EA042798B1 - THERAPEUTIC TARGETS FOR CORRECTION OF THE HUMAN DYSTROFIN GENE USING GENE EDITING AND METHODS FOR THEIR APPLICATION - Google Patents

Description

Cross-reference to related applications

This application claims priority over U.S. Provisional Application No. 62/260,712, filed November 30, 2015, and U.S. Provisional Application No. 62/330,336, filed May 2, 2016, all of which are incorporated herein by reference in in all its fullness.

Declaration of State Interest

The present invention was made with government support in accordance with the graduate scholarship program awarded by the National Science Foundation. The US Government has certain rights to the present invention.

Field of invention

The present invention relates to the field of gene expression alteration, genome engineering and genomic rearrangement of genes using regularly spaced short palindromic repeat systems based on (CRISPR)/CRISPR-associated protein (Cas) 9 systems and viral delivery systems. The present invention also relates to the field of genomic engineering and genomic rearrangement of genes in muscle such as skeletal muscle and cardiac muscle.

Background of the invention

Synthetic transcription factors have been developed to control gene expression for many different medical and scientific applications in mammalian systems, including stimulation of tissue regeneration, drug selection, compensation for genetic defects, activation of silent tumor suppressors, control of stem cell differentiation, genetic screening, and generation synthetic gene networks. These transcription factors are capable of targeting promoters or enhancers of endogenous genes or can be targeted to recognize sequences orthogonal to mammalian genomes for transgenic regulation. The most common strategies for constructing novel transcription factors targeting user-defined sequences have been based on programmable DNA-binding domains of zinc finger proteins and transcription activator-like effectors (TALEs). Both of these approaches involve the application of the principles of protein-DNA interactions of these domains with the construction of new proteins with unique DNA-binding specificity. Although these methods have been very successful in a large number of applications, the protein engineering required to manipulate protein-DNA interactions can be labor intensive and require specialized knowledge and skills.

In addition, these new proteins are not always effective. The reasons for this are not yet known, but may be related to the effects of epigenetic modifications and the state of chromatin when a protein binds to a target site in the genome. In addition, there are challenges in ensuring that these new proteins, as well as other components, are delivered to every cell. Existing methods for delivering these new proteins and many of their components involve delivery to cells in separate plasmids or vectors, resulting in highly variable levels of expression in each cell due to copy number differences. In addition, gene activation after transfection is transient due to the dissolution of plasmid DNA, and transient gene expression may not be sufficient to induce therapeutic effects. Moreover, this approach is not suitable for cell types that are not easily transfected. Thus, another limitation of these new proteins is the efficiency of transcriptional activation.

CRISPR/Cas9-based gene editing systems can be used to introduce site-specific double-strand breaks at target genomic loci. This DNA cleavage stimulates the natural DNA repair mechanism, which is reduced to one of two possible repair pathways. In the absence of a donor template, the break is repaired by non-homologous end joining (NHEJ), prone to errors in the repair pathway, resulting in small DNA insertions or deletions. This method can be used to deliberately destroy, delete or change the reading frame of target gene sequences. However, if the donor template is provided together with nucleases, then the cellular machinery performs repair of the break by homologous recombination, which is enhanced by several orders of magnitude in the presence of DNA cleavage. This method can be used to introduce specific changes to the DNA sequence at target sites. The engineered nucleases have been used to edit genes in various human stem cells and cell lines, and to edit genes in mouse livers. However, the main barrier to the implementation of these technologies is in vivo delivery to specific tissues in a manner that is efficient, effective, and promotes successful genome modification.

Hereditary genetic diseases are characterized by devastating effects on children in the United States. These diseases are currently not treatable and can only be treated by trying to alleviate the symptoms. For several decades, the field of gene therapy has promised to provide treatments for these diseases. However, technical barriers related to the safety and efficacy of delivering therapeutic genes to cells and patients have limited this approach. Duchenne muscular dystrophy (DMD) is a fatal

- 1 042798 genetic disease, clinically characterized by muscle atrophy, loss of the ability to move and death, usually in the third decade of life due to functional dystrophy. DMD is the result of congenital or spontaneous mutations in the dystrophin gene. Most of the mutations that cause DMD are the result of exon(s) deletions, shifting the translational reading frame out of frame.

Dystrophin is a key component of a protein complex that is responsible for regulating the integrity and function of muscle cells. Patients with DMD usually lose the ability to physically support themselves during childhood, gradually become weaker during adolescence, and die in their twenties and thirties. Current experimental gene therapy strategies for DMD require reintroduction of transient gene delivery vehicles or rely on the permanent incorporation of foreign genetic material into genomic DNA. Both of these methods have serious security problems. Moreover, these strategies were limited by the inability to deliver the large and complex dystrophin gene sequence. There remains a need for a more accurate and efficient gene editing tool to correct and treat patients with mutations in the dystrophin gene.

Brief description of the invention

The present invention is directed to a guide RNA (gRNA) containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 41, SEQ ID NO: 42, or its complementary subsequence.

The present invention also relates to a DNA targeting composition comprising a first gRNA and a second gRNA. The first gRNA molecule and the second gRNA molecule contain a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 41, SEQ ID NO: 42, or its complementary sequence. The first gRNA molecule and the second gRNA molecule contain targeting domains.

The present invention also relates to an isolated polynucleotide containing the above-described gRNA molecule or the above-described DNA targeting composition.

The present invention relates to a vector containing the above-described gRNA, the above-described DNA targeting composition, or the above-described isolated polynucleotide.

The present invention also relates to a vector containing the above composition for targeting DNA.

The present invention also relates to a vector encoding (a) a first guide RNA (gRNA) molecule;

(b) a second gRNA molecule; and (c) at least one Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25).

The first gRNA molecule and the second gRNA molecule contain a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 41, SEQ ID NO: 42, or its complementary sequence. The first gRNA molecule and the second gRNA molecule contain targeting domains.

The present invention also relates to a cell containing the above-described gRNA, the above-described DNA targeting composition, the above-described isolated polynucleotide, or the above-described vector.

The present invention also relates to a kit containing the above-described gRNA, the above-described DNA targeting system, the above-described isolated polynucleotide, the above-described vector or the above-described cell, and optionally instructions for use.

The present invention also relates to a method for correcting a mutant dystrophin gene in a cell. The method involves introducing into a cell the above-described gRNA, the above-described DNA targeting system, the above-described isolated polynucleotide, or the above-described vector.

The present invention also relates to a method for editing a mutant dystrophin gene in a subject's genome. The method involves administering to a subject a genome editing composition comprising the above-described gRNA, the above-described DNA targeting system, the above-described isolated polynucleotide, or the above-described vector.

The present invention also relates to a method of treating a subject in need thereof who has

- 2 042798 which has a mutant dystrophin gene. The method involves administering to the subject the above-described gRNA, the above-described DNA targeting system, the above-described isolated polynucleotide, or the above-described vector.

The present invention also relates to a modified adeno-associated virus vector for editing a mutant dystrophin gene in the genome of a subject, containing a first polynucleotide sequence encoding the above-described gRNA and a second polynucleotide sequence encoding a Cas9 molecule that recognizes a protospacer adjacent motif (PAM) or NNGRRT ( SEQ ID NO: 24), or NNGRRV (SEQ ID NO: 25).

The present invention also relates to a composition for providing a deletion of a dystrophin gene segment containing exon 51, the composition comprising (a) a first vector containing a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25); and (b) a second vector containing a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO : 25).

Each of the first and second gRNA molecules contains a targeting domain, the length of which is from 19 to 24 nucleotides, where the first vector and the second vector are designed to form the first and second double-strand breaks in the first intron and second intron flanking, respectively, exon 51 of the human DMD gene, due to which the deletion of the dystrophin gene segment containing exon 51 is ensured.

The present invention also relates to a cell containing the above composition.

The present invention also relates to a method for correcting a mutant dystrophin gene in a cell, comprising introducing into the cell (a) a first vector containing a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first Cas9 molecule that recognizes an adjacent protospacer motif (PAM) either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25); and (b) a second vector containing a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO : 25).

Each of the first gRNA molecule and the second gRNA molecule contains a targeting domain, the length of which is from 19 to 24 nucleotides, where the vector is designed to form the first and second double-strand breaks, respectively, in the first and second introns flanking exon 51 of the human dystrophin gene, due to which a deletion of the dystrophin gene segment containing exon 51 is provided.

The present invention also relates to a method of treating a subject in need thereof who has a mutated dystrophin gene. The method involves administering to the subject (a) a first vector containing a polynucleotide sequence encoding a first guide RNA molecule (gRNA) and a polynucleotide sequence encoding a first Cas9 molecule that recognizes a protospacer adjacent motif (PAM) or NNGRRT (SEQ ID NO: 24) , or NNGRRV (SEQ ID NO: 25); and (b) a second vector containing a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO : 25).

Each of the first gRNA molecule and the second gRNA molecule contains a targeting domain, the length of which is from 19 to 24 nucleotides, where the vector is designed to form the first and second double-strand breaks, respectively, in the first and second introns flanking exon 51 of the human dystrophin gene, due to which a deletion of the dystrophin gene segment containing exon 51 is provided.

The present invention also relates to a method for producing a transgenic rodent embryo that has the human dystrophin gene (hDMD) with a deletion of exon 52 (Δ52). The method involves administering to a rodent embryo the above-described gRNA, the above-described DNA targeting system, the above-described isolated polynucleotide, the above-described vector, the above-described modified adeno-associated virus-based vector, or the above-described composition, thereby ensuring the deletion of exon 52 from the human dystrophin gene, and selection of a transgenic rodent embryo , which has a deletion of exon 52 of the human dystrophin gene, where the rodent embryo contains the normal human dystrophin gene.

- 3 042798

The present invention also relates to a transgenic rodent embryo obtained using the method described above.

The present invention also relates to a transgenic rodent derived from the transgenic rodent embryo described above.

Brief description of graphic materials

In FIG. 1 shows the activity of individual JCR89 and JCR91 gRNAs that target the human dystrophin gene in HEK293T cells (wild-type dystrophin gene) and a DMD patient myoblast line (DMD 8036 and DMD 6594, each with a mutant form of the dystrophin gene) determined by analysis. surveyor.

In FIG. 2A and 2B show exon 51 deletion in genomic DNA of HEK293T cells and DMD patient myoblasts (DMD 8036 and DMD 6594) (Fig. 2A) and cDNA from DMD patient myoblast myoblasts (Fig. 2B) by co-processing SaCas9, and gRNA JCR89, and JCR91.

In FIG. 3 shows an in vivo AAV-based system for co-delivery of SaCas9 and JCR89 and JCR91 gRNA in two viral vectors to muscle tissues.

In FIG. 4 shows the detection of human exon 51 deletion in transgenic mice carrying the human DMD gene (hDMD/mdx mice) following local delivery of AAV8 viral vectors carrying SaCas9 and gRNA to the tibialis anterior (TA).

In FIG. 5 shows the detection of human exon 51 deletion in transgenic mice carrying the hDMD gene following systemic delivery of AAV8 by tail vein injection.

In FIG. 6 shows the various gRNA targets that persist between the human and rhesus monkey genomes (see gRNA sequences in Table 2). The location of each gRNA is indicated relative to exon 51 of the human dystrophin gene.

In FIG. 7 shows the activity of individual gRNAs after transfection of human HEK293T cells determined using the Surveyor assay.

In FIG. 8 shows the specificity of candidate gRNAs predicted using the CasOFFinder program (Bae et al. (2014), Bioinformatics, 30: 147 3-1475).

In FIG. 9 shows exon 51 deletion by JCR157 and JCR160 gRNA in HEK293T cells and DMD 6594 cells as determined by genomic DNA PCR.

In FIG. 10 shows the Surveyor activity of various JCR157 gRNA target lengths: 19, 20, 21, 22, and 23 nucleotides.

In FIG. 11 shows the Surveyor activity of various JCR160 gRNA target lengths: 19, 20, 21, 22, and 23 nucleotides.

In FIG. 12 shows deletions obtained by combining JCR157 and JCR160 of various lengths (21, 22 or 23 nucleotides) as determined by genomic DNA PCR.

In FIG. 13 shows targeted nuclease activity using the Surveyor in vitro assay.

In FIG. 14 shows exon 51 deletion in genomic DNA in vitro.

In FIG. 15 shows exon 51 deletion in in vitro human DMD myoblast cDNA differentiated for 7 days.

In FIG. 16 shows the splicing of exons 47-52 in myoblast cDNA from patients with DMD in vitro.

In FIG. 17 shows the construction of an A52/mdx mouse starting from a healthy hDMD/mdx mouse.

In FIG. 18 shows an in vitro guideline compliance test: individual (Surveillor analysis).

In FIG. 19 shows in vitro guideline matching: paired, where a deletion of exon 51 in the genomic DNA of HEK293T cells was created using a gRNA pair.

In FIG. 20 shows a schematic protocol for DNA microinjection.

In FIG. 21 shows a schematic crossbreeding of mice.

In FIG. 22 shows the results of genotyping founding mice.

In FIG. 23 shows a portion of the sequencing results for founding mice 7, 63, and 76.

In FIG. 24 shows a schematic of additional crossing of mice.

In FIG. 25 shows the genotyping of litter 5 (males only) from a 76 + mdx/mdx founder male. 63 represents the male founder (however, in this case, not the parent). 293 represents the HEK293T cell of the genomic control DNA.

In FIG. 26 shows the genotyping of litter 1 from a 63+mdx/mdx founder male.

In FIG. 27 shows a portion of a 392 bp sequencing read. cubs 54497 and 54498.

In FIG. 28 shows immunohistochemical staining of the heart and TA from pups 54497 and 54498.

In FIG. 29 shows that the A52/mdx mouse lacks the dystrophin protein.

In FIG. 30 shows a Western blot indicating that the A52/mdx mouse lacks the dystrophin protein, consistent with the DMD genotype, while the healthy hDMD/mdx mouse expressed dystrophin.

- 4 042798

In FIG. 31 shows the overall activity of an A52/mdx mouse compared to mdx mice and hDMD/mdx mice based on locomotor activity and visual inspection.

In FIG. 32 shows a strategy to correct the A52/mdx mouse using SaCas9 and gRNA to skip exon 51 by targeting gRNA upstream and downstream of exon 51 in the intron region for deletion.

In FIG. 33 shows in vitro recovery of dystrophin protein from an exon 51 deletion in DMD patient myoblasts (DMD 6594 cells) using SaCas9 and JCR179 and JCR183 gRNA.

In FIG. 34 shows a schematic of an A52/mdx mouse processing experiment using gRNA and the SaCas9 system.

In FIG. 35 shows an in vivo exon 51 deletion in the right TA muscle.

In FIG. 36 shows an in vivo exon 51 deletion in the right TA muscle.

In FIG. 37 shows in vivo recovery of dystrophin protein in TA-treated muscle.

In FIG. 38 shows in vivo recovery of dystrophin protein in TA-treated muscle.

In FIG. 39 shows the average of all times for the total distance travelled.

In FIG. 40 shows the average of all time points for the total number of rear-legged postures.

In FIG. 41 shows the grip strength of 16 week old untreated and treated mice.

In FIG. 42 shows the results of PCR cDNA of cardiac tissue.

In FIG. 43 shows sequencing of cDNA amplified by PCR based on the bands from FIG. 42.

Detailed description of the invention

Certain methods and engineered gRNAs described herein have been disclosed to be useful for CRISPR/CRISPR-associated (Cas)9 based gene editing systems for altering expression, genome engineering, and correcting or reducing the effects of mutations in the dystrophin gene involved in genetic diseases. , such as DMD. Opened gRNAs were generated for target sites that are more amenable to transition to clinical use. For example, the gene encoding Cas9 (SpCas9) of S. pyogenes is too large to be delivered by adeno-associated virus (AAV), a vector used for systemic muscle delivery, when all other necessary regulatory sequences are included. Instead, the opened gRNAs were selected and screened for use with S. aureus Cas9 (SaCas9), which is approximately 1 kb. less than SpCas9. Target selections have been screened to be compatible with SaCas9 targets in sequences that are conserved between the human and rhesus monkey genomes, greatly limiting the number of possible gene targets. This selection criterion was chosen to allow gRNA candidates that may be active in both humans and rhesus monkeys to facilitate preclinical testing in non-human primate models. The disclosed gRNAs that target both human and rhesus dystrophin gene sequences can be used with a CRISPR/Cas9 based system to target the intron regions around exon 51 of the human dystrophin gene, causing genomic deletions in this region, to restore functional dystrophin expression in cells from patients with DMD.

Also described herein are genetic constructs, compositions, and methods for delivering CRISPR/Cas9-based gene editing systems and multiple gRNAs for targeting the dystrophin gene. The object of the invention disclosed in the present invention also provides methods for delivering genetic constructs (eg, vectors) or compositions containing them to skeletal muscle and cardiac muscle. The vector may be an AAV, including modified AAV-based vectors. The object of the invention disclosed in the present invention describes a method for delivering active forms of drugs of this class to skeletal muscle and cardiac muscle that are effective, efficient and contribute to successful modification of the genome, and also provides a means to correct the human genome for therapeutic applications and target model species for fundamental scientific applications.

The section headings used in this section and the full disclosures in this document are provided for organizational purposes only and are not intended to be limiting.

1. Definitions.

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by a person skilled in the art. In the event of any conflict, this definition document shall prevail. Preferred methods and materials are described below, although similar or equivalent methods and materials to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned in this document are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not

- 5 042798 are intended to be limiting.

The terms contain(s), including, has, has, may, includes(s) and their variations used in this document are open transitional phrases, terms or words that do not exclude the possibility of additional actions or structures . The singular forms include references to the plural unless the context clearly indicates otherwise. The present invention also covers other embodiments that comprise, comprise, and actually consist of the embodiments or elements presented herein, whether or not they are expressly stated.

Whenever numeric ranges are mentioned in this document, each intermediate number is explicitly covered within the range to the same degree of precision. For example, in the case of the range 6-9, the numbers 7 and 8 are covered in addition to 6 and 9, and in the case of the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3 are explicitly covered. , 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

As used herein, approximately or approximately means the allowable error range for a particular level as determined by one of ordinary skill in the art, which will depend in part on how the level is measured or determined, ie. limitations in the measurement system. For example, approximately can mean 3 or more than 3 standard deviations, in accordance with practice in the art. Alternatively, approximately can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of this level. Alternatively, in particular in accordance with biological systems or processes, the term may mean within one order of magnitude, preferably 5 times and more preferably 2 times the meaning.

The terms adeno-associated virus or AAV, used interchangeably herein, refer to a small virus belonging to the Dependovirus genus of the Parvoviridae family that infects humans and certain other primate species. Currently, AAV does not cause disease, and therefore the virus elicits a very mild immune response.

As used herein, the term binding region refers to the region within the target region for a nuclease that is recognized and bound by the nuclease.

The terms cardiac muscle or cardiac muscle, used interchangeably herein, refer to the type of reflective striated muscle found in the walls and histological basis of the heart, the myocardium. The heart muscle is made up of cardiomyocytes or myocardiocytes. Myocardiocytes show banding similar to that of skeletal muscle cells, but contain only one unique nucleus, unlike multinucleated skeletal muscle. In certain embodiments, the term heart muscle condition refers to a condition associated with the heart muscle, such as cardiomyopathy, heart failure, arrhythmia, and inflammatory heart disease.

As used herein, the term coding sequence or coding nucleic acid refers to nucleic acids (an RNA or DNA molecule) that contain a nucleotide sequence encoding a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing expression in cells of the individual or mammal receiving the nucleic acid. The coding sequence may be codon-optimized.

As used herein, the term complementary sequence or complementary means that a nucleic acid can form base pairs according to Watson Crick (eg, A-T/U and C-G) or Hoogsten between nucleotides or nucleotide analogs of nucleic acid molecules. The term complementarity refers to the property shared by two nucleic acid sequences, due to which, if they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

The terms repair, genome editing, and repair, as used herein, refer to altering a mutant gene that encodes a truncated protein or no protein at all, thereby achieving the expression of a full-length functional or partially full-length functional protein. Correcting or restoring a mutated gene may involve replacing a region of a gene that has a mutation, or replacing the entire mutated gene with a copy of a gene that does not have the mutation, using a repair mechanism such as homologous recombination repair (HDR). Correcting or repairing a mutant gene may also involve repairing the frameshift mutation that causes the premature stop codon, aberrant splice acceptor site, or aberrant splice donor site by creating a double-strand break in the gene, which is then repaired using non-homologous end joining (NHEJ) . At least one base pair can be added or removed by NHEJ during repair, which can restore the appropriate reading frame and eliminate the premature stop codon. Correction or restoration of the mutant gene is also

- 6 042798 may involve the destruction of an aberrant splice acceptor site or splice donor sequence. Correction or repair of a mutated gene may also involve the removal of a non-essential segment of the gene by simultaneously exposing the same DNA strand to two nucleases in order to restore the appropriate reading frame by removing the DNA between the two nuclease target sites and repairing the break. DNA with NHEJ.

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

The term Duchenne muscular dystrophy or DMD, used interchangeably herein, refers to a recessive, fatal, X-linked disorder that results in muscle degeneration and subsequently death. DMD is a common hereditary monogenic disorder and occurs in 1 in 3500 men. DMD is the result of congenital or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. Most of the dystrophin mutations that cause DMD are exon deletions that destroy the reading frame and lead to premature termination of translation in the dystrophin gene. Patients with DMD usually lose the ability to physically support themselves during childhood, gradually become weaker during adolescence, and die in their twenties and thirties.

As used herein, the term dystrophin refers to a rod-shaped cytoplasmic protein that is part of the protein complex that connects the muscle fiber cytoskeleton to the surrounding extracellular matrix across the cell membrane. Dystrophin provides structural stability to the cell membrane dystroglycan complex, which is responsible for regulating the integrity and function of muscle cells. The dystrophin gene or DMD gene, used interchangeably herein, is formed at 2.2 million base pairs at the Xp21 locus. The primary transcript is approximately 2400 kb, while the mature mRNA is approximately 14 kb. 79 exons code for a protein made up of more than 3500 amino acids.

The term exon 51 as used herein refers to exon 51 of the dystrophin gene. Exon 51 in DMD patients is often adjacent to in-frame deletion positions and has been targeted by oligonucleotide-based exon skipping in clinical trials. Recently, an exon 51 skipping clinical trial with an Eterlipsen compound reported a significant functional benefit over 48 weeks with a mean dystrophin-positive fiber count of 47% from baseline. Mutations in exon 51 are ideally suited for robust correction through NHEJ-based genome editing.

The terms frameshift or frameshift mutation, used interchangeably herein, refer to a type of gene mutation in which the addition or deletion of one or more nucleotides causes a shift in the reading frame of codons in mRNA. A shift in reading frame can result in an amino acid sequence change during protein translation, such as a missense mutation or a premature stop codon.

The terms functional and fully functional, used interchangeably herein, describe a protein that has biological activity. The term functional gene refers to a gene transcribed into an mRNA that undergoes translation into a functional protein.

The term fusion protein as used herein refers to a chimeric protein created by joining two or more genes that originally coded for separate proteins. Translation of the hybrid gene results in a single polypeptide with functional properties derived from each of the original proteins.

The term genetic construct as used herein refers to DNA or RNA molecules that contain a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term expressed form refers to gene constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that, if present in a cell of an individual, the coding sequence will be expressed.

As used herein, the term genetic disease refers to a disease, in whole or in part, directly or indirectly caused by one or more disorders in the genome, especially a condition that is present from birth. The disorder may be a mutation, insertion, or deletion. The disorder can affect the coding sequence of a gene

- 7 042798 or its regulatory sequence. The genetic disorder may be, but is not limited to, DMD, Becker muscular dystrophy (BMD), hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited liver metabolic disorders, Lesch's syndrome - Nihana, sickle cell disease, thalassemia, xeroderma pigmentosa, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and Ty-Sachs disease.

The term homologous recombination repair or HDR, used interchangeably herein, refers to a mechanism in cells to repair double-stranded DNA damage when a homologous portion of DNA is present in the nucleus, primarily during the G2 and S phases of the cell cycle. HDR uses a donor DNA template to guide repair and can be used to create specific sequence changes in the genome, including the targeted addition of entire genes. If the donor template is provided along with a CRISPR/Cas9-based gene editing system, then the cellular machinery performs rupture repair by homologous recombination, which is enhanced by several orders of magnitude in the presence of DNA cleavage. In the event that a fragment of homologous DNA is missing, non-homologous end joining may occur instead.

The term genome editing, as used herein, refers to changing a gene. Genome editing may involve correcting or restoring a mutated gene. Genome editing may involve knocking out a gene, such as a mutated gene or a normal gene. Genome editing can be used to treat diseases or improve muscle recovery by altering a gene of interest.

The terms identical or identity, as used herein in the context of two or more nucleic acids or polypeptide sequences, means that the sequences have a certain percentage of residues that are identical in a specified region. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences in the specified region, determining the number of positions where the same residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to get percent sequence identity. In cases where two sequences are of different lengths or the alignment results in one or more steps in a checkerboard pattern, and the specified area of comparison includes only one sequence, these remainders of one sequence are included in the denominator, but not in the numerator, in the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be done manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

The terms mutant gene or mutated gene, used interchangeably herein, refer to a gene that has been subjected to a detectable mutation. The mutant gene has undergone a change, such as the loss, gain or exchange of genetic material, which affects the normal transmission and expression of the gene. The term disrupted gene as used herein refers to a mutated gene that has a mutation that causes a premature stop codon. The disrupted gene product is truncated compared to the full length undisrupted gene product.

The term non-homologous end joining (NHEJ) pathway as used herein refers to a DNA double-strand break repair pathway by direct ligation of the ends of the break without the need for a homologous template. Template-independent religation of DNA ends with NHEJ is a stochastic, error-prone repair process that introduces random microinsertions and microdeletions (insert-deletions) at the DNA break point. This method can be used to deliberately destroy, delete or change the reading frame of target gene sequences. To guide repair in NHEJ, short homologous DNA sequences, called microhomologies, are commonly used. These microhomologies are often present in single strand sticky ends at the end of double strand breaks. If the sticky ends are perfectly matched, then NHEJ usually repairs the gap accurately, however there may be inaccurate repair resulting in loss of nucleotides, but much more often the sticky ends are mismatched.

As used herein, the term normal gene refers to a gene that has not undergone changes such as loss, gain or exchange of genetic material. A normal gene undergoes normal gene transfer and gene expression.

The term nuclease-mediated NHEJ as used herein refers to NHEJ that is initiated after a nuclease, such as a Cas9 molecule, cuts double-stranded DNA.

As used herein, the terms nucleic acid or oligonucleotide or polynucleotide means at least two nucleotides covalently linked to each other. The image of a single strand also determines the sequence of the complementary strand. Thus,

- 8 042798 nucleic acid also spans the complementary strand of the depicted single strand. Many nucleic acid variants can be used for the same purpose as the specified nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complementary strands. The single strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions. Thus, the nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

The nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid may be DNA, either genomic or cDNA, RNA or a hybrid molecule, where the nucleic acid may contain combinations of deoxyribo- and ribonucleotides, base combinations including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine , isocytosine and isoguanine. Nucleic acids can be produced by chemical synthesis methods or by recombinant methods.

As used herein, the term operably linked means that the expression of a gene is under the control of the promoter to which it is spatially linked. A promoter can be located 5'-end (above) or 3'-end (below) relative to the gene under its control. The distance between a promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, this distance can be changed without loss of promoter function.

The term partially functional as used herein describes a protein that is encoded by a mutant gene and has less biological activity than a functional protein but more than a non-functional protein.

The term premature stop codon or out-of-frame stop codon, used interchangeably herein, refers to a nonsense mutation in a DNA sequence that causes a stop codon at a location not normally found in the wild-type gene. A premature stop codon can result in a truncation or shortening of the protein compared to the full length version of the protein.

As used herein, the term promoter means a molecule of synthetic or natural origin that is capable of providing, activating or enhancing the expression of a nucleic acid in a cell. The promoter may contain one or more specific transcription regulation sequences to further enhance expression and/or to alter spatial expression and/or temporal expression. The promoter may also contain distal enhancer or repressor elements, which may be located several thousand base pairs from the site of transcription initiation. The promoter can be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate the expression of a genetic component constitutively or differentially according to the cell, tissue, or organ in which expression occurs, or according to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducing agents. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lactose operon promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, human U6 promoter (hU6) and IE CMV promoter.

The term skeletal muscle as used herein refers to a type of striated muscle that is under the control of the somatic nervous system and is attached to bones by bundles of collagen fibers known as tendons. Skeletal muscle is made up of individual components known as myocytes or muscle cells, sometimes colloquially referred to as muscle fibers. Myocytes are formed by the fusion of developing myoblasts (the type of embryonic progenitor cell from which a muscle cell arises) in a process known as myogenesis. These long, cylindrical, multinucleated cells are also called muscle fibers.

The term skeletal muscle condition as used herein refers to a condition associated with skeletal muscle, such as muscular dystrophies, aging, muscle degeneration, wound healing, and muscle weakness or atrophy.

The terms subject and patient, used interchangeably herein, refer to any vertebrate animal, including, without limitation, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog). , rat and mouse, non-human primates (eg, monkeys such as cynomolgus or rhesus macaques, chimpanzees, etc.), and humans). In some embodiments, the subject may or may not be human. The subject or patient may be subjected to other forms of treatment.

The term target gene as used herein refers to any nucleotide sequence encoding a known or suspected gene product. The target gene may

- 9 042798 be a mutated gene involved in a genetic disease. In certain embodiments, the target gene is a human dystrophin gene. In certain embodiments, the target gene is a mutated human dystrophin gene.

The term target region as used herein refers to the region of a target gene for which a CRISPR/Cas9 based gene editing system has been designed for binding and cleavage.

As used herein, the term transgene refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and introduced into another distinct organism. This non-native DNA segment may retain the ability to produce RNA or protein in the transgenic organism or may alter the normal function of the genetic code of the transgenic organism. The introduction of a transgene can change the phenotype of an organism.

The term variant as used herein in relation to a nucleic acid means (i) a portion or fragment of a reference nucleotide sequence;

(ii) a sequence that is complementary to a reference nucleotide sequence or a portion thereof;

(iii) a nucleic acid that is substantially identical to the reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complementary sequence, or sequences substantially identical thereto.

A peptide or polypeptide variant differs in amino acid sequence by insertion, deletion, or conservative amino acid substitution, but retains at least one type of biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to the amino acid sequence of a reference protein that retains at least one type of biological activity. It is recognized in the art that a conservative amino acid substitution, i. replacing an amino acid with another amino acid with similar properties (eg, hydrophilicity, relative number and distribution of charged regions) usually involves little change. These minor changes can be partly identified by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol., 157: 105-132 (1982). The hydropathic index of an amino acid is determined taking into account hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted while still maintaining protein function. In one aspect, amino acids with hydropathic indices of ±2 are substituted. The hydrophobicity of amino acids can also be used to identify substitutions that will result in proteins that retain biological function. Accounting for the hydrophilicity of amino acids in the context of the peptide allows us to calculate the highest local averaged hydrophilicity of this peptide. Substitutions can be made with amino acids in which the difference in hydrophilicity values is within ±2. Both the hydrophobicity index and the hydrophilicity value of amino acids are affected by the specific side chain of that amino acid. In accordance with this observation, the compatibility of amino acid substitutions with biological function depends on the relative similarity of amino acids and, in particular, the side chains of these amino acids, which is revealed on the basis of hydrophobicity, hydrophilicity, charge, size and other properties.

The term vector as used herein means a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, and preferably is plasmid DNA. For example, the vector may encode a Cas9 protein and at least one gRNA molecule, such as a gRNA containing a targeting domain under any of SEQ ID NO: 1-19, SEQ ID NO: 41, SEQ ID NO: 42, or its complementary sequence. In some embodiments, the Cas9 protein may be characterized by the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 33, or SEQ ID NO: 45. In some embodiments, the Cas9 protein may be S. aureus Cas9, such as SaCas9 with the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 45. In some embodiments, the Cas9 protein is encoded by a nucleic acid sequence comprising the nucleic sequence under SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 , SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 43 or SEQ ID NO: 44.

Unless otherwise indicated, the scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. For example, any numerical or word designations in illustrations and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, chemistry and hybridization of protein and nucleic acids described in this document

- 10 042798 documents are those that are well known and commonly used in the art. The meaning and scope of terms should be clear and understandable; however, in the event of any implicit ambiguity, the definitions provided herein take precedence over any dictionary or external definition. In addition, unless otherwise required by context, singular terms include the plural and plural terms include the singular.

2. Genetic constructs for editing the dystrophin gene in the genome.

The present invention is directed to genetic constructs for genome editing, genomic rearrangement, or altering the expression of a dystrophin gene (eg, the human dystrophin gene). The genetic constructs include at least one gRNA that targets both human and rhesus monkey dystrophin gene sequences, such as SaCas9 compatible targets. The disclosed gRNAs can be incorporated into a CRISPR/Cas9-based gene editing system, including systems that use SaCas9, to target the intron regions around exon 51 of the human dystrophin gene, causing genomic deletions in that region, to restore functional dystrophin expression in cells from patients with DMD.

a. dystrophin gene.

The term dystrophin is a rod-shaped cytoplasmic protein that is part of the protein complex that connects the muscle fiber cytoskeleton to the surrounding extracellular matrix through the cell membrane. Dystrophin provides the structural stability of the dystroglycan complex of the cell membrane. The dystrophin gene is formed at 2.2 million base pairs at the Xp21 locus. The primary transcript is approximately 2400 kb, while the mature mRNA is approximately 14 kb. 79 exons code for a protein made up of more than 3500 amino acids. Normal skeletal muscle tissue contains only a small amount of dystrophin, but the absence of its normal expression leads to the development of severe and incurable symptoms. Some mutations in the dystrophin gene result in defective dystrophin and a severe dystrophic phenotype in affected patients. Some mutations in the dystrophin gene result in the production of a fully functional dystrophin protein and a milder dystrophic phenotype in affected patients.

DMD is the result of congenital or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. Naturally occurring mutations and their consequences are relatively well understood in the case of DMD. It is known that in-frame deletions that occur in regions of exons 45-55 (eg, exon 51) contained within the core domain can generate highly functional dystrophin proteins, and many carriers are asymptomatic or exhibit mild symptoms. Moreover, more than 60% of patients could theoretically be treated by targeting exons in a given region of the dystrophin gene (eg, targeting exon 51). Efforts have been made to restore the reading frame of impaired dystrophin in DMD patients by skipping non-essential critical exon(s) (e.g. skipping exon 51) via mRNA splicing to produce internally deleted but functional dystrophin proteins. Deletion of the inner(their) exon(s) of dystrophin (eg, deletion of exon 51) retains the corresponding reading frame, but causes a less severe manifestation of Becker muscular dystrophy or BMD. The genotype in Becker muscular dystrophy or BMD is similar to DMD in that deletions are present in the dystrophin gene. However, these deletions leave the reading frame intact. Thus, an internally truncated but partially functional dystrophin protein is created. BMD is characterized by a wide range of phenotypes, however often, if the deletions are between dystrophin exons 45-55, the phenotype is much milder compared to DMD. Thus, changing the DMD genotype to the BMD genotype is a common strategy for correcting dystrophin. There are many strategies for correcting dystrophin, many of which rely on restoration of the endogenous dystrophin reading frame. This provides a change in the genotype of the disease from DMD to Becker muscular dystrophy. Many patients with BMD have intragene deletions that retain the translational reading frame, resulting in the formation of a shorter, but in most cases functional, dystrophin protein.

In certain embodiments, modification of exon 51 (eg, deletion or deletion of exon 51, eg, with NHEJ) to restore the reading frame reduces the expression of the phenotype in subjects with DMD, including subjects with DMD with deletion mutations. In certain embodiments, exon 51 of the dystrophin gene refers to exon 51 of the dystrophin gene. Exon 51 in DMD patients is often adjacent to in-frame deletion positions and has been targeted by oligonucleotide-based exon skipping in clinical trials. In the exon 51 skipping clinical trial with the Eterlipsen Compound, a significant positive functional effect was reported at 48 weeks with a mean dystrophin-positive fiber count of 47% from baseline. Mutations in exon 51 are ideally suited for robust correction through NHEJ-based genome editing.

The vectors disclosed in the present invention can form deletions in the dystrophin gene, on

- 11 042798 an example of the human dystrophin gene. In certain embodiments, the vector is designed to form two double strand breaks (first double strand break and second double strand break) in two introns (first intron and second intron) flanking the dystrophin gene, thereby allowing the deletion of the segment of the dystrophin gene containing the target position. The dystrophin target can be an exon dystrophin target or an intra-exon dystrophin target, as described herein. Deletion of an exon target position of dystrophin may provide optimization of the dystrophin sequence in a subject suffering from Duchenne muscular dystrophy, for example, it may improve the action or activity of the encoded dystrophin protein or lead to an improvement in the subject's disease state. In certain embodiments, the removal of the exon target position of the dystrophin restores the reading frame. The exon target position of dystrophin may contain one or more exons of the dystrophin gene. In certain embodiments, the dystrophin target position contains exon 51 of a dystrophin gene (eg, the human dystrophin gene).

The genetic construct (eg, a vector) disclosed herein can mediate highly efficient gene editing in exon 51 of a dystrophin gene (eg, the human dystrophin gene). The genetic construct (eg, vector) disclosed herein restores dystrophin protein expression in DMD patient cells.

Exon 51 in DMD patients is often contiguous with in-frame deletion positions. Elimination of exon 51 from the dystrophin transcript by exon skipping can be used to treat approximately 15% of all patients with DMD. This class of dystrophin mutations is ideally suited for sustained correction by NHEJ and HDR-based genome editing. The genetic constructs (eg, vectors) described herein were designed to target modification of exon 51 in the human dystrophin gene. The genetic construct (eg, vector) disclosed in the present invention is transfected into human DMD cells and mediates efficient gene modification and conversion into the appropriate reading frame. Protein repair accompanies frame repair and is found in a large population of cells treated with a CRISPR/Cas9 based gene editing system.

b. CRISPR system.

A genetic construct (eg, a vector) disclosed herein is encoded by a CRISPR/Cas9-based gene editing system specific for a dystrophin gene (eg, the human dystrophin gene). Regularly arrayed short palindromic repeats and CRISPR, used interchangeably herein, refer to loci containing multiple short direct repeats that occur in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system involved in defense against embedding phages and plasmids that provides a form of acquired immunity. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats and serve as a memory of past exposures. Cas9 forms a complex with the 3' end of sgRNA (also referred to interchangeably herein as gRNA), and the protein-RNA pair recognizes its genomic target through complementary base pairing between the 5' end of the sgRNA sequence and a predetermined 20 bp DNA sequence ., known as the protospacer. This complex is directed to homologous loci of pathogenic DNA through the regions encoded in crRNA, i.e. protospacers and protospacer-adjacent motifs (PAM) in the pathogenic genome. The non-coding CRISPR template is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences that direct Cas nucleases to a target site (protospacer). By simply exchanging a 20 bp recognition sequence. expressed sgRNA, Cas9 nuclease can be directed to new targets in the genome. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner similar to RNAi in eukaryotic organisms.

Three classes of CRISPR systems (type I, II and III effector systems) are known. The type II effector system performs a double-strand break in the target DNA in four successive steps using a single Cas9 effector enzyme to cleave dsDNA. Compared to type I and type III effector systems, which require several different effectors to act as a complex, the type II effector system can function in an alternative environment such as eukaryotic cells. The type II effector system consists of a long pre-crRNA that is transcribed from the spacer-containing CRISPR locus, the Cas9 protein, and tracrRNA that is involved in pre-crRNA processing. At the same time, tracrRNAs hybridize with repeat regions separating pre-crRNA spacers, thereby initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event within each Cas9 spacer, producing mature crRNAs that remain associated with tracrRNA and Cas9, forming the Cas9:crRNA-tracrRNA complex.

- 12 042798

The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches for cleavage sequences corresponding to crRNA. Target recognition occurs by detecting complementarity between the protospacer sequence in the target DNA sequence and the remaining spacer sequence in the crRNA. Cas9 mediates target DNA cleavage if a protospacer adjacent motif (PAM) is also present at the 3' end of the protospacer. To target a protospacer, the sequence must be directly followed by a protospacer adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage. Different Type II systems have different PAM requirements. The S. pyogenes CRISPR system may contain the PAM sequence for this Cas9 (SpCas9) as 5'-NRG-3', where R is either A or G, characterizing the specificity of this system in human cells. The unique capability of the CRISPR/Cas9-based gene editing system is the efficient ability to simultaneously target multiple different genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, the type II system from Streptococcus pyogenes naturally prefers the use of the NGG sequence, where N can be any nucleotide, but also accepts other PAM sequences such as NAG in engineered systems (Hsu et al., Nature Biotechnology (2013) DOI : 10.1038/nbt.2647). Similarly, Cas9 derived from Neisseria meningitidis (NmCas9) typically contains the native PAM from NNNNGATT but has activity with multiple PAMs, including the highly degenerate PAM NNNNGNNN (Esvelt et al., Nature Methods (2013), DOI: 10.1038/ nmeth.2681).

The S. aureus Cas9 molecule recognizes the NNGRR (R=A or G) sequence motif (SEQ ID NO: 22) and directs cleavage of the target nucleic acid sequence 1 to 10, eg 3 to 5 bp, upstream of that sequence. In certain embodiments, the S. aureus Cas9 molecule recognizes the NNGRRN (R=A or G) sequence motif (SEQ ID NO: 23) and directs cleavage of the target nucleic acid sequence from 1 to 10, e.g., 3 to 5 bp, above this sequence. In certain embodiments, the S. aureus Cas9 molecule recognizes the NNGRRT (R=A or G) sequence motif (SEQ ID NO: 24) and directs cleavage of the target nucleic acid sequence from 1 to 10, e.g., 3 to 5 bp, above this sequence. In certain embodiments, the S. aureus Cas9 molecule recognizes the NNGRRV sequence motif (R=A or G) (SEQ ID NO: 25) and directs cleavage of the target nucleic acid sequence from 1 to 10, e.g., 3 to 5 bp, above this sequence. In the above embodiments, N may be a nucleotide residue, such as any of A, G, C, or T. Cas9 molecules can be designed to change the PAM specificity of a Cas9 molecule.

(1) Gene editing system based on CRISPR/Cas9.

An engineered form of the type II effector system from Streptococcus pyogenes has been shown to function in human cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites using a synthetically reduced guide RNA (gRNA, also used interchangeably herein as chimeric single guide RNA (sgRNA)), which is a crRNA-tracrRNA fusion construct that eliminates the need for RNase III and crRNA processing in general. This paper presents engineered CRISPR/Cas9 based systems for use in genome editing and treatment of genetic diseases. Designed systems based on CRISPR/Cas9 can be designed to target any gene, including genes involved in genetic disease, aging, tissue regeneration or wound healing. CRISPR/Cas9 based gene editing systems may include a Cas9 protein or a Cas9 fusion protein and at least one gRNA. In certain embodiments, the system includes two gRNA molecules. A Cas9 fusion protein may, for example, include a domain with an activity different from that which is endogenous to Cas9, such as a transactivation domain.

The target gene (eg, a dystrophin gene, eg, the human dystrophin gene) may be involved in cell differentiation or any other process that may require activation, or may contain a mutation, such as a frameshift or nonsense mutation. If the target gene contains a mutation that causes a premature stop codon, an aberrant splice acceptor site, or an aberrant splice donor site, then a CRISPR/Cas9-based gene editing system can be designed to recognize and bind a nucleotide sequence above or below the premature stop codon. , an aberrant splicing acceptor site, or an aberrant splicing donor site. The CRISPR-Cas9-based system can also be used to disrupt normal gene splicing by targeting splicing acceptors and donors to induce skipping of premature stop codons or repair a damaged reading frame. The CRISPR/Cas9 based gene editing system may or may not mediate off-target changes in protein-coding regions of the genome.

(a) Cas9 molecules and Cas9 fusion proteins.

A CRISPR/Cas9 based gene editing system can include the Cas9 protein or fusion

- 13 042798 Cas9 protein. The Cas9 protein is a nucleic acid cleaving endonuclease and is encoded by CRISPR loci and also participates in the type II CRISPR system. Cas9 protein can be from any species of bacteria or archaea, including but not limited to

Streptococcus pyogenes, Staphylococcus aureus (S. aureus), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp. , cycliphilus denitrifleans, Aminomonas paucivorans, Bacillus cereus, Bacillus smith!!. Bacillus thuringiensis, Bacteroides sp. _r Blastopirellula marina, Bradyrhizobium sp. , Brevibacillus laterosporus, Campylobacter coll, Campylobacter jejuni, Campylobacter lari, Candidatus Punice!spirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzas , Haemophilus sputorum , Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii , Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neiscenisseria cinereaves, Neisseria sp. , Neisseria wadsworthii, Nitrosomonas sp. , Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp. , Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp. , Subdoligranulum sp. , Tistrella mobilis, Treponema sp. or Verminephrobacter eiseniae.

In certain embodiments, the Cas9 molecule is a Cas9 protein, which is a Streptococcus pyogenes Cas9 molecule (also referred to herein as SpCas9). In certain embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule (also referred to herein as SaCas9).

A Cas9 molecule or a Cas9 fusion protein can interact with one or more gRNA molecules and together with the gRNA molecule(s) located at a site that includes a target domain and, in certain embodiments, a PAM sequence. The ability of a Cas9 molecule or a Cas9 fusion protein to recognize a PAM sequence can be determined, for example, using transformation analysis as described previously (Jinek 2012).

In certain embodiments, the ability of the Cas9 molecule or Cas9 fusion protein to interact with and cleave the target nucleic acid is dependent on the PAM sequence. The PAM sequence is the sequence in the target nucleic acid. In certain embodiments, the target nucleic acid is cleaved upstream of the PAM sequence. Cas9 molecules from different bacterial species can recognize different sequence motifs (eg, PAM sequences). In certain embodiments, the S. pyogenes Cas9 molecule recognizes the NGG sequence motif and directs cleavage of the target nucleic acid sequence 1 to 10 bp, e.g. 3 to 5 bp, upstream of that sequence (see, e.g., Mali, 2013). In certain embodiments, the S. thermophilus Cas9 molecule recognizes the sequence motif NGGNG (SEQ ID NO: 36) and/or NNAGAAW (W=A or T) (SEQ ID NO: 20) and directs the cleavage of the target nucleic acid sequence from 1 to 10 bp .o., for example, 3 to 5 bp, above these sequences (see, for example, Horvath, 2010; Deveau, 2008). In certain embodiments, the S. mutans Cas9 molecule recognizes the NGG and/or NAAR (R=A or G) sequence motif (SEQ ID NO: 21) and directs cleavage of the target nucleic acid sequence from 1 to 10 bp, e.g. from 3 up to 5 bp, above this sequence (see, for example, Deveau 2008). In certain embodiments, the S. aureus Cas9 molecule recognizes the NNGRR (R=A or G) sequence motif (SEQ ID NO: 22) and controls

- 14 042798 cleavage of the target nucleic acid sequence from 1 to 10 bp, for example from 3 to 5 bp, above this sequence. In certain embodiments, the S. aureus Cas9 molecule recognizes the NNGRRN sequence motif (R=A or G) (SEQ ID NO: 23) and directs cleavage of the target nucleic acid sequence from 1 to 10 bp, e.g., from 3 to 5 bp. o., above this sequence. In certain embodiments, the S. aureus Cas9 molecule recognizes the NNGRRT (R=A or G) sequence motif (SEQ ID NO: 24) and directs cleavage of the target nucleic acid sequence from 1 to 10 bp, e.g., from 3 to 5 bp. o., above this sequence. In certain embodiments, the S. aureus Cas9 molecule recognizes the NNGRRV sequence motif (R=A or G; V=A or C or G) (SEQ ID NO: 25) and directs 1 to 10 bp cleavage of the target nucleic acid sequence. o., for example, from 3 to 5 bp, above this sequence. In the above embodiments, N may be a nucleotide residue, eg, any of A, G, C, or T. Cas9 molecules can be designed to change the PAM specificity of the Cas9 molecule.

In certain embodiments, the vector encodes at least one Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25). In certain embodiments, at least one Cas9 molecule is a S. aureus Cas9 molecule. In certain embodiments, at least one Cas9 molecule is a mutant S. aureus Cas9 molecule.

The Cas9 protein can be mutated such that nuclease activity is inactivated. An inactivated Cas9 protein (iCas9, also referred to as dCas9) without endonuclease activity has previously been targeted to genes in bacteria, yeast, and human cells with gRNA to silence gene expression through steric hindrance. Exemplary mutations to the S. pyogenes Cas9 sequence include D10A, E762A, H840A, N854A, N863A and/or D986A. Illustrative mutations to the S. aureus Cas9 sequence include D10A and N580A. In certain embodiments, the Cas9 molecule is a mutant S. aureus Cas9 molecule. In certain embodiments, the mutant S. aureus Cas9 molecule contains the D10A mutation. The nucleotide sequence encoding this S. aureus mutant Cas9 is set forth under SEQ ID NO: 34, which is shown below.

atgaaaagga actacattct ggggctggcc atcgggatta caagcgtggg gtatgggatt attgactatg aaaaaggga cgtgatcgac gcaggcgtca gactgttcaa ggaggccaac gtggaaaaca atgagggacg gagaagcaag aggggagcca ggcgcctgaa acgacggaga aggcacagaa tccagagggt gaagaaactg ctgttcgatt acaacctgct gaccgaccat tctgagctga gtggaattaa tccttatgaa gccagggtga aaggcctgag tcagaagctg tcagaggaag agttttccgc agctctgctg cacctggcta agcgccgagg agtgcataac gtcaatgagg tggaagagga caccggcaac gagctgtcta caaaggaaca gatctcacgc aatagcaaag ctctggaaga gaagtatgtc gcagagctgc agctggaacg gctgaagaaa gatggcgagg tgaggggtc aattaatagg ttcaagacaa gcgactacgt caaagaagcc aagcagctgc tgaaagtgca gaaggcttac caccagctgg atcagagctt catcgatact tatatcgacc tgctggagac tcggagaacc tactatgagg gaccaggaga agggagcccc ttcggatgga aagacatcaa ggaatggtac gagatgctga tgggacattg cacctatttt ccagaagagc tgagaagcgt caagtacgct tataacgcag atctgtacaa cgccctgaat gacctgaaca acctggtcat caccagggat gaaaacgaga aactggaata ctatgagaag ttccagatca tcgaaaacgt gtttaagcag aagaaaaagc ctacactgaa acagattgct aggagatcc tggtcaacga aggagacatc aagggctacc gggtgacaag cactggaaaa ccagagttca ccaatctgaa agtgtatcac gatattaagg acatcacagc acggaaagaa atcattgaga acgccgaact gctggatcag attgctaaga tcctgactat ctaccagagc tccgaggaca tccaggaaga gctgactaac ctgaacagcg agctgaccca ggaagagatc gaacagatta gtaatctgaa ggggtacacc ggaacacaca acctgtccct gaaagctatc

- 15 042798

aatctgattc tggatgagct gtggcataca aacgacaatc agattgcaat ctttaaccgg ctgaagctgg tcccaaaaaa ggtggacctg agtcagcaga aagagatccc aaccacactg gtggacgatt tcattctgtc acccgtggtc aagcggagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaatgata tcattatcga gctgctaggg gagaagaaca gcaaggacgc acagaagatg atcaatgaga tgcagaaacg aaaccggcag accaatgaac gcattgaaga gattatccga actaccggga aagagaacgc aaagtacctg attgaaaaaa tcaagctgca cgatatgcag gagggaaagt gtctgtattc tctggaggcc atccccctgg aggacctgct gaacaatcca ttcaactacg aggtcgatca tattatcccc agaagcgtgt ccttcgacaa ttcctttaac aacaaggtgc tggtcaagca ggaagagaac tctaaaaagg gcaataggac tcctttccag tacctgtcta gttcagattc caagatctct tacgaaacct ttaaaaagca cattctgaat ctggccaaag gaaagggccg catcagcaag accaaaaagg agtacctgct ggaagagcgg gacatcaaca gattctccgt ccagaaggat tttattaacc ggaatctggt ggacacaaga tacgctactc gcggcctgat gaatctgctg cgatcctatt tccgggtgaa catctggat gtgaaagtca agtccatcaa cggcggttc acatcttttc tgaggcgcaa atggaagttt aaaaaggagc gcaacaaagg gtacaagcac catgccgaag atgctctgat tatcgcaaat gccgacttca tctttaagga gtggaaaaag ctggacaaag ccaagaaagt gatggagaac cagatgttcg aagagaagca ggccgaatct atgcccgaaa tcgagacaga acaggagtac aggagattt tcatcactcc tcaccagatc aagcatatca aggatttcaa ggactacaag tactctcacc gggtggataa aaagcccaac agagagctga tcaatgacac cctgtatagt acaagaaaag acgataaggg gaataccctg attgtgaaca atctgaacgg actgtacgac aaagataatg acaagctgaa aaagctgatc aacaaaagtc ccgagaagct gctgatgtac caccatgatc ctcagacata tcagaaactg aagctgatta tggagcagta cggcgacgag aagaacccac tgtataagta ctatgaagag actgggaact acctgaccaa gtatagcaaa aaggataatg gccccgtgat caagaagatc aagtactatg ggaacaagct gaatgcccat ctggacatca cagacgatta ccctaacagt cgcaacaagg tggtcaagct gtcactgaag ccatacagat tcgatgtcta tctggacaac ggcgtgtata aatttgtgac tgtcaagaat ctggatgtca tcaaaaagga gaactactat gaagtgaata gcaagtgcta cgaagaggct aaaaagctga aaaagattag caaccaggca gagttcatcg ccctcctttta caacaacgac ctgattaaga tcaatggcga actgtatagg gtcatcgggg tgaacaatga tctgctgaac cgcattgaag tgaatatgat tgacatcact taxgagagt atctggaaaa catgaatgat aagcgccccc ctcgaattat caaaacaatt gcctctaaga ctcagagtat caaaaagtac tcaaccgaca

ttctgggaaa cctgtatgag gtgaagagca aaaagcaccc tcagattatc aaaaagggc [SEQ ID NO: 34].

In certain embodiments, the mutant S. aureus Cas9 molecule contains the N580A mutation. The nucleotide sequence encoding this mutant S. aureus Cas9 molecule is set forth under SEQ ID NO: 35, which is shown below.

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atgaaaagga actacattct ggggctggac atcgggatta caagcgtggg gtatgggatt attgactatg aaaaaggga cgtgatcgac gcaggcgtca gactgttcaa ggaggccaac gtggaaaaca atgagggacg gagaagcaag aggggagcca ggcgcctgaa acgacggaga aggcacagaa tccagagggt gaagaaactg ctgttcgatt acaacctgct gaccgaccat tctgagctga gtggaattaa tccttatgaa gccagggtga aaggcctgag tcagaagctg tcagaggaag agttttccgc agctctgctg cacctggcta agcgccgagg agtgcataac gtcaatgagg tggaagagga caccggcaac gagctgtcta caaaggaaca gatctcacgc aatagcaaag ctctggaaga gaagtatgtc gcagagctgc agctggaacg gctgaagaaa gatggcgagg tgaggggtc aattaatagg ttcaagacaa gcgactacgt caaagaagcc aagcagctgc tgaaagtgca gaaggcttac caccagctgg atcagagctt catcgatact tatatcgacc tgctggagac tcggagaacc tactatgagg gaccaggaga agggagcccc ttcggatgga aagacatcaa ggaatggtac gagatgctga tgggacattg cacctatttt ccagaagagc tgagaagcgt caagtacgct tataacgcag atctgtacaa cgccctgaat gacctgaaca acctggtcat caccagggat gaaaacgaga aactggaata ctatgagaag ttccagatca tcgaaaacgt gtttaagcag aagaaaaagc ctacactgaa acagattgct aggagatcc tggtcaacga aggagacatc aagggctacc gggtgacaag cactggaaaa ccagagttca ccaatctgaa agtgtatcac gatattaagg acatcacagc acggaaagaa atcattgaga acgccgaact gctggatcag attgctaaga tcctgactat ctaccagagc tccgaggaca tccaggaaga gctgactaac ctgaacagcg agctgaccca ggaagagatc gaacagatta gtaatctgaa ggggtacacc ggaacacaca acctgtccct gaaagctatc aatctgattc tggatgagct gtggcataca aacgacaatc agattgcaat ctttaaccgg ctgaagctgg tcccaaaaaa ggtggacctg agtcagcaga aagagatccc aaccacactg gtggacgatt tcattctgtc acccgtggtc aagcggagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaatgata tcattatcga gctgctaggg gagaagaaca gcaaggacgc acagaagatg atcaatgaga tgcagaaacg aaaccggcag accaatgaac gcattgaaga gattatccga actaccggga aagagaacgc aaagtacctg attgaaaaaa tcaagctgca cgatatgcag gagggaaagt gtctgtattc tctggaggcc atccccctgg aggacctgct gaacaatcca

- 17 042798

ttcaactacg aggtcgatca tattatcccc agaagcgtgt ccttcgacaa ttcctttaac aacaaggtgc tggtcaagca ggaagaggcc tctaaaaagg gcaataggac tcctttccag tacctgtcta gttcagattc caagatctct tacgaaacct ttaaaaagca cattctgaat ctggccaaag gaaagggccg catcagcaag accaaaaagg agtacctgct ggaagagcgg gacatcaaca gattctccgt ccagaaggat tttattaacc ggaatctggt ggacacaaga tacgctactc gcggcctgat gaatctgctg cgatcctatt tccgggtgaa catctggat gtgaaagtca agtccatcaa cggcggttc acatcttttc tgaggcgcaa atggaagttt aaaaaggagc gcaacaaagg gtacaagcac catgccgaag atgctctgat tatcgcaaat gccgacttca tctttaagga gtggaaaaag ctggacaaag ccaagaaagt gatggagaac cagatgttcg aagagaagca ggccgaatct atgcccgaaa tcgagacaga acaggagtac aggagattt tcatcactcc tcaccagatc aagcatatca aggatttcaa ggactacaag tactctcacc gggtggataa aaagcccaac agagagctga tcaatgacac cctgtatagt acaagaaaag acgataaggg gaataccctg attgtgaaca atctgaacgg actgtacgac aaagataatg acaagctgaa aaagctgatc aacaaaagtc ccgagaagct gctgatgtac caccatgatc ctcagacata tcagaaactg aagctgatta tggagcagta cggcgacgag aagaacccac tgtataagta ctatgaagag actgggaact acctgaccaa gtatagcaaa aaggataatg gccccgtgat caagaagatc aagtactatg ggaacaagct gaatgcccat ctggacatca cagacgatta ccctaacagt cgcaacaagg tggtcaagct gtcactgaag ccatacagat tcgatgtcta tctggacaac ggcgtgtata aatttgtgac tgtcaagaat ctggatgtca tcaaaaagga gaactactat gaagtgaata gcaagtgcta cgaagaggct aaaaagctga aaaagattag caaccaggca gagttcatcg ccctcctttta caacaacgac ctgattaaga tcaatggcga actgtatagg gtcatcgggg tgaacaatga tctgctgaac cgcattgaag tgaatatgat tgacatcact taxgagagt atctggaaaa catgaatgat aagcgccccc ctcgaattat caaaacaatt gcctctaaga ctcagagtat caaaaagtac tcaaccgaca ttctgggaaa cctgtatgag gtgaagagca aaaagcaccc tcagattatc : aaaaagggc

[SEQ ID NO: 35].

The nucleic acid encoding the Cas9 molecule may be a synthetic nucleic acid sequence. For example, a synthetic nucleic acid molecule may be chemically modified. The synthetic nucleic acid sequence may be codon-optimized, e.g., at least one unusual codon or an uncommon codon has been replaced with a common codon. this document.

Additionally or alternatively, the nucleic acid encoding a Cas9 molecule or a Cas9 polypeptide may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

An exemplary codon-optimized nucleic acid sequence encoding the S. pyogenes Cas9 molecule is set forth under SEQ ID NO: 26, which is shown below.

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atggataaaa agtacagcat cgggctggac atcggtacaa actcagtggg gtgggccgtg attacggacg agtacaaggt accctccaaa aaatttaaag tgctgggtaa cacggacaga cactctataa agaaaaatct tattggagcc ttgctgttcg actcaggcga gacagccgaa gccacaaggt tgaagcggac cgccaggagg cggtatacca ggagaaagaa ccgcatatgc tacctgcaag aaatcttcag taacgagatg gcaaaggttg acgatagctt tttccatcgc ctggaagaat ccttttcttgt tgaggaagac aagaagcacg aacggcaccc catctttggc aatattgtcg acgaagtggc atatcacgaa aagtacccga ctatctacca ccctcaggaag aagctggtgg actactaccga taaggcggac ctcagactta ttttttggc actcgcccac atgattaaat ttagaggaca ttctttgatc gagggcgacc tgaacccgga caacagtgac gtcgataagc tgttcatcca acttgtgcag acctacaatc aactgttcga agaaaaccct ataaatgctt caggagtcga cgctaaagca atcctgtccg cgcgcctctc aaaatctaga agacttgaga atctgattgc tcagttgccc ggggaaaaga aaaatggatt gttggcaac ctgatcgcc tcagtctcgg actgacccca aatttcaaaa gtaacttcga cctggccgaa gacgctaagc tccagctgtc caaggacaca tacgatgacg acctcgacaa tctgctggcc cagattgggg atcagtacgc cgatctcttt ttggcagcaa agaacctgtc cgacgccatc ctgttgagcg atatcttgag agtgaacacc gaaattacta aagcaccct tagcgcatct atgatcaagc ggtacgacga gcatcatcag gatctgaccc tgctgaaggc tcttgtgagg caacagctcc ccgaaaaata caaggaaatc ttctttgacc aggcaaaaa cggctacgct ggctatatag atggtggggc cagtcaggag gaattctata aattcatcaa gcccattctc gagaaaatgg acggcacaga ggagttgctg gtcaaactta acagggagga ccctgctgcgg aagcagcgga ccttgacaa cgggtctatc ccccaccaga ttcatctggg cgaactgcac gcaatcctga ggaggcagga ggatttttat ccttttctta aagataaccg cgagaaaata gaaaagattc ttacattcag gatcccgtac tacgtgggac ctctcgccg gggcaattca cggtttgcct ggatgacaag gaagtcagag gagactatta caccttggaa cttcgaagaa gtggtggaca agggtgcatc tgccgtct ttcatcgagc ggatgacaaa ttttgacaag aacctccta atgagaaggt gctgcccaaa cattctctgc tctacgagta ctttaccgtc tacaatgaac tgactaaagt caagtacgtc accgagggaa tgaggaagcc ggcattcctt agtggagaac agaagaaggc gattgtagac ctgttgttca agaccaacag gaaggtgact gtgaagcaac ttaaagaaga ctactttaag aagatcgaat gtttgacag tgtggaaatt tcaggggttg aagaccgctt caatgcgtca ttggggactt accatgatct tctcaagatc ataaaggaca aagacttcct ggacaacgaa gaaaatgagg atattctcga agacatcgtc ctcaccctga ccctgttcga agacagggaa atgatagaag agcgcttgaa aacctatgcc caccctcttcg acgataaagt tatgaagcag ctgaagcgca ggagatacac aggatgggga agattgtcaa ggaagctgat caatggaatt agggataaac aggtggcaa gaccatactg gattcctca aatctgatgg cttcgccaat aggaacttca tgcaactgat tcacgatgac tctcttacct tcaaggagga cattcaaaag gctcaggtga gcgggcaggg

- 19 042798

agactcctt catgaacaca tcgcgaattt ggcaggttcc cccgctatta aaaagggcat ccttcaaact gtcaaggtgg tggatgaatt ggtcaaggta atgggcagac ataagccaga aaatattgtg atcgagatgg cccgcgaaaa ccagaccaca cagaagggcc agaaaaatag tagagagcgg atgaagagga tcgaggaggg catcaaagag ctgggatctc agattctcaa agaacaccc gtagaaaaca cacagctgca gaacgaaaaa ttgtacttgt actatctgca gaacggcaga gacatgtacg tcgaccaaga acttgatatt aatagactgt ccgactatga cgtagaccat atcgtgcccc agtccttcct gaaggacgac tccattgata acaaagtctt gacaagaagc gacaagaaca ggggtaaaag tgataatgtg cctagcgagg aggtggtgaa aaaaatgaag aactactggc gacagctgct taatgcaaag ctcattacac aacggaagtt cgataatctg acgaaagcag agagaggtgg cttgtctgag ttggacaagg cagggtttat taagcggcag ctggtggaaa ctaggcagat cacaaagcac gtggcgcaga ttttggacag ccggatgaac acaaaatacg acgaaaatga taaactgata cgagaggtca aagttatcac gctgaaaagc aagctggtgt ccgattttcg gaaagacttc cagttctaca aagttcgcga gattaataac taccatcatg ctcacgatgc gtacctgaac gctgttgtcg ggaccgcctt gataaagaag taccaaagc tggaatccga gttcgtatac ggggattaca aagtgtacga tgtgaggaaa atgatagcca agtccgagca ggagattgga aaggccacag ctaagtactt ctttttttct aacatcatga atttttttaa gacggaaatt accctggcca acggagagat cagaaagcgg ccccttatag agacaaatgg tgaaacaggt gaaatcgtct gggataaggg cagggattc gctactgtga ggaaggtgct gagtatgcca caggtaaata tcgtgaaaaa aaccgaagta cagaccggag gattttccaa ggaaagcatt ttgcctaaaa gaaactcaga caagctcatc gcccgcaaga aagattggga ccctaagaaa tacgggggat ttgactcacc caccgtagcc tattctgtgc tggtggtagc taaggtggaa aaaggaaagt ctaagaagct gaagtccgtg aaggaactct tgggaatcac tatcatggaa agatecatcct ttgaaaagaa ccctatcgat ttcctggagg ctaagggtta caaggaggtc aagaaagacc tcatcattaa actgccaaaa tactctctct tcgagctgga aaatggcagg aagagaatgt tggccagcgc cggagagctg caaaagggaa acgagcttgc tctgccctcc aaatatgtta attttctcta tctcgcttcc cactatgaaa agctgaaagg gtctcccgaa gataacgagc agaagcagct gttcgtcgaa cagcacaagc actatctgga tgaaataatc gaacaaataa gcgagttcag caaaagggtt atcctggcgg atgctaattt ggacaaagta ctgtctgctt ataacaagca ccgggataag cctattaggg aacaagccga gaatataatt cacctcttta cactcacgaa tctcggagcc ccgccgcct tcaaatactt tgatacgact atcgaccgga aacggtatac cagtaccaaa gaggtcctcg atgccaccct catccaccag

tcaattactg gcctgtacga aacacggatcgacctctctc aactgggcgg cgactag [SEQ ID NO: 26].

The corresponding amino acid sequence of the S. pyogenes Cas9 molecule is set forth under SEQ ID NO: 27, which is shown below.

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL

KRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRLEES FLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI

- 20 042798

PYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD [SEQ ID NO: 27]

Exemplary codon-optimized nucleic acid sequences encoding the S. aureus Cas9 molecule and optionally containing nuclear localization sequences (NLS) are set forth under SEQ ID NOS: 28-32, 43 and 44, which are presented below. Another exemplary codon-optimized nucleic acid sequence encoding the S. pyogenes Cas9 molecule provides nucleotides 1293-4451 under SEQ ID NO: 83.

SEQ ID NO: 28 is set forth below.

atgaaaagga actacattct ggggctggac atcgggatta caagcgtggg gtatgggatt attgactatg aaaaaggga cgtgatcgac gcaggcgtca gactgttcaa ggaggccaac gtggaaaaca atgagggacg gagaagcaag aggggagcca ggcgcctgaa acgacggaga aggcacagaa tccagagggt gaagaaactg ctgttcgatt acaacctgct gaccgaccat tctgagctga gtggaattaa tccttatgaa gccagggtga aaggcctgag tcagaagctg tcagaggaag agttttccgc agctctgctg cacctggcta agcgccgagg agtgcataac gtcaatgagg tggaagagga caccggcaac gagctgtcta caaaggaaca gatctcacgc aatagcaaag ctctggaaga gaagtatgtc gcagagctgc agctggaacg gctgaagaaa gatggcgagg tgaggggtc aattaatagg ttcaagacaa gcgactacgt caaagaagcc aagcagctgc tgaaagtgca gaaggcttac caccagctgg atcagagctt catcgatact

- 21 042798

tatatcgacc tgctggagac tcggagaacc tactatgagg gaccaggaga agggagcccc ttcggatgga aagacatcaa ggaatggtac gagatgctga tgggacattg cacctatttt ccagaagagc tgagaagcgt caagtacgct tataacgcag atctgtacaa cgccctgaat gacctgaaca acctggtcat caccagggat gaaaacgaga aactggaata ctatgagaag ttccagatca tcgaaaacgt gtttaagcag aagaaaaagc ctacactgaa acagattgct aggagatcc tggtcaacga aggagacatc aagggctacc gggtgacaag cactggaaaa ccagagttca ccaatctgaa agtgtatcac gatattaagg acatcacagc acggaaagaa atcattgaga acgccgaact gctggatcag attgctaaga tcctgactat ctaccagagc tccgaggaca tccaggaaga gctgactaac ctgaacagcg agctgaccca ggaagagatc gaacagatta gtaatctgaa ggggtacacc ggaacacaca acctgtccct gaaagctatc aatctgattc tggatgagct gtggcataca aacgacaatc agattgcaat ctttaaccgg ctgaagctgg tcccaaaaaa ggtggacctg agtcagcaga aagagatccc aaccacactg gtggacgatt tcattctgtc acccgtggtc aagcggagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaatgata tcattatcga gctgctaggg gagaagaaca gcaaggacgc acagaagatg atcaatgaga tgcagaaacg aaaccggcag accaatgaac gcattgaaga gattatccga actaccggga aagagaacgc aaagtacctg attgaaaaaa tcaagctgca cgatatgcag gagggaaagt gtctgtattc tctggaggcc tccccctgg aggacctgct gaacaatcca ttcaactacg aggtcgatca tattatcccc agaagcgtgt ccttcgacaa ttcctttaac aacaaggtgc tggtcaagca ggaagagaac tctaaaaagg gcaataggac tcctttccag tacctgtcta gttcagattc caagatctct tacgaaacct ttaaaaagca cattctgaat ctggccaaag gaaagggccg catcagcaag accaaaaagg agtacctgct ggaagagcgg gacatcaaca gattctccgt ccagaaggat tttattaacc ggaatctggt ggacacaaga tacgctactc gcggcctgat gaatctgctg cgatcctatt tccgggtgaa catctggat gtgaaagtca agtccatcaa cggcggttc acatcttttc tgaggcgcaa atggaagttt aaaaaggagc gcaacaaagg gtacaagcac catgccgaag atgctctgat tatcgcaaat gccgacttca tctttaagga gtggaaaaag ctggacaaag ccaagaaagt gatggagaac cagatgttcg aagagaagca ggccgaatct atgcccgaaa tcgagacaga acaggagtac aggagattt tcatcactcc tcaccagatc aagcatatca aggatttcaa ggactacaag tactctcacc gggtggataa aaagcccaac agagagctga tcaatgacac cctgtatagt acaagaaaag acgataaggg gaataccctg attgtgaaca

- 22 042798

atctgaacgg actgtacgac aaagataatg acaagctgaa aaagctgatc aacaaaagtc ccgagaagct gctgatgtac caccatgatc ctcagacata tcagaaactg aagctgatta tggagcagta cggcgacgag aagaacccac tgtataagta ctatgaagag actgggaact acctgaccaa gtatagcaaa aaggataatg gccccgtgat caagaagatc aagtactatg ggaacaagct gaatgcccat ctggacatca cagacgatta ccctaacagt cgcaacaagg tggtcaagct gtcactgaag ccatacagat tcgatgtcta tctggacaac ggcgtgtata aatttgtgac tgtcaagaat ctggatgtca tcaaaaagga gaactactat gaagtgaata gcaagtgcta cgaagaggct aaaaagctga aaaagattag caaccaggca gagttcatcg ccctcctttta caacaacgac ctgattaaga tcaatggcga actgtatagg gtcatcgggg tgaacaatga tctgctgaac cgcattgaag tgaatatgat tgacatcact taxgagagt atctggaaaa catgaatgat aagcgccccc ctcgaattat caaaacaatt gcctctaaga ctcagagtat caaaaagtac tcaaccgaca ttctgggaaa cctgtatgag gtgaagagca aaaagcaccc tcagattatc aaaaagggc [SEQ ID NO: 2 8] SEQ ID NO: 29 is set forth below. atgaagcgga actacatcct gggcctggac atcggcatca ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca acgaggggcag gcggagcaag agaggcgcca gaaggctgaa gcggcgggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca ccaaagagca gatcagccgg aacagcaagg ccctgaaga gaaatacgtg gccgaactgc agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga aggatatt aagggctaca gagtgaccag

- 23 042798

caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tccagcaga aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg acccggca aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac agcaagaagg gcaaccggac cccattccag tacctgagca gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgattca tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccgggaagg acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc ccgaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga agaagatcag caaccaggcc gagttttcg cctccttcta caacaacgat ctgatcaaga tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac aagaggcccc ccaggatcat taagacaatc gcctccaaga ccgagcat taagaagtac agcacagaca

ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggc

[SEQ ID NO: 29]

- 24 042798

SEQ ID NO: 30 is set forth below.

atgaagcgca actacatcct cggactggac atcggcatta ccctccgtggg atacggcatc atcgattacg aaactaggga tgtgatcgac gctggagtca ggctgttcaa agaggcgaac gtggagaaca acgaggggcg gcgctcaaag aggggggcc gccggctgaa gcgccgccgc agacatagaa tccagcgcgt gaagaagctg ctgttcgact acaaccttct gaccgaccac tccgaacttt ccggcatcaa cccatatgag gctagagtga agggattgtc ccaaaagctg tccgaggaag agttctccgc cgcgttgctc cacctcgcca agcgcagggg agtgcacaat gtgaacgaag tggaagaaga taccggaaac gagctgtcca ccaaggagca gatcagccgg aactccaagg ccctgaaga gaaatacgtg gcggaactgc aactggagcg gctgaagaaa gacggagaag tgcgcggctc gatcaaccgc ttcaagacct cggactacgt gaaggaggcc aagcagctcc tgaaagtgca aaaggcctat caccaacttg accagtcctt tatcgatacc tacatcgatc tgctcgagac tcggcggact tactacgagg gtcggggga gggctcccca ttttggttgga aggatattaa ggagtggtac gaaatgctga tggacactg cacatacttc cctgaggagc tgcggagcgt gaaatacgca tacaacgcag acctgtacaa cgcgctgaac gacctgaaca atctcgtgat cacccgggac gagaacgaaa agctcgagta ttacgaaaag ttccagatta ttgagaacgt gttcaaacag aagaagaagc cgacactgaa gcagattgcc aaggaaatcc tcgtgaacga aggagacatc aagggctatc gagtgacctc aacgggaaag ccggagttca ccaatctgaa ggtctaccac gacatcaaag acattaccgc ccggaaggag atcattgaga acgcggagct gttggaccag attgcgaaga ttctgaccat ctaccaatcc tccgaggata ttcaggaaga actcaccaac ctcaacagcg aactgaccca ggaggagata gagcaaatct ccaacctgaa gggctacacc ggaactcata acctgagcct gaaggccatc aacttgatcc tggacgagct gtggcacacc aacgataacc agatcgctat tttcaatcgg ctgaagctgg tccccaagaa agtggacctc tcacaacaaa

- 25 042798

aggagatccc tactaccctt gtggacgatt tcattctgtc ccccgtggtc aagagaagct tcatacagtc aatcaaagtg atcaatgcca ttatcaagaa atacggtctg cccaacgaca ttatcattga gctcgcccgc gagaagaact cgaaggacgc ccagaagatg attaacgaaa tgcagaagag gaaccgacag actaacgaac ggatcgaaga aatcatccgg acccggga aggaaaacgc gaagtacctg atcgaaaaga tcaagctcca tgacatgcag gaaggaaagt gtctgtactc gctggaggcc attccgctgg aggacttgct gaacaaccct tttaactacg aagtggatca tatcattccg aggagcgtgt cattcgacaa ttccttcaac aacaaggtcc tcgtgaagca ggaggaaaac tcgaagaagg gaaaccgcac gccgttccag tacctgagca gcagcgactc caagatttcc tacgaaacct tcaagaagca catcctcaac ctggcaaagg ggaagggtcg catctccaag accaagaagg aatatctgct ggaagaaaga gacatcaaca gattctccgt gcaaaaggac ttcatcaacc gcaacctcgt ggatactaga tacgctactc ggggtctgat gaacctcctg agaagactact ttagagtgaa catctggac gtgaaggtca agtcgattaa cggaggtttc acctccttcc tgcggcgcaa gtggaagttc aagaaggaac ggaacaaggg ctacaagcac cacgccgagg acgccctgat cattgccaac gccgacttca tcttcaaaga atggaagaaa cttgacaagg ctaagaaggt catggaaaac cagatgttcg aagaaaagca ggccgagtct atgcctgaaa tcgagactga acaggagtac aaggaaatct ttattacgcc acaccagatc aaacacatca aggatttcaa ggattacaag tactcacatc gcgtggacaa aaagccgaac agggaactga tcaacgacac ccctctactcc acccgggaagg atgacaaagg gaataccctc atcgtcaaca accttaacgg cctgtacgac aaggacaacg ataagctgaa gaagctcatt aacaagtcgc ccgaaaagtt gctgatgtac caccacgacc ctcagactta ccagaagctc aagctgatca tggagcagta tggggacgag aaaaacccgt tgtacaagta ctacgaagaa actgggaatt atctgactaa gtactccaag aaagataacg gccccgtgat taagaagatt aagtactacg gcaacaagct gaacgcccat ctggacatca ccgatgacta ccctaattcc cgcaacaagg tcgtcaagct gagcctcaag ccctaccggt ttgatgtgta ccttgacaat ggagtgtaca agttcgtgac tgtgaagaac cttgacgtga tcaagaagga gaactactac gaagtcaact ccaagtgcta cgaggaagca aagaagttga agaagatctc gaaccaggcc gagttcattg cctccttcta taacaacgac ctgattaaga tcaacggcga actgtaccgc gtcattggcg tgaacaacga tctcctgaac cgcatcgaag tgaacatgat cgacatcact taccgggaat acctggagaa tatgaacgac aagcgcccgc cccggatcat taagactatc gcctcaaaga cccagtcgat caagaagtac agcaccgaca

tcctgggcaa cctgtacgag gtcaaatcga agaagcaccc ccagatcatc aagaaggga

[SEQ ID NO: 30]

- 26 042798

SEQ ID NO: 31 is set forth below.

ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCAAGCGGAACTACA TCCTGGGCCTGGACATCGGCATCACCAGCGTGGGCTACGGCATCATCGACTACGAGACACGGGA CGTGATCGATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAACGAGGGCAGGCGG AGCAAGAGAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGC TGCTGTTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGC CAGAGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTG GCCAAGAGAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGCAACGAGCTGTCCA CCAGAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGGCCGAACTGCAGCT GGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAGCGACTAC GTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCA TCGACACCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAG CCCCTTCGGCTGGAAGGACATCAAAGAATGGTACGAGATGCTGATGGGCCACTGCACCTACTTC CCCGAGGAACTGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACC T GAAC AAT С T C G T GAT С AC C AG G GAG GAGAAC GAGAAG С T G GAAT AT TAG GAGAAG T T С C AGAT CATC GAGAAC G T G T T CAAG CAGAAGAAGAAGС С СAC С С T GAAGCAGAT C GС CAAAGAAAT С С T C GTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCGAGTTCACCAACC TGAAGGTGTACCACGACATCAAGGACATTACCGCCCGGAAAGAGATTATTGAGAACGCCGAGCT GCTGGATCAGATTGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAAGAACTG ACCAATCTGAACTCCGAGCTGACCCAGGAAGAGATCGAGCAGATCTCTAATCTGAAGGGCTATA CCGGCACCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTGTGGCACACCAA CGACAACCAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGTCCCAG CAGAAAGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAAGCT TCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCAT TATCGAGCTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGATGCAGAAG CGGAACCGGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGCAAAGAGAACGCCA AGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAAGGCAAGTGCCTGTACAGCCTGGA AGCCATCCCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGAGGTGGACCACATCATCCCC AGAAGCGTGTCCTTCGACAACAGCTTCAACAACAAGGTGCTCGTGAAGCAGGAAGAAAACAGCA AGAAGGGCAACCGGACCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAAAC СTTCAAGAAGCACATССTGAATСTGGCCAAGGGCAAGGGCAGAATCAGCAAGACCAAGAAAGAG TATCTGCTGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTCATCAACCGGAACC TGGTGGATACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTACTTCAGAGTGAA CAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCTGCGGCGGAAGTGG

- 27 042798

AAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCCTGATCATTGCCA ACGCCGATTTCATCTT CAAAGAG T G GAAGAAAC T G GAGAAG G С CAAAAAAGT GAT GGAAAACGA GATGTTCGAGGAAAGGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAG AT С T T CAT САС С С С С САС CAGAT CAAG GAGAT TAAG GAG T T CAAG GAG TACAAGTAGAGССАСC GGGTGGACAAGAAGCCTAATAGAGAGCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGA CAAGGGCAACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGACAATGACAAGCTG AAAAAG С T GAT СAACAAGAG С С С C GAAAAG CTGCTGATGTAC СAC CAC GAC С С С CAGAC С TAC C AGAAACT GAAGCTGATTATGGAACAGTACGGCGACGAGAAGAAT ССС С T GTACAAGTAG TAGGA GGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGTGATCAAGAAGATT AAG TAT TAG G G CAACAAAC T GAAC GCCCATCTG GACAT СAC C GAC GAC TAC С С CAACAG CAGAA ACAAGGTCGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGGACAATGGCGTGTA C AAG T T C G T GAC C G T GAAGAAT CTGGATGTGAT CAAAAAAGAAAAC TAG TAG GAAG T GAAT AG C AAGTGCTATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTTATCGCCTCCT TCTACAACAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGAACAACGA С С T G С T GAAC C G GAT C GAAG T GAACATGAT CGACAT СACС TACCGCGAGTACС T GGAAAACATG AACGACAAGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAGAGCATTAAGAAGT ACAG CACAGACAT T С T G G G CAAC С T G TAT GAAG T GAAAT С TAAGAAG СAC С С T CAGAT CAT СAA AAAGGGCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG [SEQ ID NO: 31]

SEQ ID NO: 32 is set out below.

ACCGGTGCCA CCATGTACCCC ATACGATGTT CCAGATTAGG CTTCGCCGAA GAAAAAGCGC AAGGTCGAAG CGTCCATGAA AAGGAACTAC ATTCTGGGGC TGGACATCGG GATTACAAGC GTGGGGTATG GGATTATTGA CTATGAAACA AGGGACGTGA TCGACGCAGG CGTCAGACTG TTCAAGGAGG CCAACGTGGA AAACAATGAG GGACGGAGAA GCAAGAGGGG AGCCAGGCGC CTGAAACGAC GGAGAAGGCA CAGAATCCAG AGGGTGAAGA AACTGCTGTT CGATTAGAAC CTGCTGACCG ACCATTCTG GCTGAGTGGA ATTAATCCTT ATGAAGCCAG GGTGAAAGGC CTGAGTCAG AGCTGTCAG GGAAGAGTTT TCCGCAGCTC TGCTGCACCT GGCTAAGGCGC CGAGGAGTGC ATAACGTCAA TGAGGTGGAA GAGGACACCG GCAACGAGCT GTCTACAAAG GAACAGATCT CACGCAATAG CAAAGCTCTG GAAGAGAAGT ATGTCGCAGA GCTGCAGCTG GAACGGCTGA AGAAAGATGG CGAGGTGAGA GGGTCAATTA ATAGGTTCAA GACAAGCGAC TACGTCAAAG AAGCCAAGCA GCTGCTGAAA GTGCAGAAGG CTTACCACCA GCTGGATCAG AGCTTCATCG ATACTTATAT CGACCTGCTG GAGACTCGGA GAACSTASTA TGAGGGACCA GGAGAAGGGA GCCCCTTCGG ATGGAAAAGAC ATCAAGGAAT GGTACGAGAT GCTGATGGGA CATTCACCT ATTTTCCAGA

- 28 042798

AGAGCTGAGA AGCGTCAAGT ACGCTTATAA CGCAGATCT TACAACGCCC TGAATGACCT

GAACAACCTG GTCATCACA GGGATGAAAA CGAGAAACTG GAATACTATG AGAAGTTCCA GATCATCGAA AACGTGTTTA AGCAGAAGAA AAAGCSTASA CTGAAACAGA TTGCTAAGGA GATCCTGGTC AACGAAGAGG ACATCAAGGG CTACCGGGTG ACAAGCACTG GAAAACGAGA GTTCACCAAT CTGAAAGTGT ATCACGATAT TAAGGACATC ACAGCACGGA AAGAAATCAT TGAGAACGCC GAACTGCTGG ATCAGATTGC TAAGATCCTG ACTATSTACC AGAGCTCCGA GGACATCCAG GAAGAGCTGA CTAACCTGAA CAGCGAGCTG ACCCAGGAAG AGATCGAACA GATTAGTAAT CTGAAGGGGT ACACCGGAAC ACACAACCTG TCCCTGAAAG CTATCAATCT GATTCTGGAT GAGCTGTGGC ATACAAAACGA CAATCAGATT GCAATCTTTA ACCGGCTGAA GCTGGTCCCA AAAAAGGTGG ACCTGAGTCA GCAGAAAGAG ATCCCAACCA CACTGGTGGA CGATTTCATT CTGTCACCCG TGGTCAACGCG GAGCTTCATC CAGAGCATCA AAGTGATCAA CGCCATCATC AAGAAGTACG GCCTGCCCAA TGATATCATT ATCGAGCTGG CTAGGGAGAA GAACAGCAAG GACGCACAGA AGATGATCAA TGAGATGCAG AAACGAAACC GGCAGACCAA TGAACGCATT GAAGAGATTA TCCGAACTAC CGGGAAAAGAG AACGCAAAGT ACCTGATTGA AAAAATCAAG CTGCACGATA TGCAGGAGGG AAAGTGTCTG TATTTCTCTGG AGGCCATCCC CCTGGAGGAC CTGCTGAACA ATCCATTCAA CTACGAGGTC GATCATATTA TCCCCAGAAG CGTGTCCTTC GACAATTCCT TTAACAACAA GGTGCTGGTC AAGCAGGAAG AGAACTCTAA AAAGGGCAAT AGGACTCCTT TCCAGTACCCT GTCTAGTTCA GATTCCAAGA TCTCTTACGA AACCTTTAAA AAGCACATTC TGAATCTGGC CAAAGGAAAG GGCCGCATCA GCAAGACCAA AAAGGAGTAC CTGCTGGAAG AGCGGGACAT CAACAGATTC TCCGTCCAGA AGGATTTTAT TAACCGGAAT CTGGTGGACA CAAGATACGC TACTCGCGGC CTGATGAATC TGCTGCGATC CTATTTTCCGG GTGAACAATC TGGATGTGAA AGTCAAGTCC ATCACGGCG GGTTCACATC TTTTCTGAGG CGCAAATGGA AGTTTAAAAA GGAGCCGCAAC AAAGGGTACA AGCACCATGC CGAAGATGCT CTGATTATCG CAAATGCCGA CTTCATCTTT AAGGAGTGGA AAAAGCTGGA CAAAGCCAAG AAAGTGATGG AGAACCAGAT GTTCGAAGAG AAGCAGGCCG AATCTATGCC CGAAATCGAG ACAGAACAGG AGTACAAGGA GATTTTCATC ACTCCTCACC AGATCAAGCA TATCAAGGAT TTCAAGGACT ACAAGTACTC TCACCGGGTG GATAAAAAGC CCAACAGAGA GCTGATCAAT GACACCCTGT ATAGTACAAG AAAAGACGAT AAGGGGAATA CCCTGATTGT GAACAATCTG AACGGACTGT ACGACAAAGA TAATGACAAG CTGAAAAAGC TGATCAACAA AAGTCCCGAG AAGCTGCTGA TGTACCACCA TGATCCTCAG ACATATCAGA AACTGAAGCT GATTATGGAG CAGTACGGCG ACGAGAAGAA CCCACTGTAT AAGTACTATG AAGAGACTGG GAACTACTG ACCAAGTATA GCAAAAAGGA TAATGGCCCC GTGATCAAGA AGATCAAGTA CTATGGGAAC AAGCTGAATG CCCATCTGGA CATCACAGAC GATTACCCTA ACAGTCGCAA CAAGGTGGTC AAGCTGTCAC TGAAGCCATA CAGATTCGAT GTCTATCTGG ACAACGGCGT GTATAAATTT GTGACTGTCA AGAATCTGGA TGTCATCAAA AAGGAGAACT ACTATGAAGT GAATAGCAAG TGCTACGAAG AGGCTAAAAA GCTGAAAAAG ATTAGCAACC AGGCAGAGTT CATCGCCTCC TTTTACAACA ACGACCTGAT TAAGATCAAT GGCGAACTGT ATAGGGTCAT CGGGGTGAAC AATGATCTGC TGAACCGCAT TGAAGTGAAT ATGATTGACA TCACTTACCG AGAGTATCTG GAAAACATGA ATGATAAGCG CCCCCCTCGA ATTATCAAAA CAATTGCCTC TAAGACTCAG AGTATCAAAA AGTACTCAAC CGACATTCTG GGAAACCTGT ATGAGGTGAA GAGCAAAAAG

CACCCTCAGA TTATCAAAAA GGGCTAAGAA TTC [SEQ ID NO: 32]

- 29 042798

SEQ ID NO: 43 is set forth below.

ATGGCCCCAAAAGAAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCAAGCGGAACTACA

TCCTGGGCCTGGACATCGGCATCACCAGCGTGTGGGCTACGGCATCATCGACTACGAGACACGGGA

CGTGATCGATGCCGGCGTGCGGCTGTTCAAAAGAGGCCAACGTGGAAAACAACGAGGGCAGGCGG

AGCAAGAGAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGC

TGCTGTTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGC

CAGAGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTG

GCCAAGAGAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGCAACGAGCTGTCCA

CCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGGCCGAACTGCAGCT

GGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAGCGACTAC

GTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCA

TCGACACCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAG

CCCCTTCGGCTGGAAGGACATCAAAGAATGGTACGAGATGCCTGATGGGCCACTGCACCTACTTC

CCCGAGGAACTGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACC

TGAACAATCTCGTGATCACCAGGGACGAGAACGAGAAGCTGGAATATTACGAGAAGTTCCAGAT

CATCGGAAACGTGTTCAAGCAGAAGAAGAAGCCCACCCTGAAGCAGATCGCCAAAGAAATCCTC

GTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCGAGTTCACCAACC

TGAAGGTGTACCACGACATCAAGGACATTACCGCCCGGAAAAGAGATTATTGAGAACGCCGAGCT

GCTGGATCAGATTGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAAGAACTG

ACCAATCTGAACTCCGAGCTGACCCAGGAAGAGATCGAGCAGATCTCTAATCTGAAGGGCTATA

CCGGCACCCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTGTGGCACACCAA

CGACAACCAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGTCCCAG

CAGAAAGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAAGCT TCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCAT

TATCGAGCTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGATGCAGAAG

CGGAACCGGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGCAAAGAGAACGCCA

AGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAAGGCAAGTGCCTGTACAGCCTGGA

AGCCATCCCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGAGGTGGACCACATCATCCCC

AGAAGCGTGTCCTTCGACAACAGCTTCAACAACAAGGTGCTCGTGAAGCAGGAAGAAAAACAGCA

AGAAGGGCAACCGGACCCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAAAC

CTTCAAGAAGCACATCCTGAATCTGGCCAAGGGCAAGGGCAGAATCAGCAAGACCAAGAAAGAG

TATCTGCGTGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTCATCAACCGGAACC

TGGTGGATACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTACTTCAGAGTGAA

CAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCTGCGGCGGAAGTGG

AAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCCTGATCATTGCCA

ACGCCGATTTCATCTTCAAAGAGTGGAAGAAACTGGACAAGGCCAAAAAAGTGATGGAAAACCA

GATGTTCGAGGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAG

ATCTTCATCACCCCCCCACCAGATCAAGCACATTAAGGACTTCAAGGACTACAAGTACAGCCACC

GGGTGGACAAGAAGCCTAATAGAGAGCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGA

CAAGGGCAACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGACAATGACAAGCTG

AAAAAGCTGATCAACAAGAGCCCCGAAAAGCTGCTGATGTACCACCACGACCCCCAGACCTACC

AGAAACTGAAGCTGATTATGGAACAGTACGGCGACGAGAAGAATCCCCTGTACAAGTACTACGA

GGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGTGATCAAGAAGATT

AAGTATTACGGCAACAAACTGAACGCCCATCTGGACATCACCGACGACTACCCCAACAGCAGAA

ACAAGGTCGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGGACAATGGCGTGTA

CAAGTTCGTGACCGTGAAGAATCTGGATGTGATCAAAAAAAGAAAACTACTACGAAGTGAATAGC

AAGTGCTATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTTATCGCCTCCT

TCTACAACAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGAACAACGA

- 30 042798

CCTGCTGAACCGGATCGAAGTGAACATGATCGACATCACCTACCGCGAGTACCTGGAAAACATG AACGACAAGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAGAGCATTAAGAAGT ACAGCACAGACATTCTGGGCAACCTGTATGAAGTGAAATCTAAGAAGCACCTCAGATCATCAAA AAAGGGCAAAQGCCGGGCCACGAAAAAGGCCGGCCAGGCAAA ID4

In some embodiments, the nucleotide sequence encoding the S. aureus Cas9 molecule provides for the nucleotide sequence of SEQ ID NO: 44 as shown below.

AAGCGGAACTACATCCTGGGCCTGGACATCGGCATCACCAGCGTGGGCTACGGCATCATCGACT ACGAGACACGGGACGTGATCGATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAA CGAGGGCAGGCGGAGCAAGAGAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAG AGAGTGAAGAAGCTGCTGTTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCA ACCCCTACGAGGCCAGAGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGC CCTGCTGCACCTGGCCAAGAGAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGC AACGAGCTGTCCACCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGG CCGAACTGCAGCTGGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAA GACCAGCGACTACGTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTG GACCAGAGCTTCATCGACACCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGAC CTGGCGAGGGCAGCCCCTTCGGCTGGAAGGACATCAAAGAATGGTACGAGATGCTGATGGGCCA CTGCACCTACTTCCCCGAGGAACTGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAAC GCCCTGAACGACCTGAACAATCTCGTGATCACCAGGGACGAGAACGAGAAGCTGGAATATTACG AGAAG Т Т С СAGAT CATC GAGAAC G Т G Т Т СAAG СAGAAGAAGAAG С С САС С С Т GAAG СAGAT С G С CAAAGAAATCCTCGTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCC GAG Т Т САС СААС С Т GAAG G Т G ТАС САС GACАТ СAAG GACAT TACCGCCCG GAAAGAGAT ТАТ Т G AGAACGCCGAGCTGCTGGATCAGATTGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACAT С СAG GAAGAAC Т GAC СААТ С Т GAAC ТСС GAG С Т GAC С СAG GAAGAGAT С GAG СAGAT С Т С ТААТ CTGAAGGGCTATACCGGCACCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGC TGTGGCACACCAACGACAACCAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGT GGACCTGTCCCAGCAGAAAGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTC GTGAAGAGAAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGC CCAACGACATCATTATCGAGCTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAA CGAGATGCAGAAGCGGAACCGGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGC AAAGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAAGGCAAGTGCC TGTACAGCCTGGAAGCCATCCCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGAGGTGGA С САСАТ САТ С С С СAGAAG CGTGTCCTTC GACААСAG С Т Т СААСААСAAG GTGCTCGT GAAG СAG GAAGAAAACAGCAAGAAGGGCAACCGGACCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGA TCAGCTACGAAACCTTCAAGAAGCACATCCTGAATCTGGCCAAGGGCAAGGGCAGAATCAGCAA GACCAAGAAAGAGTATCTGCTGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTC ATCAACCGGAACCTGGTGGATACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCT ACTTCAGAGTGAACAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCT GCGGCGGAAGTGGAAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCC CTGATCATTGCCAACGCCGATTTCATCTTCAAAGAGTGGAAGAAACTGGACAAGGCCAAAAAAG TGATGGAAAACCAGATGTTCGAGGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCA G GAG T AC AAAGAGAT С T T C AT С AC С С С С С AC C AGAT C AAG C AC AT T AAG GAC T T C AAG GAC TAG AAGTACAGCCACCGGGTGGACAAGAAGCCTAATAGAGAGCTGATTAACGACACCCTGTACTCCA CCCGGAAGGACGACAAGGGCAACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGA

- 31 042798

CAATGACAAGCTGAAAAAGCTGATCAACAAGAGCCCCGAAAAGCTGCTGATGTACCACCACGAC С С С СAGAC СТАС СAGAAAC Т GAAG CTGATTATG GAACAG ТAC G G С GAC GAGAAGAAT С С С С Т G Т ACAAGTACTACGAGGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGT GAT СAAGAAGAT ТAAG ТАТ ТАС G G СААСАААС Т GAAC GCCCATCTG GACАТ САС С GAC GAC ТАС CCCAACAGCAGAAACAAGGTCGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGG АСААТ G G С G Т G ТACAAG Т Т С G Т GAC С G Т GAAGAAT CTGGATGTGAT СAAAAAAGAAAAC ТАС ТА CGAAGTGAATAGCAAGTGCTATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAG TTTATCGCCTCCTTC ТАСААСААС GAT С Т GAT СAAGAT СААС G G С GAG С Т G ТATAGAG Т GAT С G G С G Т GAACААСGACС Т GС Т GAACСGGAT СGAAGТ GAACAT GAT С GACАТ САСС ТАССGСGAGТА CCTGGAAAACATGAACGACAAGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAG AGCATTAAGAAGTACAGCACAGACATTCTGGGCAACCTGTATGAAGTGAAATCTAAGAAGCACC CTCAGATCATCAAAAAGGGC [SEQ ID NO: 44]

The amino acid sequence of the S. aureus Cas9 molecule is set forth under SEQ ID NO: 33, which is shown below.

MKRNYILGLDIGIТSVGYG11DYE TRDVIDAGVRL EKEANVENNEGRRS KRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDT GNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQ LDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY NALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQIS NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP WKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTT GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAED ALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKD YKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHH DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD YPNSRNKWKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKT QSIKKYSTDILGNLYEVKSKKHPQIIKKG [SEQ ID NO: 33]

The amino acid sequence of the S. aureus Cas9 molecule is set forth under SEQ ID NO: 45, which is shown below.

KRNYILGLDIGIT SVGYG11DYE TRDVIDAGVRL FKEANVENNE GRRS KRGARRLKRRRRHRIQ RVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQL DQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYN ALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISN LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV VKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTG KENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDA LIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDY KYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHD PQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDY PNSRNKWKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQ SIKKYSTDILGNLYEVKSKKHPQIIKKG [SEQ ID NO: 45]

- 32 042798

Alternatively or additionally, the CRISPR/Cas9 based gene editing system may include a fusion protein. The fusion protein may contain two heterologous polypeptide domains, where the first polypeptide domain contains the Cas protein and the second polypeptide domain has an activity such as activity for transcription activation, activity for transcriptional repression, activity for transcript release factor, activity for histone modification, nuclease activity, nucleic acid association activity, methylase activity or demethylase activity. The fusion protein may comprise a Cas9 protein or a mutated Cas9 protein fused to a second polypeptide domain that has an activity such as transcription activation activity, transcriptional repression activity, transcription release factor activity, modification activity. histones, nuclease activity, nucleic acid association activity, methylase activity or demethylase activity.

(a) Transcriptional activation activity.

The second polypeptide domain may have transcriptional activation activity, ie. transactivating domain. For example, expression of endogenous mammalian genes, such as human genes, can be achieved by targeting the iCas9 fusion protein and the transactivation domain to mammalian promoters through a combination of gRNA. The transactivating domain may include the VP 16 protein, a variety of VP 16 proteins such as the VP48 domain, or the VP64 domain, or the kappa B NF transcription activity activator p65 domain. For example, the fusion protein may be iCas9-VP64.

(b) Transcriptional repression activity.

The second polypeptide domain may have transcriptional repression activity. The second polypeptide domain may have Kruppel box related activity such as KRAB domain, ERF domain repression activity, Mxil domain repression activity, SID4X domain repression activity, repression of the Mad-SID domain or activity against the protein that binds the TATA box. For example, the fusion protein may be dCas9-KRAB.

(c) Activity against transcript release factor.

The second polypeptide domain may have activity against a transcript release factor. The second polypeptide domain may have eukaryotic release factor 1 (ERF1) activity or eukaryotic release factor 3 (ERF3) activity.

(d) Histone modification activity.

The second polypeptide domain may have histone modification activity. The second polypeptide domain may have histone deacetylase, histone acetyltransferase, histone demethylase, or histone methyltransferase activity. The histone acetyltransferase may be p300 or CREB binding protein (CBP protein) or a fragment thereof. For example, the fusion protein may be dCas9-p300.

(e) Nuclease activity.

The second polypeptide domain may have a nuclease activity that is different from the nuclease activity of the Cas9 protein. A nuclease or protein having nuclease activity is an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of nucleic acids. Nucleases are usually further divided into endonucleases and exonucleases, although some of the enzymes may fall into both categories. Well-known nucleases are deoxyribonuclease and ribonuclease.

(f) Nucleic acid association activity.

The second polypeptide domain may have activity for association of nucleic acids or a DNA binding domain (DBD) of a nucleic acid binding protein, which is an independently folded domain of a protein that contains at least one motif that recognizes a double-stranded or single-stranded DNA molecule. The DBD may recognize a specific DNA sequence (sequence recognition) or have a general affinity for DNA. The nucleic acid association region is selected from the group consisting of the helix-turn-helix region, the leucine zipper region, the winged helix region, the winged helix-turn-helix region, the helix-loop-helix region, the immunoglobulin chain fold, the B3 domain, zinc finger domain, HMG box, Wor3 domain, TAL effector of the DNA binding domain.

(g) Methylase activity.

The second polypeptide domain may have methylase activity, which involves the transfer of a methyl group to DNA, RNA, protein, small molecule, cytosine, or adenine. The second polypeptide domain may include DNA methyltransferase.

(h) Demethylase activity.

The second polypeptide domain may have demethylase activity. Second polypeptide

- 33 042798 the domain may include an enzyme that removes methyl (CH3-) groups from nucleic acids, proteins (particularly histones) and other molecules. Alternatively, the second polypeptide can harbor a methyl group to hydroxymethylcytosine in the DNA demethylation mechanism. The second polypeptide can catalyze this reaction. For example, the second polypeptide that catalyzes this reaction may be Tet1.

(b) gRNA targeting the dystrophin gene.

The CRISPR/Cas9 based gene editing system includes at least one gRNA molecule, eg two gRNA molecules. gRNA provides targeting of the CRISPR/Cas9 based gene editing system. gRNA is a fusion of two non-coding RNAs, crRNA and tracrRNA. The sgRNA can target any desired DNA sequence by exchanging the 20 bp protospacer coding sequence that provides targeting specificity through complementary base pairing with the desired target DNA. In this case, gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the type II effector system. This duplex, which may include, for example, a 42 bp crRNA and a 75 bp tracrRNA, acts as a guide for Cas9 to cleave the target nucleic acid. The terms target region, target sequence, or protospacer, used interchangeably herein, refer to the region of a target gene (eg, dystrophin gene) that is targeted by a CRISPR/Cas9 based gene editing system. A CRISPR/Cas9 based gene editing system may include at least one gRNA where the gRNAs are targeted to different DNA sequences. Target DNA sequences may be overlapping. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different Type II systems have different PAM requirements. For example, in the type II system from Streptococcus pyogenes, the sequence NGG is used, where N can be any nucleotide. In some embodiments, the PAM sequence may be NGG, where N may be any nucleotide. In some embodiments, the PAM sequence may be NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25).

The number of gRNA molecules encoded by a genetic construct disclosed herein (e.g., an AAV vector) may be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs, at least 7 different gRNAs, at least 8 different gRNAs, at least 9 different gRNAs, at least 10 different gRNAs, at least 11 different gRNAs, at least at least 12 different gRNAs, at least 13 different gRNAs, at least 14 different gRNAs, at least 15 different gRNAs, at least 16 different gRNAs, at least 17 different gRNAs, at least 18 different gRNAs, at least 19 different gRNAs, at least 20 different gRNAs, at least 25 different gRNAs, at least 30 different gRNAs, at least 35 different gRNAs, at least 40 different gRNAs, at least 45 different gRNAs, or at least 50 different gRNAs. The number of gRNAs encoded by the vector disclosed in the present invention may be from at least 1 gRNA to at least 50 different gRNAs, from at least 1 gRNA to at least 45 different gRNAs, from at least 1 gRNA to at least at least 40 different gRNAs, from at least 1 gRNA to at least 35 different gRNAs, from at least 1 gRNA to at least 30 different gRNAs, from at least 1 gRNA to at least 25 different gRNAs, from at least at least 1 gRNA to at least 20 different gRNAs, from at least 1 gRNA to at least 16 different gRNAs, from at least 1 gRNA to at least 12 different gRNAs, from at least 1 gRNA to at least 8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs, from at least 4 gRNAs to at least 50 different gRNAs, from at least 4 different gRNAs to at least 45 different gRNAs, from at least 4 different gRNAs to at least 40 different gRNAs, from at least 4 different gRNAs to at least 35 different gRNAs, from at least 4 different gRNAs to at least 30 different gRNAs, from at least 4 different gRNAs to up to at least 25 different gRNAs, from at least 4 different gRNAs to at least 20 different gRNAs, from at least 4 different gRNAs to at least 16 different gRNAs, from at least 4 different gRNAs to at least 12 different gRNAs , at least 4 different gRNAs to at least 8 different gRNAs, from at least 8 different gRNAs to at least 50 different gRNAs, from at least 8 different gRNAs to at least 45 different gRNAs, from at least 8 different gRNAs to at least 40 different gRNAs, from at least 8 different gRNAs to at least 35 different gRNAs, from 8 different gRNAs to at least 30 different gRNAs, from at least 8 different gRNAs to at least 25 different gRNAs, from 8 different gRNAs to at least 20 different gRNAs, from at least 8 different gRNAs to at least 16 different gRNAs, or from 8 different gRNAs to at least 12 different gRNAs. In certain embodiments, a gene

- 34 042798 The tic construct (eg AAV vector) is encoded by a single gRNA molecule, i.e. the first gRNA molecule and optionally the Cas9 molecule. In certain embodiments, the first genetic construct (eg, the first AAV-based vector) is encoded by a single gRNA molecule, ie. the first gRNA molecule and optionally the Cas9 molecule, and the second genetic construct (eg, the second AAV vector) is encoded by one gRNA molecule, i.e. a second gRNA molecule and optionally a Cas9 molecule.

The gRNA molecule contains a targeting domain that is complementary to the target DNA polynucleotide sequence followed by the PAM sequence. While gRNA may contain G at the 5'end of the targeting domain or complementary polynucleotide sequence. The targeting domain of a gRNA molecule may be at least 10 base pairs, at least 11 base pairs, at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, at least at least 16 base pairs, at least 17 base pairs, at least 18 base pairs, at least 19 base pairs, at least 20 base pairs, at least 21 base pairs, at least 22 base pairs, at least 23 base pairs, at least 24 base pairs, at least 25 base pairs, at least 30 base pairs, or at least 35 base pairs complementary to the target DNA sequence followed by a PAM sequence. In certain embodiments, the length of the targeting domain of the gRNA molecule is 19-25 nucleotides. In certain embodiments, the targeting domain of the gRNA molecule is 20 nucleotides long. In certain embodiments, the targeting domain of the gRNA molecule is 21 nucleotides long. In certain embodiments, the targeting domain of the gRNA molecule is 22 nucleotides long. In certain embodiments, the targeting domain of the gRNA molecule is 23 nucleotides long.

gRNA can target the dystrophin (DMD) gene region. In certain embodiments, the gRNA may target at least one of the exons, introns, promoter region, enhancer region, transcribed region of the dystrophin gene. In certain embodiments, the gRNA molecule targets intron 50 of the human dystrophin gene. In certain embodiments, the gRNA molecule targets intron 51 of the human dystrophin gene. In certain embodiments, the gRNA molecule targets exon 51 of the human dystrophin gene. gRNA may include a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or its complementary sequence.

A single or multiplex gRNA can be engineered to restore the dystrophin reading frame by targeting a hotspot mutation site in exon 51, or as either intra-exon small inserts or deletions, or exon 51 deletion. dystrophin expression in muscle cells of a Duchenne patient in vitro. Human dystrophin has been identified in vivo following transplantation of genetically engineered patient cells into immunodeficient mice. Most importantly, the unique multiple gene-editing capabilities of the CRISPR/Cas9-based gene-editing system make it possible to efficiently generate large deletions of this mutational hotspot region, which can correct up to 62% of patient mutations with generic or patient-specific gene editing approaches. In some embodiments, candidate gRNAs are evaluated and selected based on non-target activity, target activity as measured by the Surveyor assay, and distance from the exon.

3. DNA targeting compositions.

The present invention also relates to DNA targeting compositions that contain these genetic constructs. DNA targeting compositions comprise at least one gRNA molecule (eg, two gRNA molecules) that targets a dystrophin gene (eg, the human dystrophin gene) as described above. At least one gRNA molecule can bind and recognize the target region. Target regions can be chosen directly above possible out-of-frame stop codons so that insertions or deletions during the repair process restore the dystrophin reading frame by in-frame transformation. Target regions can also be splice acceptor sites or splice donor sites such that insertions or deletions during the repair process disrupt splicing and restore the dystrophin reading frame by destroying the splice site and exonating the exon. The target regions can also be aberrant stop codons, so that insertions or deletions during the repair process restore the dystrophin reading frame, eliminating or destroying the stop codon.

In certain embodiments, a DNA targeting composition disclosed herein comprises a first gRNA and a second gRNA, wherein the first gRNA molecule and the second

- 35 042798 each gRNA molecule contains a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO : 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 , SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or its complementary sequence. In certain embodiments, the first gRNA molecule and the second gRNA molecule contain targeting domains. In certain embodiments, the first gRNA molecule is represented under SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and the second gRNA molecule is shown under SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO : 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. In certain embodiments, the first gRNA molecule is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 and the second gRNA molecule is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO : 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

In certain embodiments, the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain, which contains the nucleotide sequence set forth under SEQ ID NO: 2;

(ii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(iii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(iv) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(v) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(vi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(vii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(viii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(ix) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(x) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15;

(xi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18; and (xii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 42.

In some embodiments, the DNA targeting composition comprises the nucleotide sequence set forth in SEQ ID NO: 37 and/or the nucleotide sequence set forth in SEQ ID NO: 38.

In certain embodiments, the DNA targeting composition may further comprise at least one Cas9 molecule or a Cas9 fusion protein that is recognized by either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25) PAM. In some embodiments, the DNA targeting composition comprises the nucleotide sequence set forth under SEQ ID NO: 83 or SEQ ID NO: 84. In certain embodiments, the vector is designed to form first and second double strand breaks, respectively, in

- 36 042798 the first and second introns flanking exon 51 of the human dystrophin gene, which ensures the deletion of the dystrophin gene segment containing exon 51.

The deletion efficiency of the vectors disclosed in the present invention may be related to the size of the deletion, ie. the size of the segment deleted by the vectors. In certain embodiments, the length or size of certain deletions is determined by the distance between PAM sequences in the gene to be targeted (eg, the dystrophin gene). In certain embodiments, a specific deletion of a segment of the dystrophin gene, as defined in terms of its length and its containing sequence (e.g., exon 51), results from breaks adjacent to certain PAM sequences within the target gene (e.g., the dystrophin gene).

In certain embodiments, the size of the deletion is from about 50 base pairs to about 2000 base pairs (bp), for example from about 50 bp. to about 1999 bp, from about 50 bp to about 1900 bp, from about 50 bp to about 1800 bp, from about 50 bp to about 1700 bp, from about 50 bp to about 1650 bp, from about 50 bp to about 1600 bp, from about 50 bp up to about 1500 bp, from about 50 bp to about 1400 bp, from about 50 bp to about 1300 bp, from about 50 bp to about 1200 bp, from about 50 bp to about 1150 bp, from about 50 bp to about 1100 bp, from about 50 bp up to about 1000 bp, from about 50 bp to about 900 bp, from about 50 bp to about 850 bp, from about 50 bp to about 800 bp, from about 50 bp up to about 750 bp, from about 50 bp up to about 700 bp, from about 50 bp to about 600 bp, from about 50 bp up to about 500 bp, from about 50 bp up to about 400 bp, from about 50 bp up to about 350 bp, from about 50 bp up to about 300 bp, from about 50 bp up to about 250 bp, from about 50 bp up to about 200 bp, from about 50 bp up to about 150 bp, from about 50 bp up to about 100 bp, from about 100 bp to about 1999 bp, from about 100 bp to about 1900 bp, from about 100 bp to about 1800 bp, from about 100 bp to about 1700 bp, from about 100 bp to about 1650 bp, from about 100 bp to about 1600 bp, from about 100 bp to about 1500 bp, from about 100 bp to about 1400 bp, from about 100 bp to about 1300 bp, from about 100 bp to about 1200 bp, from about 100 bp to about 1150 bp, from about 100 bp to about 1100 bp, from about 100 bp up to about 1000 bp, from about 100 bp to about 900 bp, from about 100 bp to about 850 bp, from about 100 bp to about 800 bp, from about 100 bp up to about 750 bp, from about 100 bp to about 700 bp, from about 100 bp up to about 600 bp, from about 100 bp up to about 1000 bp, from about 100 bp up to about 400 bp, from about 100 bp up to about 350 bp, from about 100 bp up to about 300 bp, from about 100 bp up to about 250 bp, from about 100 bp up to about 200 bp, from about 100 bp up to about 150 bp, from about 200 bp to about 1999 bp, from about 200 bp to about 1900 bp, from about 200 bp to about 1800 bp, from about 200 bp to about 1700 bp, from about 200 bp to about 1650 bp, from about 200 bp to about 1600 bp, from about 200 bp up to about 1500 bp, from about 200 bp to about 1400 bp, from about 200 bp to about 1300 bp, from about 200 bp to about 1200 bp, from about 200 bp to about 1150 bp, from about 200 bp to about 1100 bp, from about 200 bp up to about 1000 bp, from about 200 bp to about 900 bp, from about 200 bp to about 850 bp, from about 200 bp to about 800 bp, from about 200 bp up to about 750 bp, from about 200 bp up to about 700 bp, from about 200 bp up to about 600 bp, from about 200 bp up to about 2000 bp, from about 200 bp up to about 400 bp, from about 200 bp up to about 350 bp, from about 200 bp up to about 300 bp, from about 200 bp up to about 250 bp, from about 300 bp to about 1999 bp, from about 300 bp to about 1900 bp, from about 300 bp to about 1800 bp, from about 300 bp to about 1700 bp, from about 300 bp to about 1650 bp, from about 300 bp to about 1600 bp, from about 300 bp up to about 1500 bp, from about 300 bp to about 1400 bp, from about 300 bp to about 1300 bp, from about 300 bp to about 1200 bp, from about 300 bp to about 1150 bp, from about 300 bp to about 1100 bp, from about 300 bp up to about 1000 bp, from about 300 bp up to about 900 bp, from approx.

- 37 042798 positively 300 b.p. to about 850 bp, from about 300 bp up to about 800 bp, from about 300 bp to about 750 bp, from about 300 bp up to about 700 bp, from about 300 bp up to about 600 bp, from about 300 bp up to about 3000 bp, from about 300 bp up to about 400 p. or from about 300 p. up to about 350 p. In certain embodiments, the size of the deletion may be about 118 bp, about 233 bp, about 326 bp, about 766 bp, about 805 bp. or approximately 1611 p.

4. Compositions for editing the genome in muscle.

The present invention is directed to genetic constructs (eg vectors) or composition thereof for editing a target gene in skeletal muscle or cardiac muscle in the genome of a subject. The composition includes a modified AAV-based vector and a nucleotide sequence encoding a CRISPR/Cas9-based gene editing system, such as a gRNA molecule and a Cas9 molecule. The composition delivers active forms of the CRISPR/Cas9 based gene editing system to skeletal muscle or cardiac muscle. The genetic constructs (eg, vectors) disclosed herein can be used to correct or reduce the effects of mutations in the dystrophin gene implicated in genetic diseases and/or other conditions associated with skeletal muscle or cardiac muscle, such as DMD. The composition may further comprise a donor DNA or a transgene. These compositions can be used in genome editing, in genome engineering, and in correcting or reducing the effects of mutations in genes involved in genetic diseases and/or other conditions associated with skeletal muscle or cardiac muscle.

a. CRISPR/Cas9 based gene editing system for targeting the dystrophin gene.

This document discloses a CRISPR/Cas9 based gene editing system specific for the dystrophin gene. A CRISPR/Cas9 based gene editing system may include Cas9 and at least one gRNA to target the dystrophin gene. The CRISPR/Cas9 based gene editing system can bind and recognize the target region. Target regions can be chosen directly above possible out-of-frame stop codons so that insertions or deletions during the repair process restore the dystrophin's reading frame by in-frame transformation. The target regions can also be splice acceptor sites or splice donor sites such that insertions or deletions during the repair process disrupt splicing and restore the dystrophin reading frame by destroying the splice site and deleting the exon. The target regions can also be aberrant stop codons, so that insertions or deletions during the repair process restore the dystrophin reading frame, eliminating or destroying the stop codon.

gRNA can target a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-19, 41, 42, or its complementary sequence. For example, the disclosed CRISPR/Cas9 based gene editing system was designed to mediate highly efficient gene editing in exon 51 of the dystrophin gene. This CRISPR/Cas9-based gene-editing system restores dystrophin protein expression in DMD patient cells. In some embodiments, the DNA targeting composition comprises the nucleotide sequence set forth under SEQ ID NO: 37, the nucleotide sequence set forth under SEQ ID NO: 38, the nucleotide sequence set forth under SEQ ID NO: 83, and/or the nucleotide sequence set forth under SEQ ID NO: 83, and/or the nucleotide sequence set forth under SEQ ID NO: 84. For example, a DNA targeting composition comprises the nucleotide sequence set forth under SEQ ID NO: 37, the nucleotide sequence set forth under SEQ ID NO: 38, and the nucleotide sequence set forth under SEQ ID NO: 83, or a composition for DNA targeting includes the nucleotide sequence set forth in SEQ ID NO: 37, the nucleotide sequence set forth in SEQ ID NO: 38, and the nucleotide sequence set forth in SEQ ID NO: 84.

b. Vectors based on adeno-associated virus.

The composition may also include a viral delivery system. In certain embodiments, the vector is an adeno-associated virus (AAV) vector. The AAV-based vector is a small virus belonging to the Dependovirus genus of the Parvoviridae family that infects humans and some other primate species. AAV-based vectors can be used to deliver a CRISPR/Cas9-based gene editing system using various construct configurations. For example, AAV-based vectors can deliver Cas9 and gRNA expression cassettes on separate vectors or on the same vector. Alternatively, if small Cas9 proteins derived from species such as Staphylococcus aureus or Neisseria meningitidis are used, both the Cas9 expression cassette and at least two gRNA expression cassettes can be combined in a single AAV-based vector with a packing limit of 4.7 That.

In certain embodiments, the AAV-based vector is a modified AAV-based vector. The modified AAV-based vector may have increased tropism for cardiac and skeletal muscle tissues. A modified AAV-based vector may be capable of delivering and expressing a gene-editing system based on

- 38 042798

CRISPR/Cas9 in a mammalian cell. For example, a modified AAV-based vector may be an AAV-SASTG vector (Piacentino et al. (2012), Human Gene Therapy, 23: 635-646). The modified AAV-based vector can deliver nucleases to skeletal and cardiac muscle in vivo. The modified AAV-based vector may be based on one or more of a number of capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9. The modified AAV vector may be based on the AAV2 pseudotype with alternative AAV muscle tropic capsids such as the AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5 and AAV/SASTG vectors, which effectively transduce skeletal muscle or cardiac muscle by systemic and local delivery (Seto et al., Current Gene Therapy (2012), 12: 139-151). The modified AAV vector may be AAV2i8G9 (Shen et al., J. Biol. Chem. (2013) 288: 28814-28823). In some embodiments, the composition comprises the nucleotide sequence set forth under SEQ ID NO: 39 and/or the nucleotide sequence set forth under SEQ ID NO: 40. In some embodiments, the composition includes a first vector containing the nucleotide sequence set forth under SEQ ID NO: 39, and a second vector containing the nucleotide sequence set forth under SEQ ID NO: 40.

5. Methods for editing the genome in muscle.

The present invention is directed to a method for editing the genome in the skeletal muscle or cardiac muscle of a subject. The method involves administering to the skeletal muscle or cardiac muscle of a subject a genome editing composition in skeletal muscle or cardiac muscle as described above. Genome editing may involve editing a mutant gene or inserting a transgene. Correction of the mutant gene may involve the removal, rearrangement or replacement of the mutant gene. Correction of the mutant gene may include nuclease-mediated NHEJ or HDR.

6. Methods for correcting a mutant gene and treating a subject.

The subject matter disclosed herein also provides methods for correcting a mutant gene (eg, a mutant dystrophin gene, eg, a mutant human dystrophin gene) in a cell, and treating a subject suffering from a genetic disease such as DMD. The method may involve introducing into a cell or subject a genetic construct (eg, a vector) disclosed in the present invention, or a composition containing it, as described above. The method may include introducing into the subject's skeletal muscle or cardiac muscle a genetic construct (e.g., a vector) disclosed in the present invention, or a composition containing it, to edit the genome in skeletal muscle or cardiac muscle, as described above. The use of a genetic construct (e.g., a vector) disclosed in the present invention, or a composition containing it, to deliver a CRISPR/Cas9-based gene editing system to skeletal muscle or cardiac muscle can restore the expression of a fully functional or partially functional protein using a repair template or donor DNA, which can replace the entire gene or region containing the mutation. A CRISPR/Cas9 based gene editing system can be used to introduce site-specific double-strand breaks at target genomic loci. Site-specific double-strand breaks are created when the CRISPR/Cas9-based gene editing system binds to target DNA sequences, thereby allowing the target DNA to be cleaved. Such DNA cleavage can stimulate the natural mechanism of DNA repair, which is reduced to one of two possible repair pathways: repair involving homologous recombination (HDR) or non-homologous end joining (NHEJ).

The present invention is directed to genome editing using a CRISPR/Cas9 based gene editing system without a repair template that can effectively correct the reading frame and restore the expression of a functional protein involved in a genetic disease. The disclosed CRISPR/Cas9-based gene editing systems can utilize repair-based repair approaches involving homologous recombination or nuclease-mediated non-homologous end joining (NHEJ), which allows efficient correction in proliferation-limited primary cell lines that may not be amenable to homologous recombination. or gene correction based on selection. This strategy combines the fast and reliable assembly of CRISPR/Cas9-based active gene editing systems with an efficient gene editing method for treating genetic diseases caused by mutations in non-essential coding regions that cause frameshifts, premature stop codons, aberrant splice donor sites, or aberrant splicing acceptor sites.

a. Nuclease-mediated non-homologous end joining.

Restoration of protein expression from an endogenous mutated gene can be carried out by NHEJ-mediated DNA repair without template involvement. In contrast to the temporal method of targeting the target gene to RNA, the correction of the target gene reading frame in the genome using a transiently expressed CRISPR/Cas9-based gene editing system can lead to a complete restoration of the expression of the target gene in each modified cell and in all its progeny. In certain embodiments, the NHEJ is nuclease-mediated NHEJ,

- 39 042798 which in certain embodiments refers to NHEJ initiated by a Cas9 molecule that cuts double stranded DNA. The method involves introducing a genetic construct (eg, a vector) disclosed in the present invention, or compositions containing it, into the skeletal muscle or cardiac muscle of a subject to edit the genome in the skeletal muscle or cardiac muscle.

Gene repair with nuclease-mediated NHEJ can correct a mutated target gene and offers several potential advantages over the HDR pathway. For example, NHEJ does not require a donor template that can cause non-specific insertional mutagenesis. Unlike HDR, NHEJ is effective at all stages of the cell cycle and can therefore be used effectively in both dividing and post-mitotic cells such as muscle fibers. This provides a robust, complete gene repair alternative to oligonucleotide-based exon skipping or pharmacologically initiated stop codon passthrough, and theoretically only one drug treatment would be required. NHEJ-based gene editing using the CRISPR/Cas9-based gene editing system, as well as other engineered nucleases, including meganucleases and zinc finger nucleases, can be combined with other existing ex vivo and in vivo platforms for cell and gene therapies in addition to described in this document, the method of electroporation of plasmids. For example, delivery of a CRISPR/Cas9-based gene editing system by mRNA-based gene transfer or as purified proteins capable of entering the cell may allow a DNA-free genome editing approach to be used, thus avoiding any possibility of insertional mutagenesis.

b. Repair involving homologous recombination.

Restoration of protein expression from an endogenous mutated gene may involve repair involving homologous recombination. The above method further involves introducing a donor matrix into the cell. The donor matrix may include a nucleotide sequence encoding a fully functional protein or a partially functional protein. For example, the donor template may include a reduced size dystrophin construct called a minidystrophin (minidis), a fully functional dystrophin construct to repair a mutant dystrophin gene, or a fragment of a dystrophin gene that, after homologous repair, results in the restoration of a mutant dystrophin gene.

c. Methods for correcting a mutant gene and treating a subject using CRISPR/Cas9.

The present invention is also directed to genome editing with a CRISPR/Cas9 based gene editing system to restore the expression of a fully functional or partially functional protein using a repair template or donor DNA that can replace the entire gene or region containing the mutation. A CRISPR/Cas9 based gene editing system can be used to introduce site-specific double-strand breaks at target genomic loci. Site-specific double-strand breaks are created when the CRISPR/Cas9-based gene editing system binds to target DNA sequences with gRNA, thereby allowing the target DNA to be cleaved. The CRISPR/Cas9 based gene editing system has the advantage of advanced genome editing due to their high rate of successful and efficient genetic modification. Such DNA cleavage can stimulate the natural mechanism of DNA repair, which is reduced to one of two possible repair pathways: repair involving homologous recombination (HDR) or non-homologous end joining (NHEJ). For example, a CRISPR/Cas9 based gene editing system targeting a dystrophin gene may include a gRNA containing the nucleic acid sequence of any of SEQ ID NOs: 1-19, 41, 42 or its complementary sequence.

The present invention is directed to genome editing using a CRISPR/Cas9 based gene editing system without a repair template that can effectively correct the reading frame and restore the expression of a functional protein involved in a genetic disease. The disclosed CRISPR/Cas9-based gene editing system can utilize repair-based repair approaches involving homologous recombination or nuclease-mediated non-homologous end joining (NHEJ), which allows efficient correction in primary cell lines with limited proliferation that may not be amenable to homologous recombination. recombination or gene correction based on selection. This strategy combines the fast and reliable assembly of a CRISPR/Cas9-based active gene editing system with an efficient gene editing method for treating genetic diseases caused by mutations in non-essential coding regions that cause frameshifts, premature stop codons, aberrant splicing donor sites, or aberrant splicing acceptor sites.

The present invention provides methods for correcting a mutated gene in a cell and treating a subject suffering from a genetic disorder such as DMD. The method may involve introducing into a cell or subject a CRISPR/Cas9 based gene editing system, a polynucleotide

- 40 042798 or a vector encoding said CRISPR/Cas9 based gene editing system or a composition containing said CRISPR/Cas9 based gene editing system as described above. The method may include introducing a CRISPR/Cas9-based gene editing system, such as introducing a Cas9 protein or a Cas9 fusion protein containing a second domain having nuclease activity, a nucleotide sequence encoding said Cas9 protein or a Cas9 fusion protein, and/or at least one gRNA, where the gRNAs target different DNA sequences. Target DNA sequences may be overlapping. The amount of gRNA introduced into the cell may be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs, at least 7 different gRNAs, at least 8 different gRNAs, at least 9 different gRNAs, at least 10 different gRNAs, at least 15 different gRNAs, at least 20 different gRNAs, at least 30 different gRNAs, or at least 50 different gRNAs described above. The gRNA may include the nucleic acid sequence of at least one of SEQ ID NOs: 1-19, 41, 42, or its complementary sequence. The method may involve repair involving homologous recombination or non-homologous end joining.

7. Ways to treat the disease.

The present invention is directed to a method of treating a disease in a subject in need thereof. The method involves introducing into a tissue or subject a genetic construct (eg, a vector) disclosed in the present invention, or a composition containing it, as described above. In certain embodiments, the implementation of the method may include introducing into the skeletal muscle or cardiac muscle of the subject of the genetic construct (eg, vector) disclosed in the present invention, or a composition containing it, as described above. In certain embodiments, the implementation of the method may include the introduction into the vein of the subject of a genetic construct (eg, vector) disclosed in the present invention, or a composition containing it, as described above. In certain embodiments, the subject suffers from a skeletal muscle or cardiac muscle condition causing degeneration or weakness or a genetic disease. For example, the subject may be suffering from Duchenne muscular dystrophy, as described above.

a. Duchenne muscular dystrophy.

As described above, the method can be used to correct the dystrophin gene and remove fully functional or partially functional protein expression of said mutated dystrophin gene. In some aspects and embodiments, the invention provides a method for reducing the effects (eg, clinical symptoms/signs) of DMD in a patient. In some aspects and embodiments, the invention provides a method for treating DMD in a patient. In some aspects and embodiments, the invention provides a method for preventing DMD in a patient. In some aspects and embodiments, the invention provides a method for preventing further progression of DMD in a patient.

8. Methods for obtaining a transgenic rodent with Δ52 hDMD.

The present invention relates to a method for obtaining a transgenic rodent embryo that has a human dystrophin gene with a deletion of exon 52. The method involves injecting a gRNA into a rodent embryo, thereby providing a deletion of exon 52 from the human dystrophin gene, and selecting a transgenic rodent embryo that has a deletion exon 52 of the human dystrophin gene, where the rodent embryo contains the normal human dystrophin gene. In some embodiments, the rodent embryo is a mouse embryo. In some embodiments, the transgenic rodent embryo is heterozygous hDMD or heterozygous hDMD-Δ52. In some embodiments, a first gRNA molecule containing a targeting domain that includes the nucleotide sequence set forth under SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that includes the nucleotide sequence set forth under SEQ ID NO: 42 , is introduced into a rodent embryo to carry out the deletion of exon 52 of the human dystrophin gene. In some embodiments, the method further comprises administering to the rodent embryo a Cas protein comprising the amino acid sequence set forth in SEQ ID NO: 27. The present invention is directed to a transgenic rodent embryo that is produced by the method. The present invention also relates to a transgenic rodent derived from a transgenic rodent embryo.

9. Constructs and plasmids.

The compositions described above may contain genetic constructs that encode a CRISPR/Cas9 based gene editing system as disclosed herein. The genetic construct, such as a plasmid, may contain a nucleic acid that encodes a CRISPR/Cas9 based gene editing system, such as a Cas9 protein and Cas9 fusion proteins and/or at least one of the gRNAs. The above compositions may contain genetic constructs that encode a modified AAV-based vector and a nucleic acid sequence,

- 41 042798 which encodes a CRISPR/Cas9 based gene editing system as disclosed herein. The genetic construct, such as a plasmid, may contain a nucleic acid that encodes a CRISPR/Cas9 based gene editing system. The compositions described above may contain genetic constructs that encode the modified lentiviral vector disclosed herein.

The genetic construct, such as a recombinant plasmid or recombinant viral particle, may contain a nucleic acid that encodes a Cas9 fusion protein and at least one gRNA. In some embodiments, the genetic construct may comprise a nucleic acid that encodes a Cas9 fusion protein and at least two different gRNAs. In some embodiments, the genetic construct may comprise a nucleic acid that encodes a Cas9 fusion protein and more than two different gRNAs. In some embodiments, the genetic construct may comprise a promoter that is operably linked to a nucleotide sequence encoding at least one gRNA molecule and/or a Cas9 molecule. In some embodiments, the promoter is operably linked to a nucleotide sequence encoding a first gRNA molecule, a second gRNA molecule, and/or a Cas9 molecule. The genetic construct may be present in the cell as a functioning extrachromosomal molecule. The genetic construct may be a linear minichromosome including a centromere, telomeres, or plasmids or cosmids.

The genetic construct may also be part of the genome of a recombinant viral vector, including recombinant lentivirus, recombinant adenovirus, and recombinant adeno-associated virus. The genetic construct may be the genetic material in attenuated live microorganisms or recombinant microbial vectors that colonize cells. Genetic constructs may contain regulatory elements for the expression of genes encoding nucleic acid sequences. The regulatory elements may be a promoter, enhancer, start codon, stop codon, or polyadenylation signal.

In certain embodiments, the genetic construct is a vector. The vector may be an adeno-associated virus (AAV) vector that encodes at least one Cas9 molecule and at least one gRNA molecule; wherein the vector is capable of expressing at least one Cas9 molecule and at least one gRNA molecule in a mammalian cell. The vector may be a plasmid. Vectors can be used for in vivo gene therapy. The vector may be recombinant. The vector may contain a heterologous nucleic acid encoding a fusion protein, such as a Cas9 fusion protein or a CRISPR/Cas9 based gene editing system. The vector may be a plasmid. The vector can be used to transfect cells with a nucleic acid encoding a Cas9 fusion protein or a CRISPR/Cas9 gene editing system, wherein the transformed host cell is cultured and maintained under conditions that express the Cas9 fusion protein or CRISPR based gene editing system. /Cas9.

Coding sequences can be optimized for stability and high levels of expression. In some cases, codons are chosen to reduce the formation of an RNA secondary structure, such as a structure formed due to intramolecular binding.

The vector may contain a heterologous nucleic acid encoding a CRISPR/Cas9 gene editing system and may further contain a start codon that may be upstream of the CRISPR/Cas9 gene editing system coding sequence, as well as a stop codon that may be downstream of the coding gene editing system sequences based on CRISPR/Cas9. The start codon and the termination codon may be in frame with the coding sequence of a CRISPR/Cas9 based gene editing system. The vector may also contain a promoter that is operably linked to the coding sequence of a CRISPR/Cas9 based gene editing system. The promoter that is operably linked to the coding sequence of the CRISPR/Cas9 gene editing system can be the simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) promoter, such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR), Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early response gene promoter, Epstein-Barr virus (EBV) promoter , a U6 promoter such as the human U6 promoter or the Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle- or skin-specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US 2004/0175727 and US 2004/0192593, the contents of which are incorporated herein in their entirety. Examples of promoters, spec.

- 42 042798 muscle-specific include the Spc5-12 promoter (described in US Patent Application Publication No. US 2004/0192593, which is incorporated herein by reference in its entirety; Hakim et al., Mol. Ther. Methods Clin. Dev. (2014), 1: 14002; and Lai et al., Hum Mol Genet. (2014), 23(12): 3189-3199), the MHCK7 promoter (described in Salva et al., Mol. Ther. (2007 ), 15: 320-329), the CK8 promoter (described in Park et al., PLoS ONE (2015), 10(4): e0124914) and the CK8e promoter (described in Muir et al., Mol. Ther. Methods Clin. Dev. (2014), 1: 14025). In some embodiments, expression of gRNA and/or Cas9 protein is controlled by tRNA.

Each of the polynucleotide sequences encoding the gRNA molecule and/or the Cas9 molecule may be operably linked to a promoter. Promoters that are operably linked to a gRNA molecule and/or a Cas9 molecule may be the same promoter. The promoters that are operably linked to the gRNA molecule and/or the Cas9 molecule may be different promoters. The promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulated promoter.

The vector may also contain a polyadenylation signal, which may be downstream of a CRISPR/Cas9 based gene editing system. The polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be the polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, CA).

The vector may also contain an enhancer upstream of the CRISPR/Cas9 based gene editing system, ie. the coding sequence of a Cas9 protein or a Cas9 fusion protein or sgRNA or a CRISPR/Cas9 based gene editing system. An enhancer may be required for DNA expression. The enhancer may be a gene enhancer for human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as one of CMV, HA, RSV, or EBV. Enhancers for performing polynucleotide function are described in US Pat. The vector may also contain a mammalian origin of replication to maintain the vector off the chromosome and to create multiple copies of the vector in the cell. The vector may also contain a regulatory sequence that may be well suited for gene expression in a mammalian or human cell into which the vector is introduced. The vector may also contain a reporter gene such as the green fluorescent protein (GFP) gene and/or a selectable marker such as the hygromycin resistance gene (Hygro).

The vector may be expression vectors or systems for producing a protein using standard techniques and publicly available starting materials included in Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is included in its entirety. through a link. In some embodiments, the vector may contain a nucleic acid sequence encoding a CRISPR/Cas9-based gene editing system, including a nucleic acid sequence encoding a Cas9 protein or a Cas9 fusion protein, and a nucleic acid sequence encoding at least one gRNA containing the sequence nucleic acid under at least one of SEQ ID NO: 1-19, 41, 42 or its complementary sequence. In some embodiments, the Cas9 protein or Cas9 fusion protein is encoded by the nucleic acid sequence of any of SEQ ID NO: 26. In some embodiments, the vector contains the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40.

10. Pharmaceutical compositions.

Disclosed in the present invention, the object of the invention provides compositions containing the above-described genetic constructs. Pharmaceutical compositions according to the present invention may be formulated according to the route of administration employed. In cases where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, free from pyrogens and do not contain particles. The use of an isotonic formulation is preferred. Typically, isotonicity additives may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the composition.

The composition may further contain a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules such as excipients, adjuvants, carriers or diluents. The pharmaceutically acceptable adjuvant may be a transfection facilitator which may include surface active agents such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, LPS analog, including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles, such like squalene and squalene, hyaluronic acid, lipids, lipo

- 43 042798 soma, calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known agents that facilitate transfection.

The transfection facilitator is a polyanion, a polycation, including poly-L-glutamate (LGS), or a lipid. The transfection facilitator is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the skeletal muscle or cardiac muscle genome-editing composition at a concentration of less than 6 mg/mL. The transfection facilitator may also include surfactants such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, an LPS analog, including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, wherein it is also possible to use hyaluronic acid when administered in combination with a genetic construct. In some embodiments, the DNA vector encoding the composition may also include a transfection facilitator such as lipids, liposomes, including lecithin liposomes, or other liposomes known in the art, as a mixture of DNA-liposomes ( see, for example, International Patent Publication No. WO 9324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection aids. Preferably, the transfection aid is a polyanion, a polycation, including poly-L-glutamate (LGS), or a lipid.

11. Delivery methods.

Provided herein is a method for delivering a genetic construct (eg, vector) or composition thereof disclosed in the present invention into a cell. Delivery of the compositions can be accomplished by transfection or electroporation of the composition, in the form of a nucleic acid molecule that is expressed in the cell and delivered to the cell surface. Nucleic acid molecules can be electroporated using the BioRad Gene Pulser Xcell or Amaxa Nucleofector IIb devices. Several different buffers can be used, including BioRad electroporation solution, Sigma phosphate buffered saline #D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N.V.). Transfections may include a transfection reagent such as Lipofectamine 2000.

Upon delivery of the genetic construct or composition disclosed herein into tissue and as a result of the vector into mammalian cells, the transfected cells will express the gRNA molecule(s) and the Cas9 molecule. The genetic construct or composition can be administered to a mammal to alter gene expression or to redesign or alter the genome. For example, the genetic construct or composition can be administered to a mammal to correct a dystrophin gene in the mammal. The mammal may be a human, a non-human primate, a cow, a pig, a sheep, a goat, an antelope, a bison, a water buffalo, a cattle, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, or a chicken, preferably a human, cow, pig or chicken.

A genetic construct (e.g., a vector) encoding a gRNA molecule(s) and a Cas9 molecule can be delivered to a mammal by DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, mediated by liposomes, using nanoparticles and/or recombinant vectors . The recombinant vector can be delivered using any viral form. The viral form may be a recombinant lentivirus, a recombinant adenovirus, and/or a recombinant adeno-associated virus.

The genetic construct (eg, vector) disclosed in the present invention, or a composition containing it, can be introduced into a cell to genetically correct a dystrophin gene (eg, a human dystrophin gene). In certain embodiments, a genetic construct (eg, a vector) disclosed in the present invention, or a composition containing it, is introduced into a myoblast from a DMD patient. In certain embodiments, a genetic construct (e.g., a vector) or composition containing it is introduced into a fibroblast cell from a DMD patient, and the genetically engineered fibroblast cell can be treated with MyoD to induce differentiation into myoblasts that can be implanted in subjects, such as a subject with injured muscles to verify that the corrected dystrophin protein is functional and/or to treat the subject. The modified cells may also be stem cells, such as induced pluripotent stem cells, bone marrow-derived progenitor cells, skeletal muscle progenitor cells, human skeletal myoblasts from DMD patients, CD ¹³³⁺ cells, mesoangioblasts, and transduced MyoD or Pax7 cells, or others. myogenic progenitor cells. For example, a CRISPR/Cas9 based gene editing system can induce neuronal or myogenic differentiation of an induced pluripotent stem cell.

12. Routes of administration.

The genetic constructs (e.g., vectors) disclosed herein, or a composition comprising them, may be administered to a subject in a variety of ways, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation,

- 44 042798 by buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal and intraarticular, or combinations thereof. In certain embodiments, a genetic construct (eg, a vector) disclosed herein or a composition is administered to a subject (eg, a subject suffering from DMD) intramuscularly, intravenously, or a combination thereof. For veterinary use, genetic constructs (eg, vectors) disclosed in the present invention, or compositions containing them, can be administered in the form of a suitable acceptable formulation in accordance with normal veterinary practice. The veterinarian can easily determine the regimen and route of administration that is most appropriate for a particular animal. The compositions can be administered by conventional syringes, needleless injection devices, ballistic transfection guns, or other physical methods such as electroporation (EP), hydrodynamics, or ultrasound.

The genetic construct (e.g., vector) disclosed in the present invention, or a composition containing it, can be delivered to a mammal using several technologies, including DNA injection (also called DNA vaccination) with and without in vivo electroporation, mediated by liposomes, using nanoparticles , recombinant vectors such as recombinant lentivirus, recombinant adenovirus and recombinant adeno-associated virus. The composition can be administered to skeletal muscle or cardiac muscle. For example, the composition may be administered to the tibialis anterior or tail.

In some embodiments, a genetic construct (e.g., a vector) disclosed in the present invention, or a composition thereof, is administered via

1) injection into the tail vein of adult mice (systemic);

2) intramuscular injections, eg local injection into a muscle, such as TA or gastrocnemius, of adult mice;

3) intraperitoneal injection into P2 mice; or

4) Injection into the facial vein in P2 (systemic) mice.

13. Cell types.

Any of these delivery methods and/or routes of administration can be used with a wide variety of cell types, such as the following cell types currently under study for DMD cell therapy, including, but not limited to, immortalized myoblasts such as wild-type myoblasts and cell lines derived from a patient with DMD, e.g. cell lines A48-50 DMD, DMD 6594 (del48-50), DMD 8036 (del48-50), C25C14, and DMD-7796, primary skin fibroblasts in DMD, induced pluripotent stem cells, progenitor cells, bone marrow-derived, skeletal muscle progenitor cells from DMD patients, CD ¹³³⁺ cells, mesoangioblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and transduced MyoD or Pax7 cells or other myogenic cells- predecessors. Immortalization of human myogenic cells can be used for clonal production of genetically corrected myogenic cells. Cells can be modified ex vivo to isolate and extend clonal populations of immortalized DMD myoblasts that include the genetically altered dystrophin gene and do not contain other nuclease-introduced mutations in protein-coding regions of the genome. Alternatively, transient in vivo delivery of CRISPR/Cas9 based systems with a non-viral or non-integrating viral transfer gene or direct delivery of purified proteins and gRNA containing cell-penetrating motifs can provide highly specific in situ correlation with minimal risk of exogenous DNA integration or without one.

14. Sets.

Provided herein is a kit that can be used to correct a mutated dystrophin gene. The kit contains at least a gRNA for correcting a mutated dystrophin gene and instructions for using a CRISPR/Cas9 based gene editing system. This document also provides a kit that can be used to edit the dystrophin gene in the genome of skeletal muscle or cardiac muscle. The kit contains genetic constructs (eg, vectors) or a composition containing them for editing the genome in skeletal muscle or cardiac muscle, as described above, and instructions for use of said composition.

The instructions included in the kits may be attached to the packaging material or may be included as a package insert. Although instructions are usually written or printed materials, they are not limited to such. The present invention contemplates any medium capable of storing such instructions and communicating them to the end user. Such media include, without limitation, electronic media (eg, magnetic disks, cassettes, cartridges, chips), optical media (eg, CD ROM), and the like. As used herein, the term instructions may include the address of an Internet site that provides instructions.

Genetic constructs (for example, vectors) or a composition containing them for the correction of mu

- 45 042798 dystrophin gene editing or editing of a dystrophin gene in the skeletal muscle or cardiac muscle genome may include a modified AAV-based vector that includes a gRNA molecule(s) and a Cas9 molecule, as described above, that specifically bind and cleave the region dystrophin gene. A CRISPR/Cas9 based gene editing system, as described above, can be included in a kit to specifically bind and target a specific region in the mutated dystrophin gene. The kit may further include donor DNA, various gRNAs, or a transgene as described above.

15. Examples.

Those skilled in the art will appreciate that other suitable modifications and adaptations of the methods of the present invention described herein are readily applicable and essential and may be carried out using suitable equivalents without departing from the scope of the present invention or the aspects and embodiments disclosed in this document. Now that a detailed description of the present invention is available, this will be more clearly disclosed by considering the following examples, which are simply meant to illustrate certain aspects and embodiments of the present invention and should not be construed as limiting the scope of the invention. Disclosures of all journal references, US patents, and publications cited herein are incorporated herein by reference in their entirety.

The present invention is characterized by many aspects, illustrated by the following non-limiting examples.

Example 1 Targeting the human dystrophin gene.

A CRISPR/Cas9 based gene editing system was used to target and secure the deletion of exon 51 in the human dystrophin gene. Cas9 from S. aureus (SaCas9), which is approximately 1 kb. less than Cas9 from S. pyogenes was used with adeno-associated virus (AAV) to deliver a CRISPR/Cas9 based gene editing system. The codon-optimized nucleic acid sequence encoding the Cas9 molecule from S. aureus is set forth under SEQ ID NO: 43 or SEQ ID NO: 44. FIG. 3 shows a schematic of an AAV-based system for in vivo co-delivery of SaCas9 and two gRNAs in two viral vectors to muscle tissues. Each vector contained a copy of SaCas9 and one gRNA driven by the CMV and hU6 promoters, respectively (PT366-179 (SEQ ID NO: 39) and PT366-183 (SEQ ID NO: 40)).

The activity of individual gRNAs targeting the human dystrophin gene, JCR89 (targets up to exon 51) and JCR91 (targets down to exon 51), which were designed for the human genome, was determined by the Surveyor assay in HEK293T cells (containing the normal variant of the dystrophin gene) and myoblast lines from DMD patients (DMD 8036 and DMD 6594, each containing a dystrophin gene mutant) (see FIG. 1). Surveyor analysis identified mismatches in genomic DNA that were indicative of insertions/deletions introduced by the CRISPR/Cas9-based gene editing system. For JCR89, the size of the band corresponding to the original form was 555 nt, and the primers used were the following: forward primer - aagttacttgtccaggcatga (SEQ ID NO: 91) and reverse primer - gaaaaacttctgccaacttttatca (SEQ ID NO: 92). The expected sizes of the bands corresponding to the shapes obtained after the cut were 134 and 421 nt. For JCR91, the size of the band corresponding to the original form was 632 nt, and the primers used were the following: forward primer - tgcaaataacaaaagtagccataca (SEQ ID NO: 93) and reverse primer - tctttagaaaggcttgaaagctg (SEQ ID NO: 94). The expected sizes of the bands corresponding to the shapes obtained after the cut were 210 and 422 nt.

HEK293T cells and DMD myoblasts (DMD 8036 and DMD 6594) were treated with SaCas9 in conjunction with JCR89 and JCR91 gRNA (SEQ ID NO: 37 and SEQ ID NO: 38). Genomic DNA was amplified with forward primer - cttcactgctggccagttta (SEQ ID NO: 95) and reverse primer - tctttagaaaggcttgaaagctg (SEQ ID NO: 94). The expected band size corresponding to the original form was 1646 nt, and the expected band size corresponding to the form with the most appropriate deletion was 766 nt (the actual size of the deletion between gRNA cut sites differed from 766 nt due to the presence of inserts/deletions). In FIG. 2A shows exon 51 deletion in genomic DNA from HEK293T cells and DMD myoblasts. In FIG. 2B shows the deletion of exon 51 in cDNA from DMD myoblasts. Without RT is a negative control in which no reverse transcriptase was added.

A CRISPR/Cas9-based gene editing system was injected into transgenic mice carrying the human DMD gene (hDMD/mdx mice) to allow exon 51 deletion. AAV8 viral vectors carrying SaCas9 and gRNA were locally delivered to the tibialis anterior (TA) muscle. See table. 1. AAV8 was injected into 3 mice: 1 mice were injected with a high dose of AAV8 into both TAs (HH), 1 mice were injected with a low dose of AAV8 into both TAs (LL) and 1 mice were injected with a low dose into both TAs (HH) and high dose into the right TA (LH). Doses are listed in column 2 of the table. 1. Mice were sacrificed through

- 46042798 weeks after treatment (weeks PT) in order to collect tissues for analysis. Nested PCR detected a deletion of exon 51 in both limbs in the HH-treated mouse, in the right TA in the LL-treated mouse, and in the right limb in the LH-treated mouse.

Table 1

Experiment Dosage AAV8 weeks Delivery RT intramuscular, nesting PCR - gDNA Δ51 HH: 6.6E11 to the front HH: both, LL: HH, LL, LH LL: 1E11 tibial 8 R,LH:R

Genomic DNA isolated from (TA) muscle

TA mouse muscles amplified in the first round

PCR reactions using forward primer: cttcactgctggccagttta (SEQ ID NO: 95) and reverse primer: tctttagaaaggcttgaaagctg (SEQ ID NO: 94). 1-3 µl of the PCR product was applied in the second round of the PCR reaction (2X gDNA PCR) using forward primer - aagttacttgtccaggcatga (SEQ ID NO: 91) and reverse primer - ttgaacatggcattgcataaA (SEQ ID NO: 96). The products of this second PCR cycle had an expected band size corresponding to the original form of 1089 nt and an expected band size corresponding to the deletion form of 323 nt (the actual size at deletion between gRNA cut sites differed from 323 nt due to the presence of inserts/deletions) . In FIG. 4 shows the results of the second PCR cycle. Lanes L show the results for the left TA muscle, which was used as a control and injected with saline. Lanes R show the results for the right TA muscle, which was injected with 2 viral vectors that were premixed in equal amounts. The CRISPR/Cas9 based gene editing system was also injected into the tail vein of hDMD/mdx mice by systemic delivery of AAV8 (see FIG. 5). Genomic DNA isolated from mouse liver (FIG. 5 - left panel) and mouse heart (FIG. 5 - right panel) was also amplified using the same protocol as for FIG. 4. The expected band, corresponding to a size of approximately 300 nucleotides, indicated a deletion of exon 51.

Additional gRNAs were generated and selected that targeted the human and rhesus monkey dystrophin gene sequences or human dystrophin gene sequences (see Figure 6 and Table 2). In table. 2 shows the main gRNA target, the recognized genomic strand, the gRNA sequence and the PAM sequence associated with the gRNA. Genomic target sequences for gRNA, indicated in the genomic plus strand, are listed in table. 3. These gRNAs were tested on cultured human cells to determine the optimal activity of gRNAs and their combinations to obtain deletions. The selected gRNAs were also ranked predominantly based on predicted specificity for the human genome (FIG. 8) and screened for optimal target sequence lengths ranging from 19 to 23 nucleotides (FIGS. 10 and 11). In FIG. 6 shows the various gRNA targets listed in Table 1. 2, which are conserved among the human and rhesus monkey genomes. The location of each gRNA is indicated relative to exon 51 of the human dystrophin gene.

- 47 042798

table 2

gRNA list

gRNA Main target A thread gRNA sequence (5' - 3') SEQ ID Consistently SEQ ID NO: awn NO: RAM JCR89 Exon 51 human DMD (above) plus AAAGATATATAAT GT CAT GAAT 1 AAGAGT 53 JCR91 Exon 51 DMD person (below) plus GCAGAATSAAAATAATAATA GTCT 2 GGGAAT 54 JCR94 Exon 52 human DMD (above) minus AACAAATATCCCTTAGTA TS 41 AGG 55 JCR99 Human exon 52 DMD (below) Exon 51 DMD minus AATGTATTTCTTCTATTC AA 42 TGG 56 JCR159 human and rhesus monkey (higher) Exon 51 DMD minus AACAATAAGTCAAATTTA ATTG 3 AAGAGT 53 JCR160 human and rhesus monkey (below) Exon 51 DMD minus GAACTGGTGGGAAATGGT CTAG 4 GAGAGT 57 JCR167 human and rhesus monkey (below) Exon 51 DMD minus TCCTTTGGTAAATAAAAG test 5 GGGAGT 58 JCR166 human and rhesus monkey (below) Exon 51 DMD minus TAGGAATCAAATGGACT GGAT 6 TTGAAT 59 JCR168 human and rhesus monkey (higher) plus TAATTCTTTCTAGAAAGA GCCT 7 CAGAGT 60 JCR170 Exon 51 DMD human and minus CTCTTGCATCTTGCACAT 8 TGGAGT 61

GTCC rhesus monkey

- 48 042798 (above)

Exon 51 DMD

JCR171 human and rhesus monkey (higher) Exon 51 DMD minus ACTTAGAGGTCTTTCTACA TASA 9 ATGAGT 62 JCR156 human and rhesus monkey (higher) Exon 51 DMD minus TCAGAGGTGAGTGGTGAG GGGA 10 AGGAAT 63 JCR157 human and rhesus monkey (higher) Exon 51 DMD minus ACACACAGCTGGGTTATC AGAG eleven GAGAGT 57 JCR176 human and rhesus monkey (higher) Exon 51 DMD minus CACAGCTGGGTTATCAGA G 12 GAGAGT 57 JCR177 human and rhesus monkey (higher) Exon 51 DMD minus ACACAGCTGGGTTATCAG AG 13 GAGAGT 57 JCR178 human and rhesus monkey (higher) Exon 51 DMD minus CACACAGCT GGGT TATSA GAG 14 GAGAGT 57 JCR179 human and rhesus monkey (higher) Exon 51 DMD minus AACACACAGCTGGGTTAT CAGAG 15 GAGAGT 57 JCR180 human and rhesus monkey (below) Exon 51 DMD minus CTGGTGGGAAATGGTCTA G 16 GAGAGT 57 JCR181 human and rhesus monkey (below) minus ACTGGTGGGAAATGGTCT AG 17 GAGAGT 57 JCR182 Exon 51 DMD human and minus AACTGGTGGGAAATGGTC TAG 18 GAGAGT 57

rhesus monkey (below) Exon 51 DMD AGAACTGGTGGGAAATGG human and JCR183 rhesus monkey minus TCTAG 19 GAGAGT 57

(below)

- 49 042798

Table 3

Target gRNA sequences

gRNA name Target sequence (plus thread 5' - 3') SEQ ID NO: Comments JCR89 AAAGATATATAATG T CAT GAAT 64 JCR91 GCAGAAT CAAAATATAATAAG T S T 65 JCR159 SAATTAAAATTTGAGTTATTTGTT 66 101 up exon 51 JCR160 C TAGAC CAT T T C C CAC CAG T T C 67 7 8 below exon 51 JCR167 AG GAC TTTTTTTAC CAAAG GA 68 1534 below exon 51 JCR166 ATCCAAGTCCATTTGATTCCTA 69 1266 below exon 51 JCR168 TAATTTTTSTAGAAAGAGCST 70 1824 above exon 51 JCR170 GGACATGTGCAAGATGCAAGAG 71 2851 upstream of exon 51 JCR171 T G TAT G TAGAAGAC CTC TAAG T 72 2947 higher exon 51 JCR156 TCCCTCACCACTCACCCTGA 73 1300 higher exon 51 JCR157 CTCTGATAACCCAGCTGTGTGT 74 1284 higher exon 51 JCR176 CTCTGATAACCCAGCTGTG 75 JCR157-19 nucleotides JCR177 CTCTGATAACCCAGCTGTGT 76 JCR157-20 nucleotides JCR178 CTCTGATAACCCAGCTGTGTG 77 JCR157-21 nucleotide JCR179 CTCTGATAACCCAGCTGTGTGTT 78 JCR157-23 nucleotide JCR180 C TAGAC SAT T T C C CAC CAG 79 JCR160-19 nucleotides JCR181 C TAGAC SAT T T C C CAC CAG T 80 JCR160-20 nucleotides JCR182 C TAGAC SAT T T C C CAC CAG T T 81 JCR160-21 nucleotide JCR183 C TAGAC SAT T T C C CAC CAG T T S T 82 JCR160-23 nucleotide

Human HEK293T cells were transfected with the individual candidate gRNAs listed in Table 1. 2. Candidate gRNA activity was determined using the Surveyor assay (see FIG. 7). For JCR160, the size of the band corresponding to the original form was 483 nt, and the primers used were the following: forward primer - cgggcttggacagaacttac (SEQ ID NO: 97) and reverse primer - ctgcgtagtgccaaaacaaa (SEQ ID NO: 98). The expected sizes of the bands corresponding to the shapes obtained after the cut were 192 and 291 nt. For JCR157, the size of the band corresponding to the original form was 631 nt, and the primers used were the following: forward primer - gagatgtcttttgcagctttcc (SEQ ID NO: 99) and reverse primer - gggaccttggtaaagccaca (SEQ ID NO: 100). The expected band sizes after cutting were 147 and 484 nt.

The specificity of candidate gRNAs was predicted using the CasOFFinder program (Bae et al. (2014), Bioinformatics, 30: 1473-1475; see Fig. 8). Candidate gRNAs were evaluated and selected based on non-target activity, target activity as measured by the Surveyor assay, and distance from the exon. JCR157 and JCR160 gRNA had a low predicted level of off-target binding

- 50 042798, and they were used for further testing.

They were transfected into HEK293T cells and DMD 6594 cells were electroporated using a modified pDO240 plasmid containing JCR157 gRNA, a modified pDO240 plasmid containing JCR160 gRNA, and a plasmid containing SaCas9 (pDO242; SEQ ID NO: 83). The predicted band size corresponding to the original form was 2451 nt, and the predicted band size corresponding to the deletion form was approximately 840-850 nt. In FIG. 9 shows the exon 51 deletion determined by PCR of genomic DNA (approximately 850 nucleotides) using the forward primer tgcctttcaatcattgtttcg (SEQ ID NO: 101) and the reverse primer agaaggcaaattggcacaga (SEQ ID NO: 102). The deletion created between the gRNA cut sites was approximately 1611 nt.

In FIG. 10 shows the activity of JCR157 gRNA against targets of various lengths (19, 20, 21, 22, and 23 nucleotides) as determined by the Surveyor assay in HEK293T cells using the primers and PCR conditions used for JCR157 in FIG. 7. In FIG. 11 shows activity of JCR160 gRNA against targets of different lengths (19, 20, 21, 22 and 23 nucleotides) as determined by Surveyor assay in HEK293T cells using forward primer - cgggcttggacagaacttac (SEQ ID NO: 97) and reverse primer. - ctgcgtagtgccaaaacaaa (SEQ ID NO: 98). The predicted stripe size corresponding to the original shape was 483 nt, and the expected stripe sizes corresponding to the cut shapes were 209 and 274 nt.

Combinations of JCR157 and JCR160 gRNA against targets of different lengths (21, 22 or 23 nucleotides) were used in HEK293T cells using the conditions used in FIG. 9. In FIG. 12 shows PCR results of genomic DNA. The combination of JCR157 and JCR160, each containing 23 nucleotide targets, provided almost a 50% deletion.

Each gRNA flanking exon 51 (JCR179 upstream and JCR183 downstream) was used alone with SaCas9 using 23 nt target sequences to demonstrate targeted nuclease activity in HEK293T (293s) and DMD6594 (DMD6594s) cells (see Fig. 13 ). For JCR179, the size of the band corresponding to the original form was 594 nt, and the primers used were the following: forward primer - tgcctttcaatcattgtttcg (SEQ ID NO: 101) and reverse primer - aaggccccaaaatgtgaaat (SEQ ID NO: 103). The expected sizes of the bands corresponding to the shapes obtained after the cut were 594 and 130 nt. For JCR183, the size of the band corresponding to the original form was 731 nt, and the primers used were the following: forward primer - gagtttggctcaaattgttactctt (SEQ ID NO: 104) and reverse primer - ctgcgtagtgccaaaacaaa (SEQ ID NO: 98). The expected sizes of the bands corresponding to the shapes obtained after the cut were 440 and 291 nt. In FIG. 13 shows targeted nuclease activity using the Surveyor in vitro assay.

They were transfected into human HEK293T cells and DMD myoblasts (DMD 6594) were electroporated using plasmids containing SaCas9 and JCR179 and JCR183 gRNA (23 nt JCR157 and JCR160 targets; SEQ ID NO: 37 and SEQ ID NO: 38) . DMD 6594 cells were immortalized myoblasts from DMD patients that initially did not contain exons 48-50. The predicted band size corresponding to the original form was 2451 nt and the predicted band size corresponding to the deletion form was approximately 823 nt. In FIG. 14 shows the in vitro deletion of exon 51 in the genomic DNA of human HEK293T cells (left panels) and DMD 6594 cells (right panels) determined by genomic DNA PCR using forward primer tgcctttcaatcattgtttcg (SEQ ID NO: 101) and reverse primer agaaggcaaattggcacaga ( SEQ ID NO: 102). The deletion created between the gRNA cut sites was approximately 1628 nt. Upper panels show a schematic representation of the target gene for upstream and downstream targeting gRNAs in HEK293T cells and DMD 6594 cells, with normally processed exons in purple and mutated exons in yellow. The middle panels show the PCR result within the deletion region of the genome, where the deletion is indicated by an asterisk. Bottom panels show genomic DNA spot digital PCR data. In HEK293T cells, gRNA and SaCas9 provided a 16% deletion, while in DMD 6594 cells they provided approximately 10% editing.

To determine if changes in genomic DNA were transcribed into RNA, RNA was isolated from DMD myoblasts, which were co-transfected with SaCas9 and both gRNAs and allowed to differentiate for 7 days (see FIG. 15). The RNA was reverse transcribed into cDNA and the cDNA was subjected to PCR amplification using standard methods known in the art. In the lower left panel of Fig. 15 shows the result of PCR amplification from exon 44 to exon 52 using forward primer tggcggcgttttcattat (SEQ ID NO: 105) and reverse primer TTCGATCCGTAATGATTGTTCTAGCC (SEQ ID NO: 106). The predicted band size corresponding to the original form was 948 nt and the predicted band size corresponding to the deletion form was approximately 715 nt. In FIG. 15 shows the deletion band only for cells treated with SaCas9 and both gRNAs. The lower right panel shows ddPCR data demonstrating cDNA editing at approximately 14%.

- 51 042798

The junction of exon 47 to 52 in cDNA from myoblasts from DMD patients was sequenced in vitro (see Fig. 16). In FIG. 16, the sequence corresponding to the bands from untreated cells (control cells A48-50) indicates that exons 47 and 51 joined as expected, while the band corresponding to the deletion form in treated cells (A48-50+ A51) indicated the connection of exon 47 to 52. Thus, the distinct absence of exon 51 and the result of the disclosed targeting system at the genomic DNA level was provided by transcription.

Example 2 Obtaining an A52/mdx mouse.

In FIG. 17 shows the scheme for obtaining an A52/mdx mouse. An hDMD/mdx mouse was obtained from the University of Leiden and manipulated to obtain a relevant DMD model in which exon 52 was deleted, this deletion resulting in a frameshift and DMD-specific genotype. The hDMD/mdx mouse contained the full length human wild-type dystrophin gene on chromosome 5 surrounded by mdx, resulting in no mouse dystrophin expression.

To obtain a relevant DMD model, a CRISPR/Cas9 editing system based on SpCas9 and gRNA was used to target and provide for the deletion of exon 52 in the human dystrophin gene. Various gRNAs targeting upstream and downstream of exon 52 were tested and checked for consistency using Surveyor analysis (see FIG. 18). For gRNAs targeting above, forward primer - ctccggaatgtctccatttg (SEQ ID NO: 87) and reverse primer TTGTGTGTCCCATGCTTGTT (SEQ ID NO: 107) were used, and the band size corresponding to the original form was 402 nt. With respect to JCR94 (AACAAATATCCCTTAGTATC (SEQ ID NO: 41)), the expected mold sizes obtained after cutting were 243 and 159 nt. For gRNAs targeting downstream, forward primer CAACGCTGAAGAACCCTGAT (SEQ ID NO: 108) and reverse primer atgagggaggactggcatc (SEQ ID NO: 88) were used, and the size of the band corresponding to the original form was 509 nt. As for JCR99 (AATGTATTTCTTCTATTCAA (SEQ ID NO: 42)), the expected sizes of the forms obtained after cutting were 346 and 163 nt gRNA pairs were tested and checked for compliance by detecting exon 51 deletion in genomic DNA from HEK293T cells, including JCR94 and JCR99 using forward primer - ctccggaatgtctccatttg (SEQ ID NO: 87) and reverse primer - atgagggagactggcatc (SEQ ID NO: 88) (see Fig. 19). The band size corresponding to the original form was 718 nt, the band size corresponding to the deletion form was 392 nt, and the deletion between the gRNA was 326 nt. Specifically, a mouse was generated by injecting JCR94 gRNA, JCR99 gRNA, and SaCas9 mRNA into mouse embryos.

In FIG. 20 shows a DNA microinjection protocol that includes the use of a BAC recombination engineering service. Day 1: Pregnant mares were treated intraperitoneally with foal mare serum gonadotropin to induce ovulation. Day 3: Pregnant mares were treated intraperitoneally with human chorionic gonadotropin. In cycle 1, 471 embryos were obtained, but only 5 plugs were observed. Used 150 embryos created by fertilization (from 14 females subjected to superovulation). Injections into the pronucleus were performed using less than 50 ng of Cas9 and 20 ng of each guide. In FIG. 21 shows a protocol for crossing mice to produce transgenic mice.

Founder mice were genotyped using the following genotyping protocol. Genomic DNA (gDNA) was extracted from mouse tail cuts using the DNEasy Blood and Tissue DNA Extraction Kit (Qiagen). For genotyping each pup, gDNA was amplified using the AccuPrime HiFi Taq kit as follows:

i) 100 ng dDNA;

ii) 2.5 µl of buffer II from the AccuPrime kit;

iii) 0.1 µl HiFi Taq from the AccuPrime kit;

iv) 1 µl JRH261 (ctccggaatgtctccatttg (SEQ ID NO: 87)) (10 µM);

v) 1 µl JRH264 (atgagggaggactggcatc (SEQ ID NO: 88)) (10 µM); and vi) water to a total volume of 25 μl.

The reactions were carried out on a thermal cycler according to the following:

i) 95° for 4 minutes;

ii) 95° for 30 s;

iii) 52° for 30 s;

iv) 68° for 1:00 min;

v) cycling steps ii)-iv) 35 times, while constantly at 4°.

The products of PCR reactions were separated on the gel (Fig. 22). The expected band sizes were 718 nt in the case of no deletion (ie, exon 52 was still present) and approximately 392 nt in the case of exon 52 deletion. As shown in FIG. 22, founder mice 7, 63, and 76 had a deletion of exon 52.

The bands of the amplified products were sequenced using primer for JRH264 (see Fig. 23) to determine the sequences of the rejoined ends of the regions subjected to

- 52 042798 healing. In FIG. 23 shows a sequenced region where letters in bold, underline and normal indicate native sequences and letters in italics indicate insertions or deletions. In the expected sequence (delta 52), bold letters are related to underlined letters. The sequences from founding mice had insertions (letters in italics) and deletions (hyphens) in this region.

Male founding mice were mated with mdx/mdx females to select for chimeric organisms (FIG. 24). Progeny derived from male 76 founder mice or male 63 founder mice and mdx/mdx females were screened and genotyped for exon 52 deletion using the conditions used in FIG. 22 (FIGS. 25 and 26, respectively). If exon 52 was deleted, the expected band size was approximately 392 nt. If exon 52 was present, the expected band size was approximately 718 nt. Pups 54497 and 54498 (from the mating pair: male founding mouse 63 + female mdx/mdx) had a deletion of exon 52 and were sequenced (FIG. 27). Pups 54497 and 54498 had 92.86% identity with each other according to a 392 bp sequencing read, with inserts/deletions being identical.

The expression level of dystrophin in a healthy hDMD/mdx mouse and A52/mdx mouse was compared by fluorescent immunohistochemistry. As shown in FIG. 28, pups 54497 and 54498 from A52/mdx mice lacked the dystrophin protein. In the case of heart staining, the exposure for the laminin probe was 100 ms, while the exposure for dystrophin was 900 ms. In the case of the TA muscle sample, the exposure time for the laminin and dystrophin probe was 2.0 s. See also FIG. 29, which shows that the A52/mdx mouse lacked the dystrophin protein. Dystrophin expression was lost in both the heart and TA muscle of the A52/mdx mouse. Several spontaneously formed revertant fibers (random splicing events or somatic mutations) were observed in TA muscle, however, this was consistent with the mdx mouse model. Western blot results also indicated that the A52/mdx mouse lacked the dystrophin protein, consistent with the characteristic DMD genotype, while the healthy hDMD/mdx mouse expressed dystrophin. See fig. thirty.

The A52/mdx mouse exhibited activity levels similar to mdx mice after the first five minutes of the open field test. In the open field test, mice were allowed to freely explore an open field arena (20x20x30 cm) for 30 minutes. Animal activity and position were automatically monitored using infrared diodes (in x, y, and z axes) coupled to Fusion activity analysis software (version 5.3, Omnitech, Columbus, Ohio) running on a computer. In FIG. 31 shows the overall activity of an A52/mdx mouse compared to mdx mice and hDMD/mdx mice based on locomotor activity and visual inspection. The distance traveled and vertical rack activities are shown in the left and right panels, respectively.

Example 3 Restoration of dystrophin expression by removing exon 51.

By deleting exon 51 in the A52/mdx mouse, one could create a genotype similar to that of Becker muscular dystrophy (BMD) and theoretically restore dystrophin expression. An A52/mdx mouse was used to demonstrate restoration of dystrophin expression by deleting exon 51 using the disclosed CRISPR/Cas9 based gene editing system. In FIG. 32 shows a strategy for corrections using SaCas9 and gRNA to skip exon 51 by targeting gRNA upstream and downstream of exon 51 in the intron region.

Standard: Plasmids containing JCR179 (above) or JCR183 gRNA (below) (SEQ ID NO: 37 and SEQ ID NO: 38, respectively) and SaCas9 were electroporated into myoblasts from DMD patients. The protein was isolated from differentiated cells and analyzed by Western blotting using an antibody to dystrophin. In FIG. 33 shows that genomic DNA can be edited to restore dystrophin protein expression, as cells treated with all 3 components (ie both gRNA and SaCas9) showed dystrophin expression.

The system was then tested on an A52/mdx mouse. In FIG. 34 shows a design of an A52/mdx mouse processing experiment using a gRNA and SaCas9 based system, including schematics of the 2 viral vectors used in the design of the experiment. Recombinant AAV8 constructs were generated using the PT366-179 (SEQ ID NO: 39) and PT366-183 (SEQ ID NO: 40) vectors and methods for preparing virus particles known in the art. These viral vectors (AAV8) were co-delivered in vivo as two viral particles. Each viral particle contained SaCas9 and one of the gRNAs (see Fig. 3). A52/mdx mice were treated with 5E11 recombinant AAV8 virus constructs. The virus was injected intramuscularly into the right TA muscle, with the left TA muscle serving as a contralateral control, and injected with PBS. After treatment, both left and right TA muscles were removed and sections were obtained from each for genomic DNA analyses. As shown in FIG. 35, PCR was performed within the region of interest, and bands corresponding to the deletion shape were noted in the case of the right TA-treated muscle on the gel shown on the left, indicating

- 53 042798 to some level of gene editing. The band corresponding to the deletion form was sequenced and the main product was the expected 3 bp ligation product from the PAM of each gRNA. In FIG. 35 shows an in vivo exon 51 deletion in the right TA muscle.

Similarly, sections of treated right TA muscle and control left TA muscle were analyzed to determine if RNA editing was provided. PCR was performed within exons 50-53 in cDNA. As shown in the gel shown on the right in FIG. 36, exon 51 and 52 were missing in two of the treated samples. The deletion band was sequenced, with the main product being the ligation of exon 50 to 53, as would be expected given that the mouse already lacked exon 52, and a CRISPR/Cas9 based gene editing system deleted exon 51 (see chromatogram of sequencing results, bottom right). In FIG. 36 shows an in vivo exon 51 deletion in the right TA muscle.

In FIG. 37 shows exemplary fluorescent immunohistochemical staining indicating the presence of a small amount of dystrophin in the PBS-injected control left TA muscle in an A52/mdx mouse. Reasons for some green staining for dystrophin in the control left TA may be reverted fibers or dead cells, which sometimes also stain green. A clear increase in the degree of green staining for dystrophin was observed in the treated right TA muscle, as shown in the right photo. In FIG. 37 shows in vivo recovery of dystrophin protein in TA-treated muscle.

Protein was extracted from the left and right TA muscles from 3 test mice, and Western blot analysis was performed. In FIG. 38 shows in vivo recovery of dystrophin protein in TA-treated muscle. In the control left TA muscles, no protein expression was observed, while all three right TA muscles showed different levels of dystrophin protein expression. The expression of the protein in the right TA mouse 1 was the strongest, while in mice 2 and 3 it was weak, but nevertheless appeared as bands. The disclosed CRISPR/Cas9 based gene editing system functioned in vivo to restore dystrophin protein expression to some extent in the A52/mdx mouse.

Physiological testing of mice. The mice to be treated (all male hDMDΔ52(hetero)/mdx(hemi) mice) were treated with recombinant AAV8 (n=10) or AAV9 (n=10) virus constructs containing SaCas9 and gRNA and compared with untreated mice (hDMD-Δ52/mdx) (n=10). Recombinant AAV constructs were generated using vectors PT366-179 (SEQ ID NO: 39) and PT366-183 (SEQ ID NO: 40) and using methods known in the art. Mice to be treated were injected with 200 μl of virus into the tail vein at 6-8 weeks of age. Mice were tested after 8 weeks.

Open field test with distance determination. The mice were allowed to freely explore the open field arena for 30 min. The activity and position of the animals were automatically monitored using infrared diodes compatible with the Fusion activity analysis software running on a computer. Data was collected continuously and grouped at 5 minute intervals. In FIG. 39 shows the mean of all time points for total distance traveled in 16 week old mice treated at 8 weeks of age compared to 16 week old mice not treated. The average of all time points for the total number of rearing postures after 16 weeks is shown in FIG. 40. Statistical methods were as follows: one-way ANOVA, comparison of the mean of each column with the mean of the raw control, and post hoc analysis using Dunnett's test (mean +/-SEM). Mice treated with AAV8 and AAV9 showed a statistically significant result in terms of greater walking distance than untreated mice of the corresponding age group (statistically significant). All treated mice showed a statistically significant result in more rearing postures compared to untreated age-matched controls.

Grip strength. Grip strength was tested in 16 week old untreated and treated mice. The mice underwent 3-5 trials, each of which was designed to test the grip strength of the front and hind legs. The average test results are shown in FIG. 41. The strength of the grip was noted as a gram-force. As shown in FIG. 41, mice treated with AAV9 showed a statistically significant result of increased forepaw grip strength compared to age-matched untreated mice. Statistical methods were as follows: two-way ANOVA, post hoc analysis using Tukey's test.

cDNA PCR. Tissues from mouse heart samples were processed using the RNEasy Plus Universal Mini Kit (Qiagen). The resulting RNA was reverse transcribed into cDNA using the Superscript VILO cDNA synthesis kit. 1 µl of cDNA was subjected to PCR amplification using DNA polymerase from the AccuPrime kit and primers for exon 48 (forward primer: gtttccagagctttacctgagaa (SEQ ID NO: 89)) and exon 54 (reverse primer: CTTTTATGAATGCTTCTCCAAG (SEQ ID NO: 90)). The expected band sizes were 997 nt in the absence of a deletion (i.e., exon 52 is still

- 54 042798 was present) and approximately 764 nt in the case of a deletion of exon 52. FIG. 42 shows results for P2 mice injected with facial vein injection of AAV9 (JA10 (P2 AAV9)) at 36-50 hours of age from birth and adult mice injected with tail vein of AAV8 (JA11 (TV AAV8 )) in the amount of 3.3-7.7E12 or AAV9 (JA12 (TV AAV9)) in the amount of 4.3-7.5E12. As shown in FIG. 42, editing occurred to varying degrees in P2 mice (mice 48-54 hours old from birth) that were treated with AAV9 and adult mice that were treated with AAV8 and AAV9, which was further confirmed by sequencing the band corresponding to the form c deletion using primer tttctgtgattttcttttggattg (SEQ ID NO: 109) binding to exon 53. FIG. 43 shows an exemplary chromatogram showing the deletion of exons 51 and 52 in sequence from the band corresponding to the deletion form from mouse 1 treated with Ja10.

The foregoing detailed description and accompanying examples are intended to be illustrative only and should not be construed as limiting the scope of the present invention, which is defined solely by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or uses of the present invention, may be made without departing from its spirit and scope.

For the sake of completeness, various aspects of the present invention are set forth in the following numbered paragraphs.

1. Guide RNA (gRNA) containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO : 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 41, SEQ ID NO: 42, or its complementary sequence.

2. DNA targeting composition comprising a first gRNA and a second gRNA, wherein the first gRNA molecule and the second gRNA molecule contain a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 41, SEQ ID NO: 42, or its complementary sequence, where the first gRNA molecule and the second gRNA molecule contain different targeting domains.

3. The DNA targeting composition of claim 2, wherein the first gRNA molecule is listed under SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 41, and the second gRNA molecule is shown under SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 42.

4. A DNA targeting composition according to claim 2 or 3, wherein the first gRNA molecule is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and the second gRNA molecule is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

5. A DNA targeting composition according to any one of claims 2-4, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 2;

(ii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(iii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(iv) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(v) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

- 55 042798 (vi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(vii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(viii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(ix) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(x) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15;

(xi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18; and (xii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 42.

6. A DNA targeting composition according to any one of claims 2 to 5, further comprising a protein (Cas) associated with short palindromic repeats in regularly arranged groups.

7. The DNA targeting composition of claim 6, wherein the Cas protein comprises a Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25).

8. A DNA targeting composition according to claim 6 or 7, wherein the Cas protein comprises a Cas9 molecule from Staphylococcus aureus comprising the amino acid sequence of SEQ ID NO: 45.

9. A DNA targeting composition according to any one of claims 2 to 8, wherein the DNA targeting composition comprises the nucleotide sequence of SEQ ID NO: 83, the nucleotide sequence of SEQ ID NO: 84, the nucleotide sequence of SEQ ID NO: 37, and /or a nucleotide sequence under SEQ ID NO: 38.

10. An isolated polynucleotide comprising a gRNA molecule according to claim 1 or a DNA targeting composition according to any one of claims 2-9.

11. A vector containing a gRNA according to claim 1, a DNA targeting composition according to any one of claims 2-9, or an isolated polynucleotide according to claim 10.

12. A vector containing a DNA targeting composition according to any one of claims 6-9.

13. Vector encoding (a) the first guide RNA molecule (gRNA);

(b) a second gRNA molecule; and (c) at least one Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25), wherein the first gRNA molecule and the second gRNA molecule contain a targeting a domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 , SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 41, SEQ ID NO: 42, or its complementary sequence, where the first gRNA molecule and the second gRNA molecule contain different targeting domains.

14. The vector of claim 13, wherein the vector is constructed to form first and second double strand breaks in the first and second introns flanking exon 51 of the human DMD gene.

15. The vector according to claim 13 or 14, where the first gRNA molecule is presented under SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 41, and the second gRNA molecule is shown under SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 42.

16. The vector according to any one of claims 13-15, wherein the first gRNA molecule is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and the second gRNA molecule is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

17. The vector according to any one of claims 13-16, wherein the first gRNA molecule and the second gRNA molecule are

- 56 042798 branes from the group consisting of (i) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 2;

(ii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(iii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(iv) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(v) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(vi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(vii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(viii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(ix) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(x) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15; and (xi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18.

18. A vector according to any one of claims 11-17, wherein the vector is a viral vector.

19. The vector of claim 18, wherein the vector is an adeno-associated virus (AAV) vector.

20. The vector of claim 19, wherein the AAV-based vector is an AAV8-based vector or an AAV9-based vector.

21. A vector according to any one of claims 11-20, wherein the vector contains a tissue-specific promoter operably linked to a nucleotide sequence encoding a first gRNA molecule, a second gRNA molecule and/or a Cas9 molecule.

22. The vector of claim 21, wherein the tissue-specific promoter is a muscle-specific promoter.

23. A cell comprising a gRNA according to claim 1, a DNA targeting composition according to any one of claims 2-9, an isolated polynucleotide according to claim 10, or a vector according to any one of claims 11-22.

24. A kit containing a gRNA according to claim 1, a DNA targeting system according to any one of claims 2-9, an isolated polynucleotide according to claim 10, a vector according to any one of claims 11-22 or a cell according to claim 23, and optionally instructions by application.

25. A method for correcting a mutant dystrophin gene in a cell, wherein the method involves introducing into the cell a gRNA according to claim 1, a DNA targeting system according to any one of claims 2-9, an isolated polynucleotide according to claim 10, or a vector according to any one of claims. 11-22.

26. A method for editing a mutant dystrophin gene in the genome of a subject, the method comprising administering to the subject a genome editing composition comprising a gRNA according to claim 1, a DNA targeting system according to any one of claims 2 to 9, an isolated polynucleotide according to claim 10, vector according to any one of paragraphs. 11-22 or cells according to item 23.

27. The method of claim 26, wherein the genome editing composition is administered to the subject intramuscularly, intravenously, or a combination thereof.

28. The method of any one of claims 25-27, wherein the correction of the mutant dystrophin gene involves nuclease-mediated non-homologous end joining.

29. A method of treating a subject in need thereof who has a mutated dystrophin gene,

- 57 042798, wherein the method comprises administering to the subject a gRNA according to claim 1, a DNA targeting system according to any one of claims 2-9, an isolated polynucleotide according to claim 10, a vector according to any one of claims 11-22, or a cell according to claim 23.

30. A modified vector based on adeno-associated virus for editing the mutant dystrophin gene in the subject's genome, containing the first polynucleotide sequence encoding the gRNA according to claim 1, and the second polynucleotide sequence encoding the Cas9 molecule, which recognizes the motif adjacent to the protospacer (PAM) or NNGRRT ( SEQ ID NO: 24), or NNGRRV (SEQ ID NO: 25).

31. The modified adeno-associated virus vector according to claim 30, wherein the modified adeno-associated virus vector contains the nucleotide sequence set forth under SEQ ID NO: 39 or SEQ ID NO: 40.

32. A composition for providing a deletion of a dystrophin gene segment containing exon 51, wherein the composition comprises (a) a first vector containing a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first Cas9 molecule that recognizes an adjacent a protospacer motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25); and (b) a second vector containing a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO : 25), where each of the first and second gRNA molecules contains a targeting domain, the length of which is from 19 to 24 nucleotides, and where the first vector and the second vector are designed to form the first and second double-strand breaks in the first intron and the second intron, flanking, respectively exon 51 of the human DMD gene, thereby providing a deletion of the segment of the dystrophin gene containing exon 51.

33. The composition according to claim 32, where the length of the segment is from about 50 p. up to about 2000 bp

34. The composition of claim 33, wherein the segment length is about 118 bp, about 233 bp, about 326 bp, about 766 bp, about 805 bp. or approximately 1611 p.

35. A composition according to any one of claims 32-34, wherein the first Cas9 molecule and the second Cas9 molecule are identical.

36. The composition of claim 35, wherein the first Cas9 molecule and the second Cas9 molecule are a Cas9 molecule from Staphylococcus aureus.

37. The composition of claim 36, wherein the first Cas9 molecule and the second Cas9 molecule are a mutant Cas9 molecule from Staphylococcus aureus.

38. A composition according to any one of claims 32-34, wherein the first Cas9 molecule and the second Cas9 molecule are different.

39. The composition of claim 38, wherein the first Cas9 molecule or the second Cas9 molecule is a Cas9 molecule from Staphylococcus aureus.

40. The composition according to any one of claims 32-39, wherein the first Cas9 molecule and/or the second Cas9 molecule provide a SaCas9 molecule containing the amino acid sequence under SEQ ID NO: 45.

41. A composition according to any one of claims 32-40, wherein the first vector and/or the second vector is a viral vector.

42. The composition of claim 41, wherein the first vector and/or the second vector is an adeno-associated virus (AAV) vector.

43. The composition of claim 42, wherein the AAV-based vector is an AAV8-based vector or an AAV9-based vector.

44. A composition according to any one of claims 32-43, wherein the dystrophin gene is a human dystrophin gene.

45. The composition according to any one of claims 32-44, where the first gRNA molecule and the second gRNA molecule contain a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO : 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or its complementary sequence, where the first gRNA molecule and the second gRNA molecule contain different targeting domains.

46. The composition according to any one of claims 32-45, where the first gRNA molecule is presented under SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and the second molecule

- 58 042798 gRNA is listed under SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

47. The composition according to any one of claims 32-46, wherein the first gRNA molecule is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and the second gRNA molecule is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

48. The composition according to any one of claims 32-47, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 2;

(ii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(iii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(iv) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(v) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(vi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(vii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(viii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(ix) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(x) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15; and (xi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18.

49. The composition according to any one of claims 32-48, wherein the first vector contains the nucleotide sequence set forth under SEQ ID NO: 39 and the second vector contains the nucleotide sequence set forth under SEQ ID NO: 40.

50. A composition according to any one of claims 32-49 for use in a medicinal product.

51. A composition according to any one of claims 32-50 for use in the treatment of Duchenne muscular dystrophy.

52. A cell containing a composition according to any one of claims 32-51.

53. A method for correcting a mutant dystrophin gene in a cell, comprising introducing into the cell (a) a first vector containing a polynucleotide sequence encoding the first guide RNA molecule (gRNA) and a polynucleotide sequence encoding the first Cas9 molecule that recognizes a motif adjacent to the protospacer (PAM ) either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25); and (b) a second vector containing a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO : 25), where each of the first gRNA molecule and the second gRNA molecule contains a targeting domain, the length of which is from 19 to 24 nucleotides, and where the vector is designed to form the first and second double-strand breaks, respectively, in the first and second introns flanking exon 51 of the gene human dystrophin, which ensures the deletion of the dystrophin gene segment containing exon 51.

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54. The method of claim 53, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1 and a second gRNA molecule containing targeting domain, which contains the nucleotide sequence set forth under SEQ ID NO: 2;

(ii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(iii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(iv) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(v) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(vi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(vii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(viii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(ix) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(x) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15; and (xi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18.

55. The method according to claim 53 or 54, wherein the mutant dystrophin gene contains a premature stop codon, a misaligned reading frame, an aberrant splice acceptor site, or an aberrant splice donor site.

56. The method of any one of claims 53-55, wherein the mutant dystrophin gene contains a frameshift mutation that causes a premature stop codon and a truncated gene product.

57. The method of any one of claims 53-55, wherein the mutant dystrophin gene is characterized by a deletion of one or more exons that provides a frameshift.

58. The method of any one of claims 53-57, wherein correcting the mutant dystrophin gene comprises deleting a premature stop codon, correcting a misaligned reading frame, or modulating splicing by misplacing a splice acceptor site or misplacing a donor splice sequence.

59. The method according to any one of claims 53-58, wherein the correction of the mutant dystrophin gene involves the deletion of exon 51.

60. The method of any one of claims 53-59, wherein the repair of the mutant dystrophin gene involves repair involving homologous recombination.

61. The method of claim 60, further comprising introducing donor DNA into the cell.

62. The method of any one of claims 53-61, wherein the correction of the mutant dystrophin gene involves nuclease-mediated non-homologous end joining.

63. The method of any one of claims 53-62, wherein the cell is a myoblast cell.

64. The method of any one of claims 53-63, wherein the cell is a cell of a subject suffering from Duchenne muscular dystrophy.

65. The method of any one of claims 53-64, wherein the cell is a myoblast of a human subject suffering from Duchenne muscular dystrophy.

66. The method according to any one of claims 53-65, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of

- 60 042798 (i) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 2;

(ii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4; and (iii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19.

67. A method of treating a subject in need thereof who has a mutant dystrophin gene, the method comprising administering to the subject (a) a first vector containing a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first Cas9 molecule which recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25); and (b) a second vector containing a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO : 25), where each of the first gRNA molecule and the second gRNA molecule contains a targeting domain, the length of which is from 19 to 24 nucleotides, and where the vector is designed to form the first and second double-strand breaks, respectively, in the first and second introns flanking exon 51 of the gene human dystrophin, which ensures the deletion of the dystrophin gene segment containing exon 51.

68. The method of claim 67, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 1 and a second gRNA molecule containing targeting domain, which contains the nucleotide sequence set forth under SEQ ID NO: 2;

(ii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(iii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(iv) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(v) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(vi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(vii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18;

(viii) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 4;

(ix) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 19;

(x) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 15; and (xi) a first gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that contains the nucleotide sequence set forth under SEQ ID NO: 18.

69. The method of claim 68 wherein the subject suffers from Duchenne muscular dystrophy.

70. The method of any one of claims 67-69, wherein the first vector and the second vector are administered to a muscle of a subject.

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71. The method of claim 70 wherein the muscle is skeletal muscle or cardiac muscle.

72. The method of claim 71 wherein the skeletal muscle is the tibialis anterior.

73. The method of any one of claims 67-72, wherein the first vector and the second vector are administered to the subject intramuscularly, intravenously, or a combination thereof.

74. A method for producing a transgenic rodent embryo having a human dystrophin gene (hDMD) with a deletion of exon 52 (Δ52), the method comprising introducing into the rodent embryo a gRNA according to claim 1, a DNA targeting system according to any one of claims 2 -9, an isolated polynucleotide according to paragraph 10, a vector according to any one of paragraphs 11-22, a modified adeno-associated virus vector according to paragraph 30 or 31, or a composition according to any one of paragraphs 32-51, thereby ensuring the deletion of exon 52 in a human dystrophin gene; and selecting a transgenic rodent embryo having an exon 52 deletion in the human dystrophin gene, wherein the rodent embryo contains a normal human dystrophin gene.

75. The method of claim 74 wherein the rodent embryo is a mouse embryo.

76. The method of claim 74 or 75, wherein the transgenic rodent embryo is heterozygous for hDMD or heterozygous for hDMD-A52.

77. The method according to any one of paragraphs 74-76, where the first gRNA molecule containing the targeting domain, which contains the nucleotide sequence set forth under SEQ ID NO: 41, and the second gRNA molecule containing the targeting domain, which contains the nucleotide sequence set forth under SEQ ID NO: 42 is introduced into a rodent embryo to effect a deletion of exon 52 of the human dystrophin gene.

78. The method according to any one of claims 74-77, further comprising introducing into the rodent embryo a Cas protein containing the amino acid sequence set forth under SEQ ID NO: 27.

79. A transgenic rodent embryo obtained using the method of any one of claims 74-78.

80. A transgenic rodent derived from a transgenic rodent embryo according to claim 79.

Приложение pDO240 с JCR179 (SEQ ID NO: 37) (gRNA выделена жирным шрифтом) ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattt tttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggt tgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagg gcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgac ggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggc gctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgcta cagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcct cttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc agggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcacta tagggcgaattgggtaccaagcttgcctatttcccatgattccttcatatttgcatatacgata caaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaat acgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatgg actatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaa aggacgaaacaccAACACACAGCTGGGTTATCAGAGgttttagtactctggaaacagaatctac taaaacaaggcaaaatgccgtgtttatctcgtcaacttgttggcgagatttttttgcggccgcc cgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatg gtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccgga agcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgct cactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgc ggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcg gtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaat caggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaa ggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgc tcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagct ccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttc gggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgc

- 62 042798 tccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaact atcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggc tacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagag ttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagca gcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgac gctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttca cctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttg gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttca tccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggcc ccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaacca gccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatt aattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgcca ttgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttccca acgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcct ccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcata attctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtc attctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataatacc gcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactct caaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttc agcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaa aagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaa gcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaaca aataggggttccgcgcacatttccccgaaaagtgccac pDO240 c JCR183 (SEQ ID NO: 38) (gRNA выделена жирным шрифтом) ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattt tttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggt tgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagg gcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgac ggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggc gctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgcta cagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcct cttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc agggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcacta tagggcgaattgggtaccaagcttgcctatttcccatgattccttcatatttgcatatacgata caaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaat acgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatgg actatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaa aggacgaaacaccAGAACTGGTGGGAAATGGTCTAGgttttagtactctggaaacagaatctac taaaacaaggcaaaatgccgtgtttatctcgtcaacttgttggcgagatttttttgcggccgcc

- 63 042798 cgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatg gtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccgga agcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgct cactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgc ggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcg gtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaat caggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaa ggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgc tcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagct ccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttc gggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgc tccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaact atcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggc tacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagag ttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagca gcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgac gctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttca cctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttg gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttca tccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggcc ccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaacca gccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatt aattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgcca ttgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttccca acgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcct ccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcata attctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtc attctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataatacc gcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactct caaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttc agcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaa aagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaa gcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaaca aataggggttccgcgcacatttccccgaaaagtgccac

PT366 c JCR179 - PT366AAV 179 (SEQ ID NO: 39) - plasmid for

- 64 042798

AAV, применяемая для работы in vivo (gRNA выделена жирным шрифтом; SaCas9 выделена верхним регистром; NLS выделена нижним регистром, жирным шрифтом и подчеркиванием) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtc gcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttc ctgcggcctctagactcgaggcgttgacattgattattgactagttattaatagtaatcaatta cggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggccc gcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagta acgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgg cagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgta ttagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggt ttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcacca aaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg cgtgtacggtgggaggtctatataagcagagctctctggctaactaccggtgccaccatggccc caaagaagaagcggaaggtcggtatccacggagtcccagcagccAAGCGGAACTACATCCTGGG CCTGGACATCGGCATCACCAGCGTGGGCTACGGCATCATCGACTACGAGACACGGGACGTGATC GATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAACGAGGGCAGGCGGAGCAAGA GAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGCTGCTGTT CGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGCCAGAGTG AAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTGGCCAAGA GAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGCAACGAGCTGTCCACCAAAGA GCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGGCCGAACTGCAGCTGGAACGG CTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAGCGACTACGTGAAAG AAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCATCGACAC CTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAGCCCCTTC GGCTGGAAGGACATCAAAGAATGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGG AACTGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAA TCTCGTGAT САС CAG G GAC GAGAAC GAGAAG С T G GAATAT TAG GAGAAG T T С CAGAT CATC GAG AAC G T G T T CAAG CAGAAGAAGAAG С С СAC С С T GAAG CAGAT C G С CAAAGAAAT С С T C G T GAAC G AAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCGAGTTCACCAACCTGAAGGT GTACCACGACATCAAGGACATTACCGCCCGGAAAGAGATTATTGAGAACGCCGAGCTGCTGGAT CAGATTGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAAGAACTGACCAATC TGAACTCCGAGCTGACCCAGGAAGAGATCGAGCAGATCTCTAATCTGAAGGGCTATACCGGCAC CCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTGTGGCACACCAACGACAAC CAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGTCCCAGCAGAAAG

- 65 042798

AGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAAGCTTCATCCA GAG CAT CAAAG T GAT GAAC G C CAT CAT CAAGAAGTACGGCCTGCCGAACGAGAT CAT TAT C GAG CTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGATGCAGAAGCGGAACC GGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGCAAAGAGAACGCCAAGTACCT GATCGAGAAGATCAAGCTGCACGACATGCAGGAAGGCAAGTGCCTGTACAGCCTGGAAGCCATC CCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGAGGTGGACCACATCATCCCCAGAAGCG TGTCCTTCGACAACAGCTTCAACAACAAGGTGCTCGTGAAGCAGGAAGAAAACAGCAAGAAGGG CAACCGGACCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAAACCTTCAAG AAGCACATCCTGAATСTGGCCAAGGGCAAGGGCAGAATCAGCAAGACCAAGAAAGAGTATСTGC TGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTCATCAACCGGAACCTGGTGGA TACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTACTTCAGAGTGAACAACCTG GACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCTGCGGCGGAAGTGGAAGTTTA AGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCCTGATCATTGCCAACGCCGA T T T CAT С T T CAAAGAGT GGAAGAAAC T GGACAAGGCCAAAAAAGT GAT GGAAAACCAGATG T T C GAGGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAGATCTTCA TCACCCCCCACCAGATCAAGCACATTAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGA CAAGAAGCCTAATAGAGAGCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGACAAGGGC AACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGACΑΆΤGACAAGCTGAAAAAGC T GAT CAACAAGAG С С С C GAAAAG CTGCTGATGTAC СAC CAC GAC С С С CAGAC С TAC CAGAAAC T GAAGCTGATTATGGAACAGTACGGCGACGAGAAGAATCCCCTGTACAAGTACTACGAGGAAACC GGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGTGATCAAGAAGATTAAGTATT ACGGCAACAAACTGAACGCCCATCTGGACATCACCGACGACTACCCCAACAGCAGAAACAAGGT CGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGGACAATGGCGTGTACAAGTTC G T GAC C G T GAAGAAT CTGGATGTGAT CAAAAAAGAAAAC TAG TAG GAAG T GAAT AG GAAG T G С T ATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTTATCGCCTCCTTCTACAA CAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGAACAACGACCTGCTG AAC C G GAT C GAAG T GAACATGAT CGACAT СACС TACCGCGAGTACС T GGAAAAGATGAACGAGA AGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAGAGCATTAAGAAGTACAGCAC AGACATTCTGGGCAACCTGTATGAAGTGAAATCTAAGAAGCACCCTCAGATCATCAAAAAGGGC aaaaqqccqqcqqccacqaaaaaqqccqqccaqqcaaaaaaqaaaaaqqgatcctacccatacg atgttccagattacgcttacccatacgatgttccagattacgcttacccatacgatgttccaga ttacgcttaagaattcctagagctcgctgatcagcctcgactgtgccttctagttgccagccat ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttc ctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggg gtggggcaggacagcaaggggggattgggaagataagaatagcaggcatgctggggaggtaccga gggcctatttcccattgcattcattactg

- 66 042798 ggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataat ttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaac ttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgAACACACA GCTGGGTTATCAGAGgttttagtactctggaaacagaatctactaaaacaaggcaaaatgccgt gtttatctcgtcaacttgttggcgagatttttgcggccgcaggaacccctagtgatggagttgg ccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgccc gggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatg cggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagta cgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctaca cttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccg gctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacg gtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaa caacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggccta ttggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgttt acaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgac acccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagaca agctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcg agacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctt agacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaat acattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaa aggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcc ttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgc acgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattg acgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactc accagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccata accatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaa ccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaa tgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggagg cggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccc tcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacaga tcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatat actttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgat

- 67 042798 aatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggta actggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccacc acttcaagaactctgtagcaccgcctacatacctcgctctgetaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcg cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccg aactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgga caggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaac gcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggc cttttgctggccttttgctcacatgt

PT366 c JCR183 (SEQ ID NO: 40) -применяемая для работы in vivo (gRNA выделена жирным шрифтом; SaCas9 выделена верхним регистром; NLS выделена нижним регистром, жирным шрифтом и подчеркиванием) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtc gcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttc ctgcggcctctagactcgaggcgttgacattgattattgactagttattaatagtaatcaatta cggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggccc gcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagta acgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttgg cagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgta ttagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggt ttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcacca aaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg cgtgtacggtgggaggtctatataagcagagctctctggctaactaccggtgccaccatggccc caaagaagaagcggaaggtcggtatccacggagtcccagcagccAAGCGGAACTACATCCTGGG CCTGGACATCGGCATCACCAGCGTGGGCTACGGCATCATCGACTACGAGACACGGGACGTGATC GATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAACGAGGGCAGGCGGAGCAAGA GAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGCTGCTGTT CGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGCCAGAGTG AAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTGGCCAAGA GAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGCAACGAGCTGTCCACCAAAGA GCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGGCCGAACTGCAGCTGGAACGG

- 68 042798

CTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAGCGACTACGTGAAAG AAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCATCGACAC CTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAGCCCCTTC GGCTGGAAGGACATCAAAGAATGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGG AACTGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAA TCTCGTGAT СAC CAG G GAC GAGAAC GAGAAG С T G GAATAT TAG GAGAAG T T С CAGAT CATC GAG AAC G T G T T CAAG CAGAAGAAGAAG С С СAC С С T GAAG CAGAT C G С CAAAGAAAT С С T C G T GAAC G AAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCGAGTTCACCAACCTGAAGGT GTACCACGACATCAAGGACATTACCGCCCGGAAAGAGATTATTGAGAACGCCGAGCTGCTGGAT CAGATTGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAAGAACTGACCAATC TGAACTCCGAGCTGACCCAGGAAGAGATCGAGCAGATCTCTAATCTGAAGGGCTATACCGGCAC CCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTGTGGCACACCAACGACAAC CAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGTCCCAGCAGAAAG AGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAAGCTTCATCCA GAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCATTATCGAG CTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGATGCAGAAGCGGAACC GGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGCAAAGAGAACGCCAAGTACCT GATCGAGAAGATCAAGCTGCACGACATGCAGGAAGGCAAGTGCCTGTACAGCCTGGAAGCCATC CCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGAGGTGGACCACATCATCCCCAGAAGCG TGTCCTTCGACAACAGCTTCAACAACAAGGTGCTCGTGAAGCAGGAAGAAAACAGCAAGAAGGG CAACCGGACCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAAACCTTCAAG AAGCACATCCTGAATCTGGCCAAGGGCAAGGGCAGAATCAGCAAGACCAAGAAAGAGTATCTGC TGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTCATCAACCGGAACCTGGTGGA TACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTACTTCAGAGTGAACAACCTG GACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCTGCGGCGGAAGTGGAAGTTTA AGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCCTGATCATTGCCAACGCCGA TTTCATCTTCAAAGAGTGGAAGAAACTGGACAAGGCCAAAAAAGTGATGGAAAACCAGATGTTC GAGGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAGATCTTCA TCACCCCCCACCAGATCAAGCACATTAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGA CAAGAAGCCTAATAGAGAGCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGACAAGGGC AACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGACAATGACAAGCTGAAAAAGC T GAT CAACAAGAG С С С C GAAAAG CTGCTGATGTAC СAC CAC GAC С С С CAGAC С TAC CAGAAAC T GAAGCTGATTATGGAACAGTACGGCGACGAGAAGAATCCCCTGTACAAGTACTACGAGGAAACC GGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGTGATCAAGAAGATTAAGTATT ACGGCAACAAACTGAACGCCCATCTGGACATCACCGACGACTACCCCAACAGCAGAAACAAGGT CGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGGACAATGGCGTGTACAAGTTC

- 69 042798

G T GAG C G T GAAGAAT CTGGATGTGAT CAAAAAAGAAAAC TAG TAG GAAG T GAATAG CAAG T G С T ATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTTATCGCCTCCTTCTACAA CAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGAACAACGACCTGCTG AAG C G GAT C GAAG T GAACAT GAT C GAGAT СAC С TAC C G C GAG TAC С T G GAAAACAT GAAC GACA AGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAGAGCATTAAGAAGTACAGCAC AGACATTCTGGGCAACCTGTATGAAGTGAAATCTAAGAAGCACCCTCAGATCATCAAAAAGGGC aaaaqqccqqcqqccacqaaaaaqqccqqccaqqcaaaaaaqaaaaaqg gatcctacccatacg atgttccagattacgcttacccatacgatgttccagattacgcttacccatacgatgttccaga ttacgcttaagaattcctagagctcgctgatcagcctcgactgtgccttctagttgccagccat ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttc ctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggg gtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggaggtaccga gggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataatt ggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataat ttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaac ttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgAGAACTGG TGGGAAATGGTCTAGgttttagtactctggaaacagaatctactaaaacaaggcaaaatgccgt gtttatctcgtcaacttgttggcgagatttttgcggccgcaggaacccctagtgatggagttgg ccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgccc gggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatg cggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagta cgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctaca cttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccg gctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacg gtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaa caacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggccta ttggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgttt acaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgac acccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagaca agctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcg agacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctt agacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaat acattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaa aggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcc ttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgc

- 70 042798 acgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattg acgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactc accagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccata accatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaa ccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaa tgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggagg cggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccc tcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacaga tcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatat actttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgat aatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggta actggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccacc acttcaagaactctgtagcaccgcctacatacctcgctctgetaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcg cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccg aactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgga caggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaac gcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgat gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggc cttttgctggccttttgctcacatgt pDO242 (SaCas9, применяемая во всех объектах с JCR89/91 и объектах с JCR157/160 для работы in vitro ; SaCas9 выделена верхним регистром) (SEQ ID NO: 83) ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattt tttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggt tgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagg gcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgac ggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggc gctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgcta cagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcct

- 71 042798 cttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc agggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcacta tagggcgaattgggtacCtttaattctagtactatgcaTgcgttgacattgattattgactagt tattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacat aacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtattta cggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacg tcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatc aatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatg ggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccatt gacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaact accggtgccaccATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGGGT ATGGGATTATTGACTATGAAACAAGGGACGTGATCGACGCAGGCGTCAGACTGTTCAAGGAGGC CAACGTGGAAAACAATGAGGGACGGAGAAGCAAGAGGGGAGCCAGGCGCCTGAAACGACGGAGA AGGCACAGAATCCAGAGGGTGAAGAAACTGCTGTTCGATTACAACCTGCTGACCGACCATTCTG AGCTGAGTGGAATTAATCCTTATGAAGCCAGGGTGAAAGGCCTGAGTCAGAAGCTGTCAGAGGA AGAGTTTTCCGCAGCTCTGCTGCACCTGGCTAAGCGCCGAGGAGTGCATAACGTCAATGAGGTG GAAGAGGACACCGGCAACGAGCTGTCTACAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGG AAGAGAAGTATGTCGCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTC AATTAATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGCTGAAAGTGCAGAAG GCTTACCACCAGCTGGATCAGAGCTTCATCGATACTTATATCGACCTGCTGGAGACTCGGAGAA CCTACTATGAGGGACCAGGAGAAGGGAGCCCCTTCGGATGGAAAGACATCAAGGAATGGTACGA GATGCTGATGGGACATTGCACCTATTTTCCAGAAGAGCTGAGAAGCGTCAAGTACGCTTATAAC GCAGATCTGTACAACGCCCTGAATGACCTGAACAACCTGGTCATCACCAGGGATGAAAACGAGA AAC T G GAATACTAT GAGAAGT T СCAGAT CATCGAAAACGT GT T TAAGCAGAAGAAAAAGСС TAC ACTGAAACAGATTGCTAAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACA AG СAC T G GAAAAC CAGAG T T СAC СAAT С T GAAAG T G TAT CAC GATAT TAAG GACAT CACAG CAC GGAAAGAAATCATTGAGAACGCCGAACTGCTGGATCAGATTGCTAAGATCCTGACTATCTACCA GAGCTCCGAGGACATCCAGGAAGAGCTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATC GAACAGATTAGTAATCTGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATCAATC TGATTСTGGATGAGCTGTGGCATACAAACGACAATCAGATTGCAATСTTTAACCGGCTGAAGCT GGTCCCAAAAAAGGTGGACCTGAGTCAGCAGAAAGAGATCCCAACCACACTGGTGGACGATTTC ATTCTGTCACCCGTGGTCAAGCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCA AGAAGTACGGCCTGCCCAATGATATCATTATCGAGCTGGCTAGGGAGAAGAACAGCAAGGACGC AC AGAAGAT GAT C AAT GAGAT G C AGAAAC GAAAC C G G C AGAC C AAT GAAC G C AT T GAAGAGAT T

- 72 042798

ATCCGAACTACCGGGAAAGAGAACGCAAAGTACCTGATTGAAAAAATCAAGCTGCACGATATGC AGGAGGGAAAGTGTCTGTATTCTCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAATCCATT СAACTAGGAGGTCGATCATATTATCCCCAGAAGCGTGTCCTTCGAGAAT T СС T T TAACAACAAG GTGCTGGTCAAGCAGGAAGAGAACTCTAAAAAGGGCAATAGGACTCCTTTCCAGTACCTGTCTA GTTCAGATTCCAAGATСTСTTACGAAACСTTTAAAAAGCACATTСTGAATСTGGCCAAAGGAAA GGGCCGCATCAGCAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACAGATTCTCC GTCCAGAAGGATTTTATTAACCGGAATCTGGTGGACACAAGATACGCTACTCGCGGCCTGATGA ATCTGCTGCGATCCTATTTCCGGGTGAACAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGG GTTCACATCTTTTCTGAGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCAC CATGCCGAAGATGCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGAGTGGAAAAAGCTGG ACAAAGCCAAGAAAGTGATGGAGAACCAGATGTTCGAAGAGAAGCAGGCCGAATCTATGCCCGA AAT C GAGACAGAACAG GAG TAGAAG GAGAT T T T CAT СAC T С С T CAC CAGAT CAAG CATAT CAAG GATTTCAAGGACTACAAGTACTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGATCAATG AC AC CCTGTATAG T AC AAGAAAAGAC GAT AAG G G GAAT AC CCTGATTGT GAAC AAT С T GAAC G G AC T G TAC GACAAAGATAAT GACAAG С T GAAAAAG С T GAT CAACAAAAG T С С C GAGAAG С T G С T G ATGTACCACCATGATCCTCAGACATATCAGAAACTGAAGCTGATTATGGAGCAGTACGGCGACG AGAAGAAC С СAC T G TATAAG TAG TAT GAAGAGAC T G G GAAC TAC С T GAC CAAG TATAG СAAAAA GGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATGGGAACAAGCTGAATGCCCATCTGGAC ATCACAGACGATTACCCTAACAGTCGCAACAAGGTGGTCAAGCTGTCACTGAAGCCATACAGAT TCGATGTCTATCTGGACAACGGCGTGTATAAATTTGTGACTGTCAAGAATCTGGATGTCATCAA AAAGGAGAAC TAG TAT GAAGT GAATAGCAAGT GC TAGGAAGAGGC TAAAAAGC T GAAAAAGAT T AGCAACCAGGCAGAGTTCATCGCCTCCTTTTACAACAACGACCTGATTAAGATCAATGGCGAAC TGTATAGGGTCATCGGGGTGAACAATGATCTGCTGAACCGCATTGAAGTGAATATGATTGACAT СAC T TAC C GAGAG TAT С T G GAAAACAT GAAT GATAAG CGCCCCCCTC GAAT TAT СAAAACAAT T G С С T С TAAGAC T CAGAG TAT CAAAAAG TACT C AACCGACAT T С T GGGAAACС T GTAT GAGGT GA AGAGCAAAAAGCACCCTCAGATTATCAAAAAGGGCagcggaggcaagcgtcctgctgctactaa gaaagctggtcaagctaagaaaaagaaaggatcctacccatacgatgttccagattacgcttaa gaattcctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttg cccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaat gaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagg acagcaagggggaggattgggaagagaatagcaggcatgctggggaggtagcggccgcCCgcgg tggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcat agctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcat aaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactg cccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgggga gaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgt

- 73 042798 tcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggg gataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccg cgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag tcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctc gtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaa gcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgt cttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatta gcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacac tagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggt agctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcaga ttacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctag atccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctg acagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccat agttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagt gctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccag ccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattg ttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgct acaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgat caaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgat cgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattct cttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcc acatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaagg atcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcat cttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggg aataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatt tatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaatag gggttccgcgcacatttccccgaaaagtgccac pJRHl (SaCas9, применяемая для всех объектов с JCR179/183, SaCas9 выделена верхним регистром ; NLS is in lower case, bold and underline) (SEQ ID NO: 84)

- 74 042798 gcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgac ggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggc gctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgcta cagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcct cttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc agggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcacta tagggcgaattgggtacctttaattctagtactatgcatgcgttgacattgattattgactagt tattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacat aacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtattta cggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacg tcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatc aatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatg ggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccatt gacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaact accggtgccaccatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagccA AGCGGAACTACATCCTGGGCCTGGACATCGGCATCACCAGCGTGGGCTACGGCATCATCGACTA CGAGACACGGGACGTGATCGATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAAC GAGGGCAGGCGGAGCAAGAGAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGA GAGTGAAGAAGCTGCTGTTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAA CCCCTACGAGGCCAGAGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCC CTGCTGCACCTGGCCAAGAGAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGCA ACGAGCTGTCCACCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGGC CGAACTGCAGCTGGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAG ACCAGCGACTACGTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGG ACCAGAGCTTCATCGACACCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACC TGGCGAGGGCAGCCCCTTCGGCTGGAAGGACATCAAAGAATGGTACGAGATGCTGATGGGCCAC TGCACCTACTTCCCCGAGGAACTGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACG С С С T GAAC GAG С T GAACAAT С T C G T GAT GAG GAG G GAG GAGAAC GAGAAG С T G GAATAT TAG GA GAAG T T С C AGAT CATC GAGAAC G T G T T C AAG C AGAAGAAGAAG С С С AC С С T GAAG C AGAT C G С C AAAGAAATCCTCGTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCG AG T T СAC СAAC С T GAAG G T G TAC CAC GACAT CAAGGACAT TACCGCCCGGAAAGAGATTAT T GA GAACGCCGAGCTGCTGGATCAGATTGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATC CAG GAAGAAC T GAC CAAT C T GAAC T C C GAG C T GAC C CAG GAAGAGAT C GAG CAGAT C T C TAAT C

- 75 042798

TGAAGGGCTATACCGGCACCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCT GTGGCACACCAACGACAACCAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTG GACCTGTCCCAGCAGAAAGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCG TGAAGAGAAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCC CAACGACATCATTATCGAGCTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAAC GAGATGCAGAAGCGGAACCGGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGCA AAGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAAGGCAAGTGCCT GTACAGCCTGGAAGCCATCCCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGAGGTGGAC CACATCATCCCCAGAAGCGTGTCCTTCGACAACAGCTTCAACAACAAGGTGCTCGTGAAGCAGG AAGAAAACAGCAAGAAGGGCAACCGGACCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGAT CAGCTACGAAACCTTCAAGAAGCACATCCTGAATCTGGCCAAGGGCAAGGGCAGAATCAGCAAG ACCAAGAAAGAGTATCTGCTGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTCA TCAACCGGAACCTGGTGGATACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTA CTTCAGAGTGAACAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCTG CGGCGGAAGTGGAAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCC TGATCATTGCCAACGCCGATTTCATCTTCAAAGAGTGGAAGAAACTGGACAAGGCCAAAAAAGT GATGGAAAACCAGATGTTCGAGGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAG GAG T AC AAAGAGAT С T T C AT С AC С С С С С AC C AGAT C AAG C AC AT T AAG GAC T T C AAG GAC TACA AGTACAGCCACCGGGTGGACAAGAAGCCTAATAGAGAGCTGATTAACGACACCCTGTACTCCAC CCGGAAGGACGACAAGGGCAACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGAC AATGACAAGCTGAAAAAGCTGATCAACAAGAGCCCCGAAAAGCTGCTGATGTACCACCACGACC С С CAGAC С TAC CAGAAAC T GAAG CTGATTATG GAACAG TAC G G C GAC GAGAAGAAT CCCCTGTA CAAGTACTACGAGGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGTG AT CAAGAAGAT TAAG TAT TAC G G CAACAAAC T GAAC GCCCATCTG GACAT СAC C GAC GAC TAC C CCAACAGCAGAAACAAGGTCGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGGA СAAT G G C G T G TAGAAG T T C G T GAC C G T GAAGAAT CTGGATGTGAT CAAAAAAGAAAAC TAG TAG GAAGTGAATAGCAAGTGCTATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGT TTATCGCCTCCTTCTACAACAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGG C G T GAACAAC GAC С T G С T GAAC C G GAT C GAAG T GAACAT GAT C GACAT СAC С TAC C G C GAG TAG С T G GAAAACAT GAAC GACAAGAG G С С С С С CAG GAT CAT TAAGACAAT C G С С T С CAAGAC С CAGA GCATTAAGAAGTACAGCACAGACATTCTGGGCAACCTGTATGAAGTGAAATCTAAGAAGCACCC T CAGAT CAT CAAAAAG G G Caaaaqqccqqcqqccacqaaaaaqqccqqccaqqcaaaaaaqaaa aaqggatcctacccatacgatgttccagattacgcttaagaattcctagagctcgctgatcage ctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgacc

- 76 042798 gaatagcaggcatgctggggaggtagcggccgcccgcggtggagctccagcttttgttcccttt agtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgtta tccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaa tgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgt cgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctc ttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct cactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgag caaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggct ccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagga ctataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgc cgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacg ctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacg acttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgc tacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgc getctgetgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca ccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctca agaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaaggg attttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtt ttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtga ggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtag ataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccac gctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtgg tcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagt tcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgt cgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccat gttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagat gcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgag ttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctc atcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagtt cgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgg gtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga atactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcg gatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaa agtgccac

- 77 042798

NLS sequence in PT366 (SEQ ID NO: 85)

AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG pDO203 - typical scaffold for gRNA cloning with SpCas9;

JCR94 и JCR99 помещали в сайт, выделенный жирным шрифтом, для тестирования в клетках, с последующим получением mRNA для получения мыши hDMD-flenbTa52/mdx (SEQ ID NO: 86) ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattt tttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggt tgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagg gcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgac ggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggc gctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgcta cagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcct cttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc agggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcacta tagggcgaattgggtaccgagggcctatttcccatgattccttcatatttgcatatacgataca aggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatac gtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggac tatcatatgcttatcgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaag gacgaaacaccGGGTCTTCGAGAAGACCTgttttagagctagaaatagcaagttaaaataaggc tagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttccgcggtggagctcca gcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcc tgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaa gcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttcc agtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggttt gcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcgg cgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcag gaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggc gtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtgg cgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgct ttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgt gtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgag gtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggaca

- 78 042798 gtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgat ccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcag aaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa aactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaa attaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacca atgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctga ctccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatga taccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggc cgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaa gctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg tggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagt tасаtgatcccccatgttgtgcaaaaaagcggttagetccttcggtcctccgatcgttgtсада agtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtca tgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtg tatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcaga actttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgc tgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttacttt caccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcg acacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggtt attgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcg cacatttccccgaaaagtgccac

Claims (18)

1. A DNA targeting composition comprising a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain encoded by a polynucleotide containing a nucleotide sequence, presented in SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain, which is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (ii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (iii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (iv) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; (v) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (vi) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (vii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; (viii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing - 79 042798 nucleotide sequence shown in SEQ ID NO: 4; (ix) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (x) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15; (xi) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; and (xii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: :42. 2. The DNA targeting composition of claim 1, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO : 1, and a second gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (ii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (iii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; and (iv) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: :42. 3. A DNA targeting composition according to claim 1 or 2, further comprising a protein associated with regularly spaced short palindromic repeats (Cas). 4. The DNA targeting composition of claim 3, wherein the Cas protein comprises a Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25). 5. An isolated polynucleotide comprising a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (ii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (iii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (iv) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; (v) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; - 80 042798 (vi) a first gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14 and a second gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (vii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; (viii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (ix) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (x) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15; (xi) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; and (xii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: :42. 6. The isolated polynucleotide of claim 5, wherein the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) a first gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (ii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (iii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; and (iv) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: :42. 7. An isolated polynucleotide according to claim 5 or 6, further encoding a protein associated with regularly spaced short palindromic repeats (Cas). 8. The isolated polynucleotide of claim 7, wherein the Cas protein comprises a Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25). 9. A vector containing an isolated polynucleotide according to any one of claims 5-8. 10. The vector of claim 9, wherein the vector is constructed to form first and second double strand breaks in the first and second introns flanking exon 51 of the human DMD gene. 11. A vector according to claim 9 or 10, wherein the vector is a viral vector. 12. A vector according to any one of claims 9-11, wherein the vector contains a tissue-specific promoter operably linked to a nucleotide sequence encoding a first gRNA molecule, a second gRNA molecule and/or a Cas9 molecule. 13. A cell comprising a DNA targeting composition according to any one of claims 1-4, or an isolated polynucleotide according to any one of claims 5-8, or a vector according to any one of claims 9-12. 14. A kit for providing a deletion of a dystrophin gene segment containing exon 51 or exon 52, where the kit contains a composition for targeting DNA according to any one of claims 1-4, or an isolated polynucleotide according to any one of claims 1-4, or a vector according to any from claims 9-12, or a cage according to claim 13. - 81 042798 15. The kit according to claim 14, further containing instructions for use. 16. A method of providing a deletion of a dystrophin gene segment containing exon 51 or exon 52 in a cell, the method comprising introducing into the cell a DNA targeting composition according to any one of claims 1-4, or an isolated polynucleotide according to any one of claims 5-8 , or a vector according to any one of claims 9-12. 17. A method of providing a deletion of a dystrophin gene segment containing exon 51 or exon 52 in a subject, the method comprising administering to the subject a DNA targeting composition according to any one of claims 1 to 4, or an isolated polynucleotide encoding a DNA targeting composition according to any from claims 5 to 8, or a vector according to any of claims 9 to 12, or a cell according to claim 13. 18. A composition for providing a deletion of a dystrophin gene segment containing exon 51 or exon 52, wherein the composition comprises (a) a first vector containing a polynucleotide sequence encoding a first guide RNA (gRNA) molecule and a polynucleotide sequence encoding a first Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO: 25); and (b) a second vector containing a polynucleotide sequence encoding a second gRNA molecule and a polynucleotide sequence encoding a second Cas9 molecule that recognizes a protospacer adjacent motif (PAM) of either NNGRRT (SEQ ID NO: 24) or NNGRRV (SEQ ID NO : 25), where each of the first and second gRNA molecules contains a targeting domain, the length of which is from 19 to 24 nucleotides, and where the first vector and the second vector are designed to form the first and second double-strand breaks, respectively, in the first intron and second intron, flanking exon 51 or exon 52 of the human DMD gene, thereby providing a deletion of the dystrophin gene segment containing exon 51 or exon 52, and where the first gRNA molecule and the second gRNA molecule are selected from the group consisting of (i) the first gRNA molecule containing a targeting domain , which is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1, and a second gRNA molecule containing a targeting domain, which is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (ii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (iii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (iv) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; (v) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (vi) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; (vii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; (viii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (ix) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 19; - 82 042798 (x) the first gRNA molecule containing the targeting domain, which is encoded by the polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 14, and the second gRNA molecule containing the targeting domain, which is encoded by the polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 15; (xi) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 11, and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 18; and (xii) a first gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 41 and a second gRNA molecule containing a targeting domain that is encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: :42.