUGAGCUCUGAGUGGA
1783








DMD


UCUGAGCUCUGAGUGGAAGG
1784








DMD


CCGUUUACUUCAAGAGCUGA
1785








DMD


AAGCAGCCUGACCUAGCUCC
1786








DMD


GCUCCUGGACUGACCACUAU
1787








DMD


CCCUCAGCUCUUGAAGUAAA
1788








DMD


GUCAGUCCAGGAGCUAGGUC
1789








DMD


UAGUGGUCAGUCCAGGAGCU
1790








DMD


GCUCCAAUAGUGGUCAGUCC
1791








DMD


UGGCCAAAGACCUCCGCCAG
1792








DMD


GUGGCAGACAAAUGUAGAUG
1793








DMD


UGUAGAUGUGGCAAAUGACU
1794








DMD


CUUGGCCCUGAAACUUCUCC
1795








DMD


CAGAGAAUAUCAAUGCCUCU
1796








DMD


CAGAGAAUAUCAAUGCCUCU
1797








DMD


CAUUUGUCUGCCACUGGCGG
1798








DMD


CUACAUUUGUCUGCCACUGG
1799








DMD


CAUCUACAUUUGUCUGCCAC
1800








DMD


AUAAUCCCGGAGAAGUUUCA
1801








DMD


UAUCAUCUGCAGAAUAAUCC
1802








DMD


UGUUAUCAUGUGGACUUUUC
1803








DMD


UGAUAUAUCAUUUCUCUGUG
1804








DMD


UUUAUGAAUGCUUCUCCAAG
1805








DMD


UUCUCCAGGCUAGAAGAACAA
1806








DMD


CUGCUCUUUUCCAGGUUCAAG
1807








DMD


GUCUGUUUCAGUUACUGGUGG
1808








DMD


UCCAGUUUCAUUUAAUUGUUU
1809








DMD


CUUAUGGGAGCACUUACAAGC
1810








DMD


UUGCUUCAUUACCUUCACUGG
1811








DMD


UUGUGUCACCAGAGUAACAGU
1812








DMD


AGUAACCACAGGUUGUGUCAC
1813








DMD


UUCAAAUUUUGGGCAGCGGUA
1814








DMD


CAAGAGGCUAGAACAAUCAUU
1815








DMD


UUGUACUUCAUCCCACUGAUU
1816








DMD


CUUCAGAACCGGAGGCAACAG
1817








DMD


CAACAGUUGAAUGAAAUGUUA
1818








DMD


GCCAAGCUUGAGUCAUGGAAG
1819








DMD


CUUGGUUUCUGUGAUUUUCUU
1820








DMD


UCAUUUCACAGGCCUUCAAGA
1821








DMD


CAGAAAUAUUCGUACAGUCUC
1822








DMD


CAAUUACCUCUGGGCUCCUGG
1823








DMD


GATACTAGGGTGGCAAATAG
1824








DMD


GTGTTCTTAAAAGAATGGTG
1825








DMD


GTCAAGAACAGCTGCAGAAC
1826








DMD


GCAGTTGAATGAAATGTTAA
1827








DMD


GATACTAGTGTGGCTCATAG
1828








DMD


GATACGATGGTGGCAAATCG
1829








DMD


GATACTAGGGTGGGGAATAA
1830








DMD


TTTTTCTTAAAAGAATGGTA
1831








DMD


TTGATCTTAGAAGAATGGTG
1832








DMD


GTTTTCTTGAAAAAATGGTG
1833








DMD


CTGTTCTTAAAAGGTTGGTG
1834








DMD


GAGTTCTTCAAAGAATAGTG
1835








DMD


TCTAGGGCAGCTGCAGAAC
1836








DMD


TCATTCACAGCTGCAGAAC
1837








DMD


CAAAGAATAGCTGCAGAAC
1838








DMD


TCAAGAACAGCTGCAGCAG
1839








DMD


TCAAGAACAGCTGCATCAC
1840








DMD


CAGTTACATGAAATGTTAA
1841








DMD


CATTTTAATGAAATGTTAA
1842








DMD


AAGTTGAATGAAATTTTAA
1843








DMD


CAGTGGAATAAAATGTTAA
1844








DMD


AAAGATATATAATGTCATGAAT
1845








DMD


GCAGAATCAAATATAATAGTCT
1846








DMD


AACAAATATCCCTTAGTATC
1847








DMD


AATGTATTTCTTCTATTCAA
1848








DMD


AACAATAAGTCAAATTTAATTG
1849








DMD


GAACTGGTGGGAAATGGTCTA
1850








G









DMD


TCCTTTGGTAAATAAAAGTCCT
1851








DMD


TAGGAATCAAATGGACTTGGAT
1852








DMD


TAATTCTTTCTAGAAAGAGCCT
1853








DMD


CTCTTGCATCTTGCACATGTCC
1854








DMD


ACTTAGAGGTCTTCTACATACA
1855








DMD


TCAGAGGTGAGTGGTGAGGGG
1856








A









DMD


ACACACAGCTGGGTTATCAGA
1857








G









DMD


CACAGCTGGGTTATCAGAG
1858








DMD


ACACAGCTGGGTTATCAGAG
1859








DMD


CACACAGCTGGGTTATCAGAG
1860








DMD


AACACACAGCTGGGTTATCAG
1861








AG









DMD


CTGSTGGGARATGGTCTAG
1862








DMD


ACTGGTGGGAAATGGTCTAG
1863








DMD


AACTGGTGGGAAATGGTCTAG
1864








DMD


AGAACTGGTGGGAAATGGTCT
1865








AG









DMD


ATATCTTCTTAAATACCCGA
1866








DMD


AGTCTCACAAAACTGCAGAG
1867








DMD


TACTTATGTATTTTAAAAAC
1868








DMD


GAATAATTTCTATTATATTACA
1869








DMD


TTCGAAAATTTCAGGTAAGCCG
1870








DMD


TCATTTCTAAAAGTCTTTTGCC
1871








DMD


TTTGAGACACAGTATAGGTTAT
1872








DMD


ATATAATAGAAATTATTCAT
1873








DMD


TAATATGCCCTGTAATATAA
1874








DMD


TGATATCATCAATATCTTTG
1875








DMD


GCAATTAATTGGAAAATGTG
1876








DMD


CTTTAAGCTTAGGTAAAATCA
1877








DMD


CAGTAATGTGTCATACCTTC
1878








DMD


CAGGGCATATTATATTTAGA
1879








DMD


CAAAAGCCAAATCTATTTCA
1880








DMD


ATGCTTTGGTGGGAAGAAGTA
1881








GAGGA









DMD


ATGCTTTGGTGGGAAGAATAG
1882








AGGAC









DMD


TTGTGACAAGCTCACTAATTAG
1883








G









DMD


AAGTTTGAAGAACTTTTACCAG
1884








G









DMD


AGGCAGCGATAAAAAAAACCT
1885








GG









DMD


GCTTTGGTGGGAAGAAGTAGA
1886








GG









DMD


GCTGGGTGTCCCATTGAAA
1887








DMD


CAGCCGCTCGCTGCAGCAG
1888








DMD


TGGAGAGTTTGCAAGGAGC
1889








DMD


GTTTATTCAGCCGGGAGTC
1890








DMD


CGCCAGGAGGGGTGGGTCTA
1891








DMD


CCTTGGTGAGACTGGTAGA
1892








DMD


GTCTTCAGGTTCTGTTGCT
1893








DMD


ATATTCCTGATTTAAAAGT
1894








DMD


TTAAAAGTCGGCTGGTAGC
1895








DMD


CGGGCCGGGGGCGGGGTCC
1896








DMD


GCCCGAGCCGCGTGTGGAA
1897








DMD


CCTTCATTGCGGCGGGCTG
1898








DMD


CCGACCCCTCCCGGGTCCC
1899








DMD


CAGGACCGCGCTTCCCACG
1900








DMD


TGCACCCTGGGAGCGCGAG
1901








DMD


CCGCACGCACCTGTTCCCA
1902








DMD


AAAACAGCGAGGGAGAAAC
1903








DMD


TTAACTTGATTGTGAAATC
1904








DMD


AAAACAATGCATATTTGCA
1905








DMD


AAAATCCAGTATTTTAATG
1906








DMD


ACCCAGCACTGCAGCCTGG
1907








DMD


AACTTATGCGGCGTTTCCT
1908








DMD


TCACTTTAAAACCACCTCT
1909








DMD


GCATCTTTTTCTCTTTAAT
1910








DMD


TGTACTCTCTGAGGTGCTC
1911








DMD


ACGCAGATAAGAACCAGTT
1912








DMD


CATCAAGTCAGCCATCAGC
1913








DMD


GAGTCACCCTCCTGGAAAC
1914








DMD


GCTAGGGATGAAGAATAAA
1915








DMD


TTGACCAATAGCCTTGACA
1916








DMD


TGCAAATATCTGTCTGAAA
1917








DMD


AAATTAGCAGTATCCTCTT
1918








DMD


CCTGGGCTCCGGGGCGTTT
1919








DMD


GGCCCCTGCGGCCACCCCG
1920








DMD


CTCCCTCCCTGCCCGGTAG
1921








DMD


AGGTTTGGAAAGGGCGTGC
1922








DMD


GATTGGCTTTGATTTCCCTA
1923








DMD


GTGTAGAGTAAGTCAGCCTATG
1924








G









DMD


GCCTACTCAGACTGTTACTC
1925








DMD


GTTGGACAGAACTTACCGACTG
1926








G









DMD


GCAGTTGCCTAAGAACTGGT
1927








DMD


GGGGCTCCACCCTCACGAGT
1928








DMD


GTTTGCTTCGCTATAAAACGAG
1929








G









DMD


GTCTGAGGATGGGGCCGCAAT
1930








GG









DMD


GGATCTGTCAAATCGCCTGCAG
1931








G









DMD


GCCAGGATGGCATTGGGCAGC
1932








GG









DMD


GCTGAATCTGCGGTGGCAGGA
1933








GG









DMD


GTTCTTTTGTTCTTCTAGCCTGG
1934








DMD


GGAAAAGCTTGAGCAAGTCAA
1935








GG









DMD


GGAAGAGTTGCCCCTGCGCCA
1936








GG









DMD


GACAAATCTCCAGTGGATAAA
1937








GG









DMD


GTGTTTCTCAGGTAAAGCTCTG
1938








G









DMD


GGAAGGACCATTTGACGTTAA
1939








GG









DMD


GAACTGCTATTTCAGTTTCCTG
1940








G









DMD


GCCAGCCACTCAGCCAGTGAA
1941








GG









DMD


GGTATGCTTTTCTGTTAAAGAG
1942








G









DMD


GCTCCTGGACTGACCACTATTG
1943








G









DMD


GGAACAGAGGCGTCCCCAGTT
1944








GG









DMD


GGAGGCTAGAACAATCATTAC
1945








GG









DMD


GACAAGAACACCTTCAGAACC
1946








GG









DMD


GGGTTTCTGTGATTTTCTTTTGG
1947








DMD


GGGCCAAAGACCTCCGCCAGT
1948








GG









DMD


GTTGGAGAAGCATTCATAAAA
1949








GG









DMD


GTCGCTCACTCACCCTGCAAAG
1950








G









DMD


GAAAAGAGCTGATGAAACAAT
1951








GG









DMD


GTACACTTTTCAAAATGCTTTG
1952








G









DMD


GGAGATGATCATCAAGCAGAA
1953








GG









DMD


GCTTTGAAAGAGCAATAAAAT
1954








GG









DMD


GCACAAAAGTCAAATCGGAAT
1955








GG









DMD


GATTTCAATATAAGATTCGGAG
1956








G









DMD


GCTTAAGCAATCCCGAACTCTG
1957








G









DMD


GCCTTCTTTATCCCCTATCG
1958








DMD


GAGGCCAAACCTCGGCTTACN
1959








NGRR









DMD


GTTCGAAAATTTCAGGTAAGNN
1960








GRR









DMD


GGCAGAACAGGAGATAACAGN
1961








NGRRT









DMD


GGCGGCCCTCGCCCTTCTCTGG
1962








GGAT









DMD


GTAGTGATCGTGGATACGAGA
1963








GG









DMD


GTACAGCCCTCGGTGTATATTG
1964








G









DMD


GGGAAGGAATTAAGCCCGAAT
1965








GG









DMD


GGGAACAGCTTTCGTAGTTGAG
1966








G









DMD


GATAAAGTCCAGTGTCGATCAG
1967








G









DMD


GAAAACCAGAGCTTCGGTCAA
1968








GG









DMD


GGAGTCTTCTGGGCAGGCTTAA
1969








AGGCTAACCTGG









DMD


GTCGGGTGAGCATGTCTTTAAT
1970








CTACCTCGATGG









DMD


GGTGTCACCAGAGTAACAGTCT
1971








GAGT









DMD


GTGATCATCAAGCAGAAGGTA
1972








TGAG









DMD


GAACTTCGAAAATTTCAGGTAA
1973








GCCGAGG









DMD


GGAAACTCATCAAATATGCGTG
1974








TTAGTGT









DMD


GTCATTTACACTAACACGCATA
1975








TTTGATG









DMD


GGAATGAAACTCATCAAATAT
1976








GCGTGTTA









DMD


GTCATCAATATCTTTGAAGGAC
1977








TCTGGGT









DMD


GTGTTTTCATAGGAAAAATAGG
1978








CAAGTTG









DMD


GAATTGGAAAATGTGATGGGA
1979








AACAGATA









DMD


GATGATCATCAAGCAGAAGGT
1980








ATGAGAAA









DMD


GAGATGATCATCAAGCAGAAG
1981








GTATGAGA









DMD


GCATTTTTTCTCATACCTTCTGC
1982








TTGATG









DMD


GTCCTACTCAGACTGTTACTCT
1983








GGTGACA









DMD


GACAGGTTGTGTCACCAGAGTA
1984








ACAGTCT









DMD


GTTATCATTTTTTCTCATACCTT
1985








CTGCTT









DMD


GTTGCCTAAGAACTGGTGGGA
1986








AATGGTCT









DMD


GAAACAGTTGCCTAAGAACTG
1987








GTGGGAAA









DMD


GTTTCCCACCAGTTCTTAGGCA
1988








ACTGTTT









DMD


GTGGCTTTGATTTCCCTAGGGT
1989








CCAGCTT









DMD


GTAGGGAAATCAAAGCCAATG
1990








AAACGTTC









DMD


GGACCCTAGGGAAATCAAAGC
1991








CAATGAAA









DMD


GTGAGGGCTCCACCCTCACGAG
1992








TGGGTTT









DMD


GAAGGATTGAGGGCTCCACCCT
1993








CACGAGT









DMD


GGCTCCACCCTCACGAGTGGGT
1994








TTGGTTC









DMD


GTATCCCCTATCGAGGAAACCA
1995








CGAGTTT









DMD


GGATAAAGAAGGCCTATTTCAT
1996








AGAGTTG









DMD


GAGGCCTTCTTTATCCCCTATC
1997








GAGGAAA









DMD


GTGAGGGCTCCACCCTCACGAG
1998








TGGGT









DMD


GGATAAAGAAGGCCTATTTCAT
1999








AGAGT









DMD


CACCGCAGCCGCTCGCTGCAGC
2000








AG









DMD


AAACCTGCTGCAGCGAGCGGC
2001








TGC









DMD


CACCGGCTGGGTGTCCCATTGA
2002








AA









DMD


AAACTTTCAATGGGACACCCAG
2003








CC









DMD


CACCGGTTTATTCAGCCGGGAG
2004








TC









DMD


AAACGACTCCCGGCTGAATAA
2005








ACC









DMD


CACCGTGGAGAGTTTGCAAGG
2006








AGC









DMD


AAACGCTCCTTGCAAACTCTCC
2007








AC









DMD


CACCGCCCTCCAGACTTTCCAC
2008








CT









DMD


AAACAGGTGGAAAGTCTGGAG
2009








GGC









DMD


CACCGAATTTTCTTCCAAGTTC
2010








TC









DMD


AAACGAGAACTTGGAAGAAAA
2011








TTC









DMD


CACCGCTGCGGAGAGAAGAAA
2012








GGG









DMD


AAACCCCTTTCTTCTCTCCGCA
2013








GC









DMD


CACCGAGAGCCACCCCCTGGCT
2014








CC









DMD


AAACGGAGCCAGGGGGTGGCT
2015








CTC









DMD


CACCGCGAAGCCAACCGCGGC
2016








GGG









DMD


AAACCCCGCCGCGGTTGGCTTC
2017








GC









DMD


CACCGAGAGGGAAGACGATCG
2018








CCC









DMD


AAACGGGCGATCGTCTTCCCTC
2019








TC









DMD


CACCGCCCCTTTAACTTTCCTC
2020








CG









DMD


AAACCGGAGGAAAGTTAAAGG
2021








GGC









DMD


CACCGGCAGCCCCGCTTCCTTC
2022








AA









DMD


AAACTTGAAGGAAGCGGGGCT
2023








GCC









DMD


CACCGCGAGAGCGAGAGGAGG
2024








GAG









DMD


AAACCTCCCTCCTCTCGCTCTC
2025








GC









DMD


CACCGGAGAGAGCTTGAGAGC
2026








GCG









DMD


AAACCGCGCTCTCAAGCTCTCT
2027








CC









DMD


CACCGGGTGGAGGGGGCGGGG
2028








CCC









DMD


AAACGGGCCCCGCCCCCTCCAC
2029








CC









DMD


CACCGGGTATCCACGTAAATCA
2030








AA









DMD


AAACTTTGATTTACGTGGATAC
2031








CC









DMD


CACCGCCAATCACTGGCTCCGG
2032








TC









DMD


AAACGACCGGAGCCAGTGATT
2033








GGC









DMD


CACCGGGCGCCCGAGGGAAGA
2034








AGA









DMD


AAACTCTTCTTCCCTCGGGCGC
2035








CC









DMD


CACCGGGGTGGGGGTACCAGA
2036








GGA









DMD


AAACTCCTCTGGTACCCCCACC
2037








CC









DMD


CACCGCCGGGGACAGAAGAGA
2038








GGG









DMD


AAACCCCTCTCTTCTGTCCCCG
2039








GC









DMD


CACCGGAGAGAGAGTGGGAGA
2040








AGC









DMD


AAACGCTTCTCCCACTCTCTCT
2041








CC









DMD


CACCGAAAGTAACTGTCAAAT
2042








GCG









DMD


AAACCGCATTTGACAGTTACTT
2043








TC









DMD


CACCGTTAACCAGAGCGCCCA
2044








GTC









DMD


AAACGACTGGGCGCTCTGGTTA
2045








AC









DMD


CACCGCGTCGGAGCTGCCCGCT
2046








AG









DMD


AAACCTAGCGGGCAGCTCCGA
2047








CGC









DMD


TGTACTCTCTGAGGTGCTC
2048








DMD


ACGCAGATAAGAACCAGTT
2049








DMD


CATCAAGTCAGCCATCAGC
2050








DMD


GAGTCACCCTCCTGGAAAC
2051








DMD


CCTGGGCTCCGGGGCGTTT
2052








DMD


GGCCCCTGCGGCCACCCCG
2053








DMD


CTCCCTCCCTGCCCGGTAG
2054








DMD


AGGTTTGGAAAGGGCGTGC
2055








DMD


ACTCCACTGCACTCCAGTCT
2056








DMD


TCTGTGGGGGACCTGCACTG
2057








DMD


GGGGCGCCAGTTGTGTCTCC
2058








DMD


ACACCATTGCCACCACCATT
2059








DMD


CAATGACCCCTTCATTGACC
2060








DMD


TTGATTTTGGAGGGATCTCG
2061








DMD


GGAATCCATGGAGGGAAGAT
2062








DMD


TGTTCTCGCTCAGGTCAGTG
2063








DMD


CTCTCTGCTCCTTTGCCACA
2064








DMD


GTGCTCTTCGGGTTTCAGGA
2065








DMD


CGAAAGAGAAAGCGAACCAGT
2066








ATCGAGAAC









DMD


CGTTGTGCATAGTCGCTGCTTG
2067








ATCGC









DMD


UAGAAGAUCUGAGCUCUGAG
2068








DMD


AGAUCUGAGCUCUGAGUGGA
2069








DMD


UCUGAGCUCUGAGUGGAAGG
2070








DMD


CCGUUUACUUCAAGAGCUGA
2071








DMD


AAGCAGCCUGACCUAGCUCC
2072








DMD


GCUCCUGGACUGACCACUAU
2073








DMD


CCCUCAGCUCUUGAAGUAAA
2074








DMD


GUCAGUCCAGGAGCUAGGUC
2075








DMD


UAGUGGUCAGUCCAGGAGCU
2076








DMD


GCUCCAAUAGUGGUCAGUCC
2077








DMD


UGGCCAAAGACCUCCGCCAG
2078








DMD


GUGGCAGACAAAUGUAGAUG
2079








DMD


UGUAGAUGUGGCAAAUGACU
2080








DMD


CUUGGCCCUGAAACUUCUCC
2081








DMD


CAGAGAAUAUCAAUGCCUCU
2082








DMD


CAGAGAAUAUCAAUGCCUCU
2083








DMD


CAUUUGUCUGCCACUGGCGG
2084








DMD


CUACAUUUGUCUGCCACUGG
2085








DMD


CAUCUACAUUUGUCUGCCAC
2086








DMD


AUAAUCCCGGAGAAGUUUCA
2087








DMD


UAUCAUCUGCAGAAUAAUCC
2088








DMD


UGUUAUCAUGUGGACUUUUC
2089








DMD


UGAUAUAUCAUUUCUCUGUG
2090








DMD


UUUAUGAAUGCUUCUCCAAG
2091








DMD


UUCUCCAGGCUAGAAGAACAA
2092








DMD


CUGCUCUUUUCCAGGUUCAAG
2093








DMD


GUCUGUUUCAGUUACUGGUGG
2094








DMD


UCCAGUUUCAUUUAAUUGUUU
2095








DMD


CUUAUGGGAGCACUUACAAGC
2096








DMD


UUGCUUCAUUACCUUCACUGG
2097








DMD


UUGUGUCACCAGAGUAACAGU
2098








DMD


AGUAACCACAGGUUGUGUCAC
2099








DMD


UUCAAAUUUUGGGCAGCGGUA
2100








DMD


CAAGAGGCUAGAACAAUCAUU
2101








DMD


UUGUACUUCAUCCCACUGAUU
2102








DMD


CUUCAGAACCGGAGGCAACAG
2103








DMD


CAACAGUUGAAUGAAAUGUUA
2104








DMD


GCCAAGCUUGAGUCAUGGAAG
2105








DMD


CUUGGUUUCUGUGAUUUUCUU
2106








DMD


UCAUUUCACAGGCCUUCAAGA
2107








DMD


CAGAAAUAUUCGUACAGUCUC
2108








DMD


CAAUUACCUCUGGGCUCCUGG
2109








DMD


GAACUUCUAUUUAAUUUUG
2110








DMD


AUUUCAGGUAAGCCGAGGUU
2111








DMD


UCUUAAUAAUGUUUCACUGU
2112








DMD


AUAAUUUCUAUUAUAUUACA
2113








DMD


UUUCAUUCAUAUCAAGAAGA
2114








DMD


AUAGUUUAAAGGCCAAACCU
2115








DMD


UGUGAAAAAAUAUAGUUUAA
2116








DMD


CGAAAAUUUCAGGUAAGCCG
2117








DMD


CAAAAACCCAAAATATTTTAGC
2118








T









DMD


CCTTTTTGGTATCTTACAGGAA
2119








C









DMD


CCGCTGCCCAATGCCATCCTGG
2120








A









DMD


TTTTTCCTTTTATTCTAGTTGAA
2121








DMD


TTGATCCATATGCTTTTACCTG
2122








C









DMD


TCAACAGATCTGTCAAATCGCC
2123








T









DMD


TTCTTCTTTCTCCAGGCTAGAA
2124








G









DMD


GTTCTTCTAGCCTGGAGAAAGA
2125








A









DMD


CAAATCCTGCATTGTTGCCTGT
2126








A









DMD


CTGTTAAAGAGGAAGTTAGAA
2127








GA









DMD


AAAATTTTTATATTACAGAATA
2128








T









DMD


TTGTAGACTATCTTTTATATTCT
2129








DMD


TTTTGCATTTTAGATGAAAGAG
2130








A









DMD


AACATCTTCTCTTTCATCTAAA
2131








A









DMD


TTTTGAACATCTTCTCTTTCATC
2132








DMD


CAAAAACCCAAAATATTTTAGC
2133








T









DMD


GCTTGTGTTTCTAATTTTTCTTT
2134








DMD


ACTTATTGTTATTGAAATTGGC
2135








T









DMD


TACCATGTATTGCTAAACAAAG
2136








T









DMD


GTATCAATTCACACCAGCAAGT
2137








T









DMD


CTCCTCTGTAAAGTGGCGATTA
2138








T









DMD


TTTAAAATGAAGATTTTCCACC
2139








A









DMD


AAATGAAGATTTTCCACCAATC
2140








A









DMD


CCACCAATCACTTTACTCTCCT
2141








A









DMD


CCACCAGTTCTTAGGCAACTGT
2142








T









DMD


CATTAATTTATATCCTTGATTAT
2143








DMD


GTTGTTGTTGTTAAGGTCAAAG
2144








T









DMD


AAATTACCCTAGATCTTAAAGT
2145








T









DMD


GCCTCTGATTAGGGTGGGGGCG
2146








TG









DMD


TCACAGGCTCCAGGAAGGGTTT
2147








GG









DMD


CCCAGGGGGGCCTCTTTCGGAA
2148








GG









DMD


GGAAGGCTCTCTTGGTGATGGA
2149








GA









DMD


AAGCTAGTCTAGTGCAAGCTAA
2150








CA









DMD


CTGGCCTATGTTATTACCTGTA
2151








TG









DMD


TGGCCTATGTTATTACCTGTAT
2152








GG









DMD


TTCCATTCTAATGGGTGGCTGT
2153








T









DMD


CTCCTCTGTAAAGTGGCGAT
2154








DMD


TTCCATTCTAATGGGTGGCT
2155








DMD


GTATCAATTCACACCAGCAA
2156








DMD


TACCATGTATTGCTAAACAA
2157








DMD


ACTTATTGTTATTGAAATTG
2158








DMD


GCTTGTGTTTCTAATTTTTC
2159








DMD


CAAAAACCCAAAATATTTTA
2160








DMD


TTTAAAATGAAGATTTTCCA
2161








DMD


AAATGAAGATTTTCCACCAA
2162








DMD


CCACCAATCACTTTACTCTC
2163








DMD


CCACCAGTTCTTAGGCAACT
2164








DMD


CATTAATTTATATCCTTGAT
2165








DMD


AGTTATAGCTCTCTTTCAAT
2166








DMD


ATGTATAACAATTCCAACAT
2167








DMD


AAATTACCCTAGATCTTAAA
2168








DMD


GTTGTTGTTGTTAAGGTCAA
2169








DMD


GCTTGTGTTTCTAATTTTTC
2170








DMD


TAATTTTTCTTTTTCTTCTT
2171








DMD


GCAAAAAGGAAAAAAGAAGA
2172








DMD


GGGTTTTTGCAAAAAGGAAA
2173








DMD


AGCTCCTACTCAGACTGTTA
2174








DMD


TGCAAAAACCCAAAATATTT
2175








DMD


TGTCACCAGAGTAACAGTCT
2176








DMD


CTTAGTAACCACAGGTTGTG
2177








DMD


TAGTTTGGAGATGGCAGTTT
2178








DMD


GAGATGGCAGTTTCCTTAGT
2179








DMD


CTTGATGTTGGAGGTACCTG
2180








DMD


ATGTTGGAGGTACCTGCTCT
2181








DMD


TAACTTGATCAAGCAGAGAA
2182








DMD


TCTGCTTGATCAAGTTATAA
2183








DMD


TAAAATCACAGAGGGTGATG
2184








DMD


ATATCCTCAAGGTCACCCAC
2185








DMD


ATGATCATCTCGTTGATATC
2186








DMD


TCATACCTTCTGCTTGATGA
2187








DMD


TCATTTTTTCTCATACCTTC
2188








DMD


TGCCAACTTTTATCATTTTT
2189








DMD


AATCAGAAAGAAGATCTTAT
2190








DMD


ATTTCCCTAGGGTCCAGCTT
2191








DMD


GCTCAAATTGTTACTCTTCA
2192








DMD


AGCTCCTACTCAGACTGTTA
2193








DMD


ATTCTAGTACTATGCATCTT
2194








DMD


ACTTAAGTTACTTGTCCAGG
2195








DMD


CCAAGGTCCCAGAGTTCCTA
2196








DMD


TTTCCCTGGCAAGGTCTGAA
2197








DMD


GCTCATTCTCATGCCTGGAC
2198








DMD


TTTAGCAATACATGGTAGAA
2199








DMD


AGCCAAACTCTTATTCATGA
2200








DMD


TAACAATGTGGATACTTTGT
2201








DMD


GUGUUAUUACUUGCUACUGCA
2202








DMD


GUGUAUUGCUUGUACUACUCA
2203








DMD


GUUUAAAUGUAAAUAGCUCAG
2204








DMD


GAAUUUUCAAUGAUGUUCUGG
2205








G









DMD


GAACUGGUGGGAAAUGGUCUA
2206








G









DMD


GUUUCAUUGGCUUUGAUUUCC
2207








C









DMD


GGCAAUUCUCCUGAAUAGAAA
2208








DMD


GAUUAUACUUAGGCUGAAUAG
2209








U









DMD


GACUUCCAGAAUUAUGUGUUC
2210








DMD


GUGAGGGCCUGACACAUGGUA
2211








DMD


GUGAAGAUCAUUUCUUGGUAG
2212








DMD


GCACAGUCAGAACUAGUGUGC
2213








DMD


GAGUAAGCCCGAUCAUUAUUG
2214








DMD


GGAAGGGACAUAUUCUAUGGG
2215








DMD


GACCACAAGCUGACUUGGGGG
2216








DMD


GGAUUUGUAUCCAUUAUCUGG
2217








DMD


CUCUGCAUUGUUUUGGCCUC
2218








DMD


UCCUCCAAAGAGUAGAAUGG
2219








DMD


GCCCUAAACUUACACUGUUC
2220








DMD


AAAGAUAGAUUAGAUUGUCC
2221








DMD


GUUGCUAAAUUACAUAGUUU
2222








DMD


UGUUGCAAUAGUCAAUCAAG
2223








DMD


AUACUGAUUAAGACAGAUGA
2224








DMD


AAUACUGAUUAAGACAGAUG
2225








DMD


CUCUAUACAAAUGCCAACGC
2226








DMD


ACUUGCAUGCACACCAGCGU
2227








DMD


UUGGGCUAAUGUAGCAUAAU
2228








DMD


GCGUUGGCAUUUGUAUAGAG
2229








DMD


UGGGCUAAGUAGCAUAAUG
2230








DMD


UUUGGGCUAAUGUAGCAUAA
2231








DMD


GCUUAACUCCUUAAUAUUAA
2232








DMD


UCUUCUAUAUUAAAGCAGAU
2233








DMD


CUUCUAUAUUAAAGCAGAUU
2234








DMD


AAUAUAUAACUACCUUGGGU
2235








DMD


ACCUCCAUUCUACUCUUUGG
2236








DMD


UUUCAAUGAUAUCCAACCCA
2237








DMD


AGUACCUCCAUUCUACUCUU
2238








DMD


CUAUCCUCCAAAGAGUAGAA
2239








DMD


UUUUGCUACAUAUUUCAGGC
2240








DMD


UUUGCUACAUAUUUCAGGCU
2241








DMD


GGGUUGGAUAUCAUUGAAAA
2242








DMD


AUAUUUCAGGCUGGGUUCU
2243








DMD


UUGAAAUAUAUAACUACCUU
2244








DMD


AUUGAAAUAUAUAACUACCU
2245








DMD


GUGAGUAGUGGGGCACUUUA
2246








DMD


UGUAUGUAGAAGGUUAACUA
2247








DMD


GAGCCUAAUAAAUGUACAAU
2248








DMD


UUGUAUGUAGAAGGUUAACU
2249








DMD


CAAUUUGUUUUGAGUAACU
2250








DMD


UGCCUUCUGAAAUAGUCCAG
2251








DMD


GUUAAUAGGGAAACAGCAUA
2252








DMD


AACAAUGCAGAGUUAAUUGU
2253








DMD


GAACAUGUUGAGUAGACACA
2254








DMD


UUUAUCAUCUGUGUCUAUUC
2255








DMD


UCUUUACUUUCUUGACUAUA
2256








DMD


AAUAUUCUCAAACCUCGUUC
2257








DMD


AUUAACUGUGUUCCAGAACG
2258








DMD


UAACUGCUUCUUUGGAUGAC
2259








DMD


GACCAGAACAGUGUAAGUUU
2260








DMD


ACCAGAACAGUGUAAGUUUA
2261








DMD


CUACUUUUUCCCCACUACUG
2262








DMD


UGGAACACAGUUAAUUCACU
2263








DMD


GUGUUGUUUAACUGCUUCUU
2264








DMD


AACUGUCAGUUGCAUAUUCC
2265








DMD


CAGAAAGGAAUGCUGGUACC
2266








DMD


UCUGCCUACACAAUGAAUGG
2267








DMD


CACAGAUCAAUCCAAUUGUU
2268








DMD


UUGACAGGUGGAAAGUACAU
2269








DMD


ACAUUUUUAGGCUUGACAGG
2270








DMD


CUCUCCCAUGACAGACUCCC
2271








DMD


UUGGUAAGAGUUAUGAUAAG
2272








DMD


AACACAAAUUAAGUUCACCU
2273








DMD


AGGAUCAGUGCUGUAGUGCC
2274








DMD


GGCCGUUUAUUAUUAUUGAC
2275








DMD


UCUCAGGAUUGCUAUGCAAC
2276








DMD


CAGGAAGACAUACCAUGUAA
2277








DMD


AGCAGGGCUCUUUCAGUUUC
2278








DMD


UAACAUUUUCAGCUUGAACC
2279








DMD


UCAAGCUGAAAAUGUUACAC
2280








DMD


GUAACAUUUUCAGCUUGAAC
2281








DMD


CAGAAUGAAUUUUGGAGCAC
2282








DMD


UUUAUUAUUAUUGACUGGUG
2283








DMD


AGAAGAAUCUGACCUUUACA
2284








DMD


GCAGGGCUCUUUCAGUUUCU
2285








DMD


CUAAACAGUAGCCAGGCGUG
2286








DMD


CGCCUGGCUACUGUUUAGUG
2287








DMD


CUCCGCACUAAACAGUAGCC
2288








DMD


GUAGCCAGGCGUGUGGAUGU
2289








DMD


CUUGGCUUUGACUAUUCUGC
2290








DMD


AGUAGCCAGGCGUGUGGAUG
2291








DMD


UCCUCCCACAUCCACACGCC
2292








DMD


UUGGCUUUGACUAUUCUGCU
2293








DMD


AUAAUGUCUCUGGCUUGUAA
2294








DMD


UGGUACCCGGCAGCUCUCUG
2295








DMD


GUGGGAGGAACCUCAAAGAG
2296








DMD


UGACUAUUCUGCUGGGAACA
2297








DMD


CUCUCUGAGGAAUGUUCCCU
2298








DMD


AACAUUCCUCAGAGAGCUGC
2299








DMD


AUUCUGAAGCUCCAAACAAU
2300








DMD


UAAAUUACUCUGCUAAAGUA
2301








DMD


AGUACAAACCAGGUUUGUAC
2302








DMD


AUAUCCUUCCAGUACAAACC
2303








DMD


CAAACCAGGUUUGUACUGGA
2304








DMD


GGCAGCUAAAGCAUCACUGA
2305








DMD


AUCUCUGAGUAGUACAAACC
2306








DMD


GUGUCCCAUUCUCUUUGACU
2307








DMD


UGUGUCCCAUUCUCUUUGAC
2308








DMD


UUCUGAAUGUUGAACAAGUA
2309








DMD


GUCUCCCAGUCAAAGAGAAU
2310








DMD


AUUCUCUUUGACUGGGAGAC
2311








DMD


UCUUUGACUGGGAGACAGGC
2312








DMD


GUGGUGUCCUUUGAAUAUGC
2313








DMD


AGAUUGUCCAGGAUAUAAUU
2314








DMD


UUAGCAACCAAAUUAUAUCC
2315








DMD


GUUGAAAUUAAACUACACAC
2316








DMD


AUCUUUACCUGCAUAUUCAA
2317








DMD


GUGUCCUUUGAAUAUGC
2318








DMD


UUGUCCAGGAUAUAAUU
2319








DMD


GCAACCAAAUUAUAUCC
2320








DMD


GAAAUUAAACUACACAC
2321








DMD


UUUACCUGCAUAUUCAA
2322








DMD


UACACAUUUUUAGGCUUGAC
2323








DMD


CAUUCCUGGGAGUCUGUCAU
2324








DMD


UGUAUGAUGCUAUAAUACCA
2325








DMD


GUGGAAAGUACAUAGGACCU
2326








DMD


UCUUAUCAUAACUCUUACCA
2327








DMD


ACAUUUUUAGGCUUGAC
2328








DMD


UCCUGGGAGUCUGUCAU
2329








DMD


AUGAUGCUAUAAUACCA
2330








DMD


GAAAGUACAUAGGACCU
2331








DMD


UAUCAUAACUCUUACCA
2332








DMD


GAGTTCCTACTCAGACTGTTAC
2333








TC









DMD


GTGAGTTCCTACTCAGACTGTT
2334








ACTC









DMD


GTCTGAGTTCCTACTCAGACTG
2335








TTACTC









DMD


AAAGATATATAATGTCATGAAT
2336








DMD


GCAGAATCAAATATAATAGTCT
2337








DMD


AACAAATATCCCTTAGTATC
2338








DMD


AATGTATTTCTTCTATTCAA
2339








DMD


AACAATAAGTCAAATTTAATTG
2340








DMD


GAACTGGTGGGAAATGGTCTA
2341








G









DMD


TCCTTTGGTAAATAAAAGTCCT
2342








DMD


TAGGAATCAAATGGACTTGGAT
2343








DMD


TAATTCTTTCTAGAAAGAGCCT
2344








DMD


CTCTTGCATCTTGCACATGTCC
2345








DMD


ACTTAGAGGTCTTCTACATACA
2346








DMD


TCAGAGGTGAGTGGTGAGGGG
2347








A









DMD


ACACACAGCTGGGTTATCAGA
2348








G









DMD


CACAGCTGGGTTATCAGAG
2349








DMD


ACACAGCTGGGTTATCAGAG
2350








DMD


CACACAGCTGGGTTATCAGAG
2351








DMD


AACACACAGCTGGGTTATCAG
2352








AG









DMD


CTGSTGGGARATGGTCTAG
2353








DMD


ACTGGTGGGAAATGGTCTAG
2354








DMD


AACTGGTGGGAAATGGTCTAG
2355








DMD


AGAACTGGTGGGAAATGGTCT
2356








AG









DMD


ATATCTTCTTAAATACCCGA
2357








DMD


AGTCTCACAAAACTGCAGAG
2358








DMD


TACTTATGTATTTTAAAAAC
2359








DMD


GAATAATTTCTATTATATTACA
2360








DMD


TTCGAAAATTTCAGGTAAGCCG
2361








DMD


TCATTTCTAAAAGTCTTTTGCC
2362








DMD


TTTGAGACACAGTATAGGTTAT
2363








DMD


ATATAATAGAAATTATTCAT
2364








DMD


TAATATGCCCTGTAATATAA
2365








DMD


TGATATCATCAATATCTTTG
2366








DMD


GCAATTAATTGGAAAATGTG
2367








DMD


CTTTAAGCTTAGGTAAAATCA
2368








DMD


CAGTAATGTGTCATACCTTC
2369








DMD


CAGGGCATATTATATTTAGA
2370








DMD


CAAAAGCCAAATCTATTTCA
2371








DMD


ATGCTTTGGTGGGAAGAAGTA
2372








GAGGA









DMD


ATGCTTTGGTGGGAAGAATAG
2373








AGGAC









DMD


TTGTGACAAGCTCACTAATTAG
2374








G









DMD


AAGTTTGAAGAACTTTTACCAG
2375








G









DMD


AGGCAGCGATAAAAAAAACCT
2376








GG









DMD


GCTTTGGTGGGAAGAAGTAGA
2377








GG









VII. EXAMPLES

The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.


Example 1

Genomic editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (DMD). Most of these mutations are clustered in “hotspots.” There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots were screened. Correction of DMD mutations by exon skipping is referred to herein as “myoediting.” In proof-of-concept studies, myoediting was performed in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. Thus, abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.


Identification of Optimal Guide RNAs to Target 12 Different Exons Associated with Hotspot Regions of DMD Mutations


A list of the top 12 exons that, when skipped, can potentially restore the dystrophin open reading frame in most of the hotspot regions of DMD mutations is shown in Table 5. As an initial step toward correcting a majority of human DMD mutations by exon skipping, pools of guide RNAs were screened to target the top 12 exons of the human DMD gene (FIGS. 1A and 1B). Three to six PAM sequences (NAG or NGG) were selected to target the 3′ or 5′ splice sites, respectively, of each exon (FIG. 1A and Table 5). These guide RNAs were cloned in plasmid SpCas9-2A-GFP. Indels that remove essential splice donor or acceptor sequences allow for skipping of the corresponding target exon. On the basis of the frequency of known DMD mutations, these guide RNAs would be predicted to be capable of rescuing dystrophin function in up to 60% of DMD patients.


To test the feasibility and efficacy of this strategy in the human genome, human embryonic kidney 293 cells (239 cells) were used to target the splice acceptor site of exon 51 (FIG. 1C). Transfected 293 cells were sorted by green fluorescent protein (GFP) expression, and gene editing efficiency was detected by the mismatch-specific T7E1 endo-nuclease assay (FIG. 6A). The ability of three guide RNAs (Ex51-g1, Ex51-g2, and Ex51-g3) to target the splice acceptor site of exon 51 is shown in Table 5 and FIG. 2B. In GFP-positive sorted 293 cells, Ex51-g3 showed high editing activity, whereas Ex51-g1 and Ex51-g2 had no detectable activity. Next, cleavage efficiency of guide RNAs, which target the top 12 exons, including exons 51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8, and 55, was evaluated. One or two guide RNAs with the highest efficiency of editing of each exon are shown in FIG. 1C. The selected guide RNAs for exons 51, 45, and 55 use NAG as the PAM (Table 5). Genomic polymerase chain reaction (PCR) products from the myoedited top 12 exons were cloned and sequenced (FIG. 5A and Table 20). Indels were observed that removed essential splice sites or shifted the open reading frame (FIG. 5A). In brain and kidney tissues, an N-terminally truncated form of dystrophin (Dp140) is transcribed from an alternative promoter in intron 44. Skipping of six targeted exons (exons 51, 53, 46, 52, 50, and 55) in Dp140 mRNA was confirmed in 293 cells by sequencing of reverse transcription PCR (RT-PCR) products (FIG. 5B).









TABLE 20







Sequence of primers for top 12 exons.


PCR/T7E1 and RT-PCR primers











Exon

SEQ ID

SEQ ID


#
PCR/T7E1
NO:
RT-PCR
NO:





51
F: TTCCCTGGCAAGGTCTGA
2427
F-E47: CCCAGAAGAGCAAGATAAACTTGAA
2451



R: ATCCTCAAGGTCACCCACC
2428
R-E52: CTCTGTTCCAAATCCTGCATTGT
2452





45
F: GTCTTTCTGTCTTGTATCCTTTGG
2429





R: AATGTTAGTGCCTTTCACCC
2430







53
F: GGGAAATCAGGCTGATGGGT
2431
F-E52: CAAGACCAGCAATCAAGAGGCTAG
2453



R: GTCTACTGTTCATTTCAGC
2432
R-E54: TCATGTGGACTTTTCTGGTATCATC
2454





44
F: GCAGGAAACTATCAGAGTG
2433





R: ACACCTTGCTGTTACGAT
2434







46
F: CCACCAAACCTGGCAAAT
2435
F-E45: GAACTCCAGGATGGCATTGG
2455



R:
2436
R-E52: CTCTGTTCCAAATCCTGCATTGT
2456



GAACTATGAATAACCTAATGGGC






AG








52
F: TTCTTACTCAAGGCATTCAGAC
2437
F-E51: GAAACTGCCATCTCCAAACTAGAAA
2457



R: GGTCACCACACCCATCAAT
2438
R-E54: TTCTCCAAGAGGCATTGATATTCTC
2458





50
F: TGCCTGGAGAAAGGGTTT
2439
R-E47: CCCAGAAGAGCAAGATAAACTTGAA
2459



R: GCACAGTCAATAACACAAAGGT
2440
R-E52: CTCTGTTCCAAATCCTGCATTGT
2460





43
F: AGCGATCCACTCTCTCAGGATG
2441





R:
2442





GCACCTCAATGCCCCAATCTGATT






TACG








 6
F: GGGTCTAATATGGCAGAATCCA
2443





R:
2444





GTTGTAAAGTAGGACATGATCTG






G








 7
F: AGGACTATGGGCATTGGTT
2445





R:
2446





GTGTAGAAATGACAAGTCTCAGA






TG








 8
F:
2447





GAAAGCTACTCTGTTAGATGGGCT






AG






R: GGCTTTGTATATATACACGTG
2448







55
F: GCAGCATCAAAGACAAGCA
2449
F-E52: CAAGACCAGCAATCAAGAGGCTAG
2461



R: TCCTTACGGGTAGCATCC
2450
R-E56: GAGAGACTTTTTCCGAAGTTCAC
2462









Correction of Diverse DMD Patient Mutations by Myoediting

To evaluate the effectiveness of a single-guide RNA to correct different types of human DMD mutations by exon skipping, three DMD iPSC lines with representative types of DMD mutations were obtained: a large deletion (termed Del; lacking exons 48 to 50), a pseudo-exon mutation (termed pEx; caused by an intronic point mutation), and a duplication mutation (termed Dup). Briefly, peripheral blood mono-nuclear cells (PBMCs) obtained from whole blood were cultured and then reprogrammed into iPSCs using recombinant Sendai viral vectors expressing reprogramming factors. Cas9 and guide RNAs for correction or bypass of the mutations in iPSC myoediting on an iPSC line (also known as Del) from a DMD patient with a large deletion of exons 48 to lines were introduced into cells by nucleofection. Pools of treated cells or single clones were then differentiated into induced cardiomyocytes (iCMs) using standardized conditions. Purified iCMs were used to generate 3D-EHM and to perform functional assays (FIG. 2A).


Correction of a Large Deletion Mutation

It is estimated that ˜60 to 70% of DMD cases are caused by large deletions of one or more exons. Myoediting was performed on an iPSC line from a DMD patient with a large deletion of exons 48 to 50 in a hotspot. The large deletion creates a frameshift mutation and introduces a premature stop codon in exon 51, as shown in FIG. 2B. Destruction of the splice acceptor in exon 51 will, in principle, allow for splicing of exons 47 to 52, thereby reconstituting the open reading frame (FIG. 2B and FIG. 6B). Theoretically, skipping exon 51 can potentially correct ˜13% of DMD patients. Optimized guide RNA Ex51-g3 and Cas9 (FIG. 2C) were nucleofected into this iPSC line, resulting in successful destruction of the splice acceptor or reframing of exon 51 by NHEJ, as demonstrated by genomic sequencing, and restoration of the open reading frame (FIG. 6B). The pool of myoedited and DMD iPSCs (Del-Cor.) was differentiated into iCMs and rescue of in-frame dystrophin mRNA expression was confirmed by sequencing of RT-PCR products from amplification of exons 47 to 52 (FIG. 2D and FIG. 6C).


Correction of a Pseudo-Exon Mutation

To further extend this approach to rare mutations, attempts were made to correct a point mutation within iPSCs from a DMD patient (also known as pEx), who has a spontaneous point mutation in intron 47 (c.6913-4037T>G). This point mutation generates a novel RNA splicing acceptor site (YnNYAG) and results in a pseudo-exon of exon 47A (FIG. 2E), which encodes a premature stop signal. Two guide RNAs (Ex47A-g1 and Ex47A-g2) were designed to precisely target the mutation (FIG. 2F and FIG. 7A and 7B). As shown in FIG. 2G, myoediting abolished the cryptic splice acceptor site and permanently skipped the pseudo-exon, restoring full-length dystrophin protein in the corrected cells (pEx-Cor.). The efficacy of exon skipping was tested by RT-PCR in these DMD iCMs (FIG. 2G). Sequencing of the RT-PCR products confirmed that exon 47 was spliced to exon 48 (FIG. 7C).


It is noteworthy that Ex47A-g2 targets only the mutant allele because the wild-type intron lacks the PAM sequence (NAG) for SpCas9. Moreover, the T>G mutation in this patient creates a disease-specific PAM sequence (AG) for Cas9. It is also noteworthy that this type of correction restores the normal dystrophin protein without any internal deletions (FIGS. 7B and 7C).


Correction of a Large Duplication Mutation

Exon duplications account for ˜10 to 15% of identified DMD-causing mutations. Myoediting was tested on an iPSC line (also known as Dup) from a DMD patient with a large duplication (exons 55 to 59), which disrupts the dystrophin open reading frame (FIG. 2H). Whole-genome sequencing and analysis the copy number variation profile in cells from this patient was performed and identified the precise insertion site in intron 54 (FIG. 2H). This insertion site (In59-In54 junction) was confirmed by PCR (FIG. 8A and Table 4).


It was hypothesized that the 5′ flanking sequence of the duplicated exon 55 is identical such that one guide RNA targeting this region should be able to make two DSBs and delete the entire duplicated region (exons 55 to 59; ˜150 kb). To test this hypothesis, three guide RNAs (In54-g1, In54-g2, and In54-g3) were designed to target sequences near the junction of intron 54 and exon 55 (FIG. 2I). The efficiency of DNA cutting with these guide RNAs was evaluated in 293 cells by T7E1 (FIG. 8B). Guide RNA In54-g1 was selected for subsequent experiments on Dup iPSCs. Genomic PCR products from the myoedited Dup iPSC mixture were cloned and sequenced (FIG. 8C).


To confirm the correction of the duplication mutation, the pool of treated DMD iPSCs (also known as Dup-Cor.) was differentiated into cardiomyocytes. mRNA with duplicated exons was semiquantified by RT-PCR using the duplication-specific primers (Ex59F, a forward primer in exon 59, and Ex55R, a reverse primer in exon 55) and normalized to expression of the b-actin gene (FIG. 2J and Table 4). As expected, the duplication-specific RT-PCR band was absent in wild-type (WT) cells and was decreased dramatically in Dup-Cor. cells. To confirm this result, RT-PCR on the duplication borders of exon 53 to Ex55 and Ex59 to exon 60 (FIG. 8D) was performed. The intensity of duplication-specific upper bands was decreased in corrected iCMs. Single colonies were picked from the treated mixture of cells. Duplication-specific PCR primers (F2-R1) were used to screen the corrected colonies (FIG. 8E). PCR results of three representative corrected colonies (Dup-Cor. #4, #6, and #26) and the uncorrected control (Dup) are shown in FIG. 8E. The absence of a duplication-specific PCR band in colonies 4, 6, and 26 confirmed the deletion of the duplicated DNA region.


Restoration of Dystrophin Protein in Patient-Derived iCMs by Myoediting


Next, the restoration and stable expression of dystrophin protein in single clones and pools of treated iCMs was confirmed by immunocytochemistry (FIG. 3A to 3C, and FIGS. 6D, 7D, and 8F) and Western blot analysis (FIG. 24, D to F). Even without clonal selection and expansion, most of the iCMs in Del-Cor., pEx-Cor., and Dup-Cor. were dystrophin-positive (FIG. 3A to 3C, and FIGS. 6D, 7D, and 8F). From mixtures of myoedited Del iPSCs, two clones (#16 and #27) were picked and differentiated into cardiomyocytes. Clone #27, which has a higher dystrophin expression level, was selected for subsequent experiments (also known as Del-Cor-SC). One selected clone for corrected pEx (#19) was used for further studies (also known as pEx-Cor-SC). Two selected clones for corrected Dup (#26 and #6) were differentiated into iCMs. Clone #6 was used for functional assay experiments (also known as Dup-Cor-SC). Dystrophin protein expression levels of the corrected iCMs were estimated to be comparable to WT cardiomyocytes (50 to 100%) by immunocytochemistry and Western blot analysis (FIG. 3).


Restoration of Function of Patient-Derived iCMs by Myoediting


In addition to measuring dystrophin mRNA and protein expression by biochemical methods, functional analysis to the macroscale was used, using 3D-EHM derived from normal, DMD, and corrected DMD iCMs. Briefly, iPSCs-derived cardiomyocytes were metabolically purified by glucose deprivation. Purified cardiomyocytes were mixed with human foreskin fibroblasts (HFFs) at a 70%:30% ratio. The cell mixture was reconstituted in a mixture of bovine collagen and serum-free medium. After 4 weeks in culture, contraction experiments were performed (FIG. 4A).


EHMs from eight iPSC lines were tested: (i) WT, (ii) uncorrected Del, (iii) Del-Cor-SC, (iv) uncorrected pEx, (v) pEx-Cor., (vi) pEx-Cor-SC, (vii) uncorrected Dup, and (viii) Dup-Cor-SC. Functional phenotyping of DMD and corrected DMD cardiomyocytes in EHM revealed a contractile dysfunction in all DMD EHMs (Del, pEx, and Dup) compared to WT EHMs (FIG. 4B to 4E). A more pronounced contractile dysfunction was seen in Del compared with pEx and Dup EHM. Force of contraction (FOC) was markedly reduced in DMD EHMs and was significantly improved in corrected DMD EHMs (Del-Cor-SC, pEx-Cor-SC, and Dup-Cor-SC) (FIG. 4B to 4E) with completely restored cardiomyocyte maximal inotropic capacity in Dup-Cor-SC (FIGS. 4D and 4E).


Because current gene therapy delivery methods are only able to affect a portion of the heart muscle, an obvious question is what percentage of corrected cardiomyocytes is needed to rescue the phenotype of DCM. To address this question, DMD cells (Del) and corrected DMD cells (Del-Cor-SC) were precisely mixed to simulate a wide range of “therapeutic efficiency” (10 to 100%) in EHM (FIG. 4F). This revealed that 30 to 50% of cardiomyocytes need to be repaired for partial (30%) or maximal (50%) rescue of the contractile phenotype (FIG. 4F). These findings are consistent with previous in vivo studies showing that mosaic dystrophin expression in 50% cardiomyocytes in carrier mice resulted in a near-normal cardiac phenotype. Our findings show that contractile dysfunction was efficiently restored in corrected DMD EHM to a comparable level of WT EHM. Myoediting is thus a highly specific and efficient approach to rescue clinical phenotypes of DMD in EHM.


Discussion

The DMD gene is the largest known gene in the human genome, encompassing 2.6 million base pairs and encoding 79 exons. The large size and complicated structure of the DMD gene contribute to its high rate of spontaneous mutation. There are ˜3000 documented mutations in humans, which include large deletions or duplications (˜77%), small indels (˜12%), and point mutations (˜9%). These mutations mainly affect exons; however, intronic mutations can alter the splicing pattern and cause the disease, as shown here for the pEx mutation.


To potentially simplify the correction of diverse DMD mutations by CRISPR/Cas9 gene editing, guide RNAs were identified that are capable of skipping the top 12 exons, which account for ˜60% of DMD patients. Thus, it is not necessary to design individual guides for each DMD mutation or excise large genomic regions with pairs of guide RNAs.


Rather, patient mutations can be grouped such that skipping of individual exons can restore dystrophin expression in large numbers of patients. In the proof-of-concept study described in Example 1, the optimized myoediting approach using only one guide RNA efficiently restored the DMD open reading frame in a wide spectrum of mutation types, including large deletions, point mutations, and duplications, which cover most of the DMD population. Even relatively large and complex deletions can be corrected by a single cut in the DNA sequence that eliminates a splice acceptor or donor site without the requirement for multiple guide RNAs to direct simultaneous cutting at distant sites with ligation of DNA ends. Although exon-skipping mainly converts DMD to milder BMD, for a subset of patients with duplication or pseudo-exon mutations, myoediting can eliminate the mutations and restore the production of normal dystrophin protein, as we have shown in this study for pEx and Dup mutations.


Dilated cardiomyopathy, characterized by contractile dysfunction and ventricular chamber enlargement, is one of the main causes of death in DMD patients. However, because of the marked interspecies differences in cardiac physiology and anatomy, as well as the natural history of the disease, the shortened longevity of these animals (˜2 years), and the small size of their hearts ( 1/3000 the size of the human heart), cardiomyopathy is not generally observed in mouse models of DMD at the young age. To overcome limitations and shortcomings of 2D cell culture systems and small animal models, human iPSC-derived 3D-EHM was used to show that dystrophin mutations impaired cardiac contractility and sensitivity to calcium concentration. Contractile dysfunction was observed in DMD EHM, resembling the DCM clinical phenotype of DMD patients. Contractile dysfunction was partially-to-fully restored in corrected DMD EHM by myoediting. Thus, genome editing represents an effective means of eliminating the genetic cause and correcting the muscle and cardiac abnormalities associated with DMD. The data presented herein further demonstrate that EHM serves as a suitable preclinical tool to approximate therapeutic efficiency of myoediting.


Human CRISPR clinical trials received approval in China and the United States. One key concern for the CRISPR/Cas9 system is specificity because off-target effects may cause unexpected mutations in the genome. Multiple approaches have been developed to evaluate possible off-target effects, including (i) in silico prediction of target sites and testing them by deep sequencing and (ii) unbiased whole-genome sequencing. In addition, several new approaches have been reported to minimize potential off-target effects and/or to improve the specificity of the CRISPR/Cas9 system, including titration of dosage of Cas9 and guide RNA, paired Cas9 nickases, truncated guide RNAs, and high-fidelity or enhanced Cas9. Although most studies have used in vitro cell culture systems, we and others did not observe off-target effects in our previous studies of germline editing and post-natal editing in mice. According to a recent study of gene editing in human preimplantation embryos, off-target mutations were also not detected in the edited genome. Although comprehensive and extensive analysis of off-target effects is beyond the scope of this study, we are aware that it will eventually be important to thoroughly evaluate possible off-target effects of individual guide RNAs before potential therapeutic application.


Materials and Methods

Plasmids.


The pSpCas9(BB)-2A-GFP (PX458) plasmid containing the human codon-optimized SpCas9 gene with 2A-EGFP and the backbone of guide RNA was a gift from F. Zhang (plasmid #48138, Addgene). Cloning of guide RNA was carried out according to the Feng Zhang Lab CRISPR plasmid instructions (addgene.org/crispr/zhang/).


Transfection and Cell Sorting of Human 293 Cells.


Cells were transfected by Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions, and the cells were incubated for a total of 48 to 72 hours. Cell sorting was performed by the Flow Cytometry Core Facility at University of Texas (UT) Southwestern Medical Center. Transfected cells were dissociated using trypsin-EDTA solution. The mixture was incubated for 5 min at 37° C., and 2 ml of warm Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum was added. The resuspended cells were transferred into a 15-ml Falcon tube and gently triturated 20 times. The cells were centrifuged at 1300 rpm for 5 min at room temperature. The medium was removed, and the cells were resuspended in 500 ml of phosphate-buffered saline (PBS) supplemented with 2% bovine serum albumin (BSA). Cells were filtered into a cell strainer tube through its mesh cap. Sorted single cells were separated into microfuge tubes into GFP+ and GFP-cell populations.


Human iPSC Maintenance, Nucleofection, and Differentiation.


The DMD iPSC line Del was purchased from Cell Bank RIKEN BioResource Center (cell no. HPS0164). The WT iPSC line was a gift from D. Garry (University of Minnesota). Other iPSC lines (pEx and Dup) were generated and maintained by UT Southwestern Wellstone Myoediting Core. Briefly, PBMCs obtained from DMD patients' whole blood were cultured and then reprogrammed into iPSCs using recombinant Sendai viral vectors expressing reprogramming factors (Cytotune 2.0, Life Technologies). iPSC colonies were validated by immuno-cytochemistry, mycoplasma testing, and teratoma formation. Human iPSCs were cultured in mTeSRTM1 medium (STEMCELL Technologies) and passaged approximately every 4 days (1:18 split ratio). One hour before nucleofection, iPSCs were treated with 10 mM ROCK inhibitor (Y-27632) and dissociated into single cells using Accutase (Innovative Cell Technologies Inc.). Cells (1×106) were mixed with 5 mg of SpCas9-2A-GFP plasmid and nucleofected using the P3 Primary Cell 4D-Nucleofector X kit (Lonza) according to manufacturer's protocol. After nucleofection, iPSCs were cultured in mTeSRTM1 medium supplemented with 10 mM ROCK inhibitor, penicillin-streptomycin (1:100) (Thermo Fisher Scientific), and primosin (100 mg/ml; InvivoGen). Three days after nucleofection, GFP+ and GFP− were sorted by fluorescence-activated cell sorting, as described above, and subjected to PCR and T7E1 assay.


Isolation of Genomic DNA from Sorted Cells.


Protease K (20 mg/ml) was added to DirectPCR Lysis Reagent (Viagen Biotech Inc.) to a final concentration of 1 mg/ml. Cells were centrifuged at 4° C. at 6000 rpm for 10 min, and the supernatant was discarded. Cell pellets kept on ice were resuspended in 50 to 100 ml of DirectPCR/protease K solution and incubated at 55° C. for >2 hours or until no clumps were observed. Crude lysates were incubated at 85° C. for 30 min and then spun for 10 s. NaCl was added to a final concentration of 250 mM, followed by the addition of 0.7 volumes of isopropanol to precipitate DNA. The DNA was centrifuged at 4° C. at 13,000 rpm for 5 min, and the supernatant was discarded. The DNA pellet was washed with 1 ml of 70% EtOH and dissolved in water. The DNA concentration was measured using a NanoDrop instrument (Thermo Fisher Scientific).


Amplifying Targeted Genomic Regions by PCR.


PCR assays contained 2 ml of GoTaq polymerase (Promega), 20 ml of 5× green GoTaq reaction buffer, 8 ml of 25 mM MgCl2, 2 ml of 10 mM primer, 2 ml of 10 mM deoxynucleotide triphosphate, 8 ml of genomic DNA, and double-distilled H2O (ddH2O) to 100 ml. PCR conditions were as follows: 94° C. for 2 min, 32× (94° C. for 15 s, 59° C. for 30 s, and 72° C. for 1 min), 72° C. for 7 min, and then held at 4° C. PCR products were analyzed by 2% agarose gel electrophoresis and purified from the gel using the QIAquick PCR Purification kit (Qiagen) for direct sequencing. These PCR products were subcloned into pCRII-TOPO vector (Invitrogen) according to the manufacturer's instructions. Individual clones were picked, and the DNA was sequenced.


T7E1 Analysis of PCR Products.


Mismatched duplex DNA was obtained by denaturation/renaturation of 25 ml of the genomic PCR samples using the following conditions: 95° C. for 10 min, 95° to 85° C. (−2.0° C./s), 85° C. for 1 min, 85° to 75° C. (−0.3° C./s), 75° C. for 1 min, 75° to 65° C. (−0.3° C./s), 65° C. for 1 min, 65° to 55° C. (−0.3° C./s), 55° C. for 1 min, 55° to 45° C. (−0.3° C./s), 45° C. for 1 min, 45° to 35° C. (−0.3° C./s), 35° C. for 1 min, 35° to 25° C. (−0.3° C./s), 25° C. for 1 min, and then held at 4° C.


Following denaturation/renaturation, the following was added to the samples: 3 ml of 10×NEBuffer 2, 0.3 ml of T7E1 (New England Biolabs), and ddH2O to 30 ml. Digested reactions were incubated for 1 hour at 37° C. Undigested PCR samples and T7E1-digested PCR products were analyzed by 2% agarose gel electrophoresis.


Whole-Genome Sequencing.


Whole-genome sequencing was performed by submitting the blood samples to Novogene Corporation. Purified genomic DNA (1.0 mg) was used as input material for the DNA sample preparation. Sequencing libraries were generated using TruSeq Nano DNA HT Sample Preparation kit (Illumina) following the manufacturer's instructions. Briefly, the DNA sample was fragmented by sonication to a size of 350 bp. The DNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing with further PCR amplification. The libraries were sequenced on an Illumina sequencing platform, and paired-end reads were generated.


Isolation of RNA.


RNA was isolated from cells using TRIzol RNA isolation reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.


Cardiomyocyte Differentiation and Purification.


iPSCs were adapted and maintained in TESR-E8 (STEMCELL Technologies) on 1:120 Matrigel in PBS-coated plates and passaged using EDTA solution (Versene, Thermo Fisher Scientific) twice weekly. For cardiac differentiation, iPSCs were plated at 5×104 to 1×105 cells/cm2 and induced with RPMI, 2% B27, 200 mM L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (Asc; Sigma-Aldrich), activin A (9 ng/ml; R&D Systems), BMP4 (5 ng/ml; R&D Systems), 1 mM CHIR99021 (Stemgent), and FGF-2 (5 ng/ml; Miltenyi Biotec) for 3 days; following another wash with RPMI medium, cells were cultured from days 4 to 13 with 5 mM IWP4 (Stemgent) in RPMI supplemented with 2% B27 and 200 mM Asc. Cardiomyocytes were metabolically purified by glucose deprivation from days 13 to 17 in glucose-free RPMI (Thermo Fisher Scientific) with 2.2 mM sodium lactate (Sigma-Aldrich), 100 mM b-mercaptoethanol (Sigma-Aldrich), penicillin (100 U/ml), and streptomycin (100 mg/ml). Cardiomyocyte purity was 92±2% from 15 independent differentiation runs (one to three for each cell line).


EHM Generation.


To generate defined, serum-free EHM, purified cardiomyocytes were mixed with HFFs (American Type Culture Collection) at a 70%:30% ratio. The cell mixture was reconstituted in a mixture of pH-neutralized medical-grade bovine collagen (0.4 mg per EHM; LLC Collagen Solutions) and concentrated serum-free medium [2×RPMI, 8% B27 without insulin, penicillin (200 U/ml), and streptomycin (200 mg/ml)] and cultured for 3 days in Iscove medium with 4% B27 without insulin, 1% nonessential amino acids, 2 mM glutamine, 300 mM ascorbic acid, IGF1 (100 ng/ml; AF-100-11), FGF-2 (10 ng/ml; AF-100-18B), VEGF165 (5 ng/ml; AF-100-20), TGF-b1 (5 ng/ml; AF-100-21C; all growth factors are from PeproTech), penicillin (100 U/ml), and streptomycin (100 mg/ml). After a 3-day condensation period, EHM were transferred to flexible holders to support auxotonic contractions. Analysis was carried out after a total EHM culture period of 4 weeks.


Analysis of Contractile Function.


Contraction experiments were performed under isometric conditions in organ baths at 37° C. in gassed (5% CO2/95% 02) Tyrode's solution (containing 120 mM NaCl, 1 mM MgCl2, 0.2 mM CaCl2, 5.4 mM KCl, 22.6 mM NaHCO3, 4.2 mM NaH2PO4, 5.6 mM glucose, and 0.56 mM ascorbate). EHM were electrically stimulated at 1.5 Hz with 5-ms square pulses of 200 mA. EHMs were mechanically stretched at intervals of 125 mm until the maximum systolic force amplitude (FOC) was observed according to the Frank-Starling law. Responses to increasing extracellular calcium (0.2 to 4 mM) were investigated to determine maximal inotropic capacity. Where indicated, forces were normalized to muscle content (sarcomeric α-actinin-positive cell content, as determined by flow cytometry).


Flow Cytometry of EHM-Derived Cells.


Single-cell suspensions of EHM were prepared as described previously and fixed in 70% ice-cold ethanol. Fixed cells were stained with Hoechst 3342 (10 mg/ml; Life Technologies) to exclude cell doublets. Cardiomyocytes were identified by sarcomeric a-actinin staining (clone EA-53, Sigma-Aldrich). Cells were run on a LSRII SORP cytometer (BD Biosciences) and analyzed using the DIVA software. At least 10,000 events were analyzed per sample.


Immunostaining.


iPSC-derived cardiomyocytes were fixed with acetone and subjected to immunostaining. Fixed cardiomyocytes were blocked with serum cocktail (2% normal horse serum/2% normal donkey serum/0.2% BSA/PBS), and incubated with dystrophin antibody (1:800; MANDYS8, Sigma-Aldrich) and troponin-I antibody (1:200; H170, Santa Cruz Biotechnology) in 0.2% BSA/PBS. Following overnight incubation at 4° C., they were incubated with secondary antibodies [biotinylated horse anti-mouse immunoglobulin G (IgG) (1:200; Vector Laboratories) and fluorescein-conjugated donkey anti-rabbit IgG (1:50; Jackson ImmunoResearch)] for 1 hour. Nuclei were counter-stained with Hoechst 33342 (Molecular Probes).


EHM cryosections to be immunostained were thawed, further air-dried, and fixed in cold acetone (10 min at −20° C.). Sections were briefly equilibrated in PBS (pH 7.3) and then blocked for 1 hour with serum cocktail (2% normal horse serum/2% normal donkey serum/0.2% BSA/PBS). Blocking cocktail was decanted, and dystrophin/troponin primary antibody cocktail [mouse anti-dystrophin, MANDYS8 (1:800; Sigma-Aldrich) and rabbit anti-troponin-I (1:200; H170, Santa Cruz Bio-technology)] in 0.2% BSA/PBS was applied without intervening wash. Following overnight incubation at 4° C., unbound primary antibodies were removed with PBS washes, and sections were probed for 1 hour with secondary antibodies [biotinylated horse anti-mouse IgG (1:200; Vector Laboratories) and rhodamine donkey anti-rabbit IgG (1:50; Jackson ImmunoResearch)] diluted in 0.2% BSA/PBS. Unbound secondary antibodies were removed with PBS washes, and final dystrophin labeling was carried out with a 10-min incubation of the sections with fluorescein-avidin-DCS (1:60; Vector Laboratories) diluted in PBS. Unbound rhodamine was removed with PBS washes, nuclei were counterstained with Hoechst 33342 (2 mg/ml; Molecular Probes), and slides were coverslipped with Vectashield (Vector Laboratories).


Western Blot Analysis.


Western blot analysis for human iPSC-derived cardiomyocytes was performed, using antibodies to dystrophin (ab15277, Abcam; D8168, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (MAB374, Millipore), and cardiac myosin heavy chain (ab50967, Abcam). Goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) were used for described experiments.


All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.


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Claims
  • 1. A method for editing a mutant dystrophin gene in a cardiomyocyte, the method comprising contacting the cardiomyocyte with: a Cas9 nuclease, or a sequence encoding a Cas9 nuclease, anda gRNA, or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene.
  • 2. The method of claim 1, wherein the gRNA targets a splice donor or splice acceptor site of exon 51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8, or 55.
  • 3. The method of claim 1, wherein the gRNA comprises or targets a sequence of any one of SEQ ID NOs. 60-705, 712-862, 947-2377.
  • 4. The method of claim 1, wherein a vector comprises the gRNA, or a sequence encoding the gRNA.
  • 5. The method of claim 4, wherein the vector is a viral vector or a non-viral vector.
  • 6. The method of claim 5, wherein the viral vector is an adeno-associated viral (AAV) vector.
  • 7. The method of claim 6, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.
  • 8. The method of claim 5, wherein the non-viral vector is a plasmid.
  • 9. The method of claim 5, wherein the non-viral vector is a nanoparticle.
  • 10. The method of claim 1, wherein a first vector comprises the gRNA, or a sequence comprising the gRNA, and a second vector comprises the Cas9, or a sequence comprising the Cas9.
  • 11. The method of claim 10, wherein the first vector and the second vector are AAVs.
  • 12. The method of claim 1, wherein the mutant dystrophin gene comprises a point mutation.
  • 13. The method of claim 12, wherein the point mutation is a pseudo-exon mutation.
  • 14. The method of claim 1, wherein the mutant dystrophin gene comprises a deletion.
  • 15. The method of claim 1, wherein the mutant dystrophin gene comprises a duplication mutation.
  • 16. The method of claim 1, wherein the Cas9 nuclease is isolated or derived from a Streptococcus pyogenes (spCas9).
  • 17. The method of claim 1, wherein the Cas9 nuclease is isolated or derived from a Staphylococcus aureus (saCas9).
  • 18. A cardiomyocyte produced according to the method of claim 1, wherein the cardiomyocyte expresses a dystrophin protein.
  • 19. The cardiomyocyte of claim 18, wherein the cardiomyocyte is derived from an induced pluripotent stem cell (iPSC).
  • 20. A composition comprising a therapeutically effective amount of the cardiomyocyte of claim 18.
  • 21. A method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of claim 20.
  • 22. The method of claim 21, wherein the therapeutically effective amount at least partially or completely restores cardiac contractility in the patient.
  • 23. An induced pluripotent stem cell (iPSC) comprising: a Cas9 nuclease, or a sequence encoding a Cas9 nuclease, anda gRNA, or a sequence encoding a gRNA,wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene.
  • 24. A composition comprising a cardiomyocyte derived from the iPSC of claim 23.
  • 25. A method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of claim 24.
  • 26. The method of claim 25, wherein the therapeutically effective amount at least partially or completely restores cardiac contractility in the patient.
  • 27. A method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject: a Cas9 nuclease, or a sequence encoding a Cas9 nuclease, anda gRNA, or a sequence encoding a gRNA, wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene;wherein the administering restores dystrophin expression in at least 10% of the subject's cardiomyocytes.
  • 28. The method of claim 27, wherein the gRNA targets a splice donor or splice acceptor site of exon 51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8, or 55.
  • 29. The method of claim 27, wherein the gRNA comprises or targets a sequence of any one of SEQ ID NOs. 60-705, 712-862, or 947-2377.
  • 30. The method of claim 27, wherein a vector comprises the gRNA, or a sequence encoding the gRNA.
  • 31. The method of claim 30, wherein the vector is a viral vector or a non-viral vector.
  • 32. The method of claim 31, wherein the viral vector is an adeno-associated viral (AAV) vector.
  • 33. The method of claim 32, wherein the AAV vector is selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.
  • 34. The method of claim 31, wherein the non-viral vector is a plasmid.
  • 35. The method of claim 31, wherein the non-viral vector is a nanoparticle.
  • 36. The method of claim 27, wherein a first vector comprises the gRNA, or a sequence encoding the gRNA, and a second vector comprises the Cas9, or a sequence encoding the Cas9.
  • 37. The method of claim 36, wherein the first vector and the second vector are AAVs.
  • 38. The method of claim 27, wherein the mutant dystrophin gene comprises a point mutation.
  • 39. The method of claim 38, wherein the point mutation is a pseudo-exon mutation.
  • 40. The method of claim 27, wherein the mutant dystrophin gene comprises a deletion.
  • 41. The method of claim 27, wherein the mutant dystrophin gene comprises a duplication mutation.
  • 42. The method of claim 27, wherein the Cas9 nuclease is isolated or derived from a Streptococcus pyogenes (spCas9).
  • 43. The method of claim 27, wherein the Cas9 nuclease is isolated or derived from a Staphylococcus aureus Cas9 (saCas9).
  • 44. The method of claim 27, wherein the subject suffers from dilated cardiomyopathy.
  • 45. The method of claim 27, wherein the administering restores dystrophin expression in at least 30% of the subject's cardiomyocytes.
  • 46. The method of claim 27, wherein the administering at least partially rescues cardiac contractility.
  • 47. The method of claim 27, wherein the administering restores dystrophin expression in at least 50% of the subject's cardiomyocytes.
  • 48. The method of claim 27, wherein the administering completely rescues cardiac contractility.
  • 49. A method for treating or preventing Duchene Muscular Dystrophy (DMD) in a subject in need thereof, the method comprising: contacting an induced pluripotent stem cell (iPSC) with a Cas9 nuclease, or a sequence encoding a Cas9 nuclease, anda gRNA, or a sequence encoding a gRNA,wherein the gRNA targets a splice donor or splice acceptor site of the dystrophin gene;differentiating the iPSC into a cardiomyocyte; andadministering the cardiomyocyte to the subject.
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser. No. 62/624,748, filed Jan. 31, 2018, which is incorporated by reference herein in its entirety for all purposes.

FEDERAL FUNDING SUPPORT CLAUSE

This invention was made with government support under grants no. HL-130253, HL-077439, DK-099653, and AR-067294 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2019/015988 1/31/2019 WO 00
Provisional Applications (1)
Number Date Country
62624748 Jan 2018 US