Materials and methods for the treatment of Duchenne muscular dystrophy

Abstract

To provide materials and methods for the treatment of Duchenne muscular dystrophy. [Solution] This application relates to a patient with Duchenne muscular dystrophy (DMD), ex This application presents materials and methods for treatment both in vivo and in vivo. In addition, this application presents materials and methods for editing the dystrophin gene in cells by genome editing. This disclosure presents a method for addressing the genetic basis of DMD. By using genome manipulation tools that create permanent changes in the genome that can repair the dystrophin reading frame and restore the activity of the dystrophin protein in just a few treatments, the resulting therapy can correct the underlying gene deletion that causes the disease.

Description

[Technical Field] 【0001】 Technical field This application presents materials and methods for treating patients with Duchenne muscular dystrophy (DMD) both ex vivo and in vivo. In addition, this application presents materials and methods for editing the dystrophin gene in cells by genome editing. 【0002】 Related applications This application claims the interests of U.S. Provisional Application No. 62 / 247,484, filed on 28 October 2015, and U.S. Provisional Application No. 62 / 324,064, filed on 18 April 2016, both of which are incorporated herein by reference in their entirety. 【0003】 Incorporating sequence lists by reference This application is for a computer-readable format (filename: 160101PCT sequence listing _ST25: 286,928,896 bytes; ASCII text file; 2016) This includes a sequence listing (created on October 28, 2018) which is incorporated in whole by reference herein and forms part of this disclosure. [Background technology] 【0004】 background Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disorder that occurs in approximately 1 in 4,000 live male births. Patients are generally diagnosed by age 4 and are wheelchair-bound by age 10. Most patients do not live beyond age 25 due to heart failure and / or respiratory failure. Existing treatments are, at best, palliative. The most common treatment for DMD is steroids, which are used to slow muscle loss. However, because most DMD patients begin steroid administration at a young age, the treatment delays puberty and further contributes to a reduced quality of life for the patients. 【0005】 DMD is caused by mutations in the dystrophin gene (X chromosome: 31,117,228~33,344,609 (Genome Reference Consortium: GRCh38 / hg38)). Dystrophin, with a genomic region spanning 2.2 megabases, is the second largest human gene. The dystrophin gene contains 79 exons, which are processed into 11,000 base pair mRNA, which is translated into a 427 kDa protein. Functionally, dystrophin acts as a linker between actin filaments and the extracellular matrix within muscle fibers. The N-terminus of dystrophin is an actin-binding domain, while the C-terminus interacts with transmembrane scaffolds that anchor muscle fibers to the extracellular matrix. During muscle contraction, dystrophin provides structural support that allows muscle tissue to resist mechanical forces. DMD is caused by a wide variety of mutations in the dystrophin gene that result in premature stop codons, and therefore in the form of truncated dystrophin protein. The truncated dystrophin protein lacks a C-terminus and therefore cannot provide the structural support necessary to resist the stress of muscle contraction. As a result, muscle fibers are torn apart, leading to muscle wasting. 【0006】 Becker muscular dystrophy (BMD) is a less severe form of muscular dystrophy compared to DMD. BMD is also caused by mutations in the dystrophin gene, but the mutations in BMD preserve the dystrophin leading frame. The BMD dystrophin protein contains an internal deletion, but retains both the N-terminal and C-terminal portions. Therefore, the dystrophin protein in BMD, though shorter than the wild-type protein, can still function as a linker between actin filaments and the extracellular matrix. In fact, depending on the size of the internal deletion, BMD patients may exhibit only mild symptoms. Consequently, most research efforts focus on converting severe DMD phenotypes into less severe BMD phenotypes. 【0007】 Genome manipulation refers to strategies and techniques for targeted and specific modification of an organism's genetic information (genome). Genome manipulation has a wide range of possible applications, particularly in the area of ​​human health; correction of genes containing harmful mutations; and is a highly active research area, for example, for applications exploring gene function. Early techniques developed for inserting genes into living cells, such as genetic engineering, were often limited by the random nature of insertion of new sequences into the genome. New genes were typically placed blindly, sometimes inactivating, disrupting, or even causing severely undesirable effects on the function of other genes. Furthermore, these techniques generally lacked any degree of reproducibility, as there was no guarantee that the new sequences would be inserted in the same location in two different cells. More recent genome manipulation strategies, such as ZFN, TALEN, HE, and MegaTAL, enable modification of specific regions of DNA, thereby increasing the accuracy of correction or insertion compared to earlier techniques and providing some degree of reproducibility. Nevertheless, such recent genome manipulation strategies still have limitations. 【0008】 Multiple studies suggest that genomic manipulation is an attractive strategy for treating DMD. One of the earliest methods involved manipulating a mini-dystrophin gene that is less than 4kb and can be packaged into an adeno-associated virus (AAV) vector. This was done using a mouse model (Wang, B., J. Li, and X. Xiao, Proc Natl Acad Sci U). In SA, 2000, Vol. 97 (No. 25): pp. 13714-1379 (Watchko, J. et al., Hum Gene Ther, 2002, Vol. 13 (No. 12): pp. 1451-1460) and in a dog model (Wang, Z. et al., Mol Ther, 2012, Vol. 20 (No. 8): pp. 1501-1507), experiments were conducted. This is a gene replacement therapy that has been investigated in detail, and a Phase I clinical trial suggested that there are problems associated with the immune response to non-self synthetic epitopes (Mendell, JR et al., N Engl J Med, 2010, vol. 363 (no. 15): pp. 1429-1437). 【0009】 More recently, oligo-mediated exon skipping has been used to repair the reading frame within cells in DMD patients. In this strategy, short oligos block the splicing signal found in pre-mRNA, facilitating single-exon skipping. Single-exon skipping allows the transcription mechanism to bypass early stop codons and produce proteins with intact N and C termini. Phase I / II clinical trials have shown that weekly injections of antisense oligos induce exon skipping and dystrophin-positive fibers (Cirak, S. et al., Lancet, 2011, vol. 378 (no. 9791): pp. 595-605). However, a major limitation of this type of treatment is that the drug targets pre-mRNA rather than genomic loci, requiring repeated administration throughout the patient's life. Ongoing Phase II / III clinical trials are evaluating the delivery of AAV-mediated exon-skipping oligos for sustained expression, as well as the delivery of multiple antisense oligos to facilitate multi-exon-skipping strategies. Despite the efforts of researchers and healthcare professionals worldwide attempting to address DMD, and despite the potential of genomic manipulation, there remains an urgent need to develop safe and effective treatments for DMD, the most prevalent and debilitating genetic disorder. [Prior art documents] [Non-patent literature] 【0010】 [Non-Patent Document 1] Wang, B., J. Li, and X. Xiao, Proc Natl Acad Sci U S A, 2000, 97(25): 13714-13719 [Non-Patent Document 2] Watchko, J. et al., Hum Gene Ther, 2002, 13(12): 1451-1460 [Non-Patent Document 3] Wang, Z. et al., Mol Ther, 2012, 20(8): 1501-1507 [Non-Patent Document 3] Mendell, J.R. et al., N Engl J Med, 2010, 363(15): 1429-1437 [Non-Patent Document 4] Cirak, S. et al., Lancet, 2011, 378(9791): 595-605 [Summary of the Invention] [Means for Solving the Problems] 【0011】 Abstract This disclosure presents a method for addressing the genetic basis of DMD. By using a genome engineering tool that creates a permanent change in the genome that can repair the reading frame of dystrophin and restore the activity of dystrophin protein with only a single treatment, the resulting therapy can correct the underlying gene defect that causes the disease. 【0012】 In this specification, ex vivo and in vivo cell methods for creating permanent changes to the genome by genome editing to delete, insert, or replace (delete and insert) one or more exons within the dystrophin gene or abnormal intron splice acceptor or donor sites, repair the dystrophin reading frame, and restore the activity of the dystrophin protein, which can be used to treat Duchenne muscular dystrophy (DMD), are presented. This specification also presents components, kits, and compositions for performing such methods. Cells produced by such methods are also presented. 【0013】 In this specification, a method for editing the dystrophin gene in human cells by genome editing, comprising introducing one or more deoxyribonucleic acid (DNA) endonucleases into the human cells to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene, the SSBs or DSBs resulting in a permanent deletion, insertion, or replacement of one or more exons within the dystrophin gene or abnormal intron splice acceptor or donor sites, and leading to the repair of the dystrophin reading frame and the restoration of the activity of the dystrophin protein, is presented. The human cells can be muscle cells or muscle progenitor cells. 【0014】 This specification also presents an ex vivo method for treating a patient (e.g., a human) with Duchenne muscular dystrophy (DMD), comprising: i) creating DMD patient-specific induced pluripotent stem cells (iPSCs); ii) editing the dystrophin gene within or near the iPSCs; iii) differentiating the genome-edited iPSCs into Pax7+ muscle progenitor cells; and iv) implanting the Pax7+ muscle progenitor cells into the patient. 【0015】 The steps for creating patient-specific induced pluripotent stem cells (iPSCs) may include: a) isolating somatic cells from the patient; and b) introducing a set of pluripotency-related genes into the somatic cells to induce them to become pluripotent stem cells. The somatic cells may be fibroblasts. The set of pluripotency-related genes is one or more genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC. 【0016】 The step of editing within or near the dystrophin gene of an iPSC may include introducing one or more deoxyribonucleic acid (DNA) endonucleases into the iPSC to produce one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene, resulting in the permanent deletion, insertion, or replacement of one or more exons or abnormal intron splice acceptor or donor sites within or near the dystrophin gene, thereby resulting in the repair of the dystrophin reading frame and the repair of the activity of the dystrophin protein. 【0017】 The steps for differentiating genome-edited iPSCs into Pax7+ myoprimordial cells may include contacting the genome-edited iPSCs with a specific culture medium formulation containing a small molecule drug; overexpression of the trans gene; or serum depletion. 【0018】 The step of implanting Pax7+ myoprimordial cells into a patient may include implanting the Pax7+ myoprimordial cells into the patient by local injection into the desired muscle. 【0019】 This specification also presents in vivo methods for treating patients (e.g., humans) with Duchenne muscular dystrophy (DMD), comprising the step of editing the dystrophin gene in the patient's cells. The cells may be muscle cells or muscle progenitor cells. 【0020】 The step of editing dystrophin in a patient's cells may include introducing one or more deoxyribonucleic acid (DNA) endonucleases into the patient's cells to produce one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the dystrophin gene, resulting in the permanent deletion, insertion, or replacement of one or more exons or abnormal intron splice acceptor or donor sites in or near the dystrophin gene, thereby resulting in the repair of the dystrophin reading frame and the repair of the activity of the dystrophin protein. 【0021】 One or more DNA endonucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr 3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonucleases; these may be homologs, recombinants of these naturally occurring molecules, codon-optimized versions of these, modified forms of these, and any combination of the foregoing. 【0022】 The method may include the step of introducing one or more polynucleotides encoding one or more DNA endonucleases into a cell. The method may include the step of introducing one or more ribonucleic acids (RNAs) encoding one or more DNA endonucleases into a cell. One or more polynucleotides or one or more RNAs may be one or more modified polynucleotides or one or more modified RNAs. One or more DNA endonucleases may be one or more proteins or polypeptides. 【0023】 The method may further include the step of introducing one or more guide ribonucleic acids (gRNAs) into cells. The one or more gRNAs are single-molecule guide RNAs (sgRNAs). The one or more gRNAs, or one or more sgRNAs, are one or more modified gRNAs, or one or more modified sgRNAs. One or more DNA endonucleases can be pre-complexed with the one or more gRNAs, or one or more sgRNAs. 【0024】 The method may further include the step of introducing a polynucleotide donor template containing at least a portion of the wild-type dystrophin gene or cDNA into cells. At least a portion of the wild-type dystrophin gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or may include at least a portion of the dystrophin gene or the entire cDNA.At least a portion of the wild-type dystrophin gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, Exon 43, Exon 44, Exon 45, Exon 46, Exon 47, Exon 48, Exon 49, Exon 50, Exon 51, Exon 52, Exon 53, Exon 54, Exon 55, Exon 56, Exon 57, Exon 58, Exon 59, Exon 60, Exon 61, Exon 62, Exon 63, Exon 64, Exon 65, Exon 66, Exon 67, Exon 68, Exon 69, Exon 70, Exon 71, Exon 72, Exon 73, Exon 74, Exon 75, Exon 76, Exon 77, Exon 78, Exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA. The donor template can be a single-stranded or double-stranded polynucleotide. 【0025】 The method may further include the step of introducing one or more guide ribonucleic acids (gRNAs) into cells. The one or more DNA endonucleases may be one or more Cas9 or Cpf1 endonucleases that produce an SSB or DSB, which is a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs) consisting of a first SSB or DSB break in the 5' locus and a second SSB or DSB break in the 3' locus, resulting in a permanent deletion or replacement of one or more exons or abnormal intron splice acceptor or donor sites between the 5' and 3' locus in or near the dystrophin gene, resulting in the repair of the dystrophin leading frame and the repair of the dystrophin protein activity. One gRNA may create a pair of SSBs or DSBs. A single gRNA may contain a spacer sequence complementary to the 5' locus, the 3' locus, or the segment between the 5' and 3' locus. The first gRNA may contain a spacer sequence complementary to the 5' locus segment, and the second gRNA may contain a spacer sequence complementary to the 3' locus segment. 【0026】 One or more gRNAs can be one or more single-molecule guide RNAs (sgRNAs). One or more gRNAs, or one or more sgRNAs, can be one or more modified gRNAs, or one or more modified sgRNAs. One or more DNA endonucleases can be pre-complexed with one or more gRNAs, or one or more sgRNAs. 【0027】 A deletion in chromosomal DNA may exist between the 5' locus and the 3' locus. 【0028】 A deletion can be a deletion of a single exon. A deletion of a single exon can be a deletion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, or exon 53. The 5' locus can be proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53. The 3' locus can be proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53. The 5' locus can be proximal to the 5' boundary of a single exon selected from the group consisting of exons 2, 8, 43, 44, 45, 46, 50, 51, 52, and 53, and the 3' locus can be proximal to these 3' boundaries. Proximal to the exon boundaries may include splice donors and acceptors around adjacent introns. 【0029】 A deletion can be a multiexon deletion. A multiexon deletion can be a deletion of exons 45-53 or exons 45-55. The 5' locus can be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55. The 3' locus can be proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55. The 5' locus can be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55, and the 3' locus can be proximal to these 3' boundaries. Proximity to the exon boundary may include splice donors and acceptors around adjacent introns. 【0030】 A substitution of chromosomal DNA may exist between the 5' locus and the 3' locus. The substitution may be a single exon substitution. A single exon substitution may be a substitution of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 5' locus may be proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 3' locus may be proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 5' locus may be proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70, and the 3' locus may be proximal to these 3' boundaries. Proximal to the exon boundary may include splice donors and acceptors around adjacent introns or adjacent exons. 【0031】 The replacement can be a multiexon replacement. A multiexon replacement can be a replacement of exons 45-53 or exons 45-55. The 5' locus can be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 or exons 45-55. The 3' locus can be proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 or exons 45-55. The 5' locus can be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 or exons 45-55, and the 3' locus can be proximal to these 3' boundaries. Proximity to an exon boundary may include splice donors and acceptors around adjacent introns or adjacent exons. 【0032】 The method may further include the step of introducing a polynucleotide donor template containing at least a portion of the wild-type dystrophin gene or cDNA into cells, the replacement being carried out by homology-driven repair (HDR). 【0033】 At least a portion of the wild-type dystrophin gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or may include at least a portion of the dystrophin gene or the entire cDNA.At least a portion of the wild-type dystrophin gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, This may include exons 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the dystrophin gene or the entire cDNA. 【0034】 The method may further include the step of introducing a guide ribonucleic acid (gRNA) and a polynucleotide donor template containing at least a portion of the wild-type dystrophin gene into a cell. One or more DNA endonucleases may be one or more Cas9 or Cpf1 endonucleases that produce an SSB or DSB in or near the dystrophin gene that facilitates the insertion of a novel sequence derived from the polynucleotide donor template into the chromosomal DNA in the locus, resulting in the permanent insertion or correction of one or more exons or abnormal intron splice acceptor or donor sites in or near the dystrophin gene, thereby repairing the dystrophin leading frame and restoring the activity of the dystrophin protein. The gRNA may contain a spacer sequence complementary to the segment of the locus. 【0035】 The method may further include the step of introducing one or more guide ribonucleic acids (gRNAs) and a polynucleotide donor template containing at least a portion of the wild-type dystrophin gene into a cell. One or more DNA endonucleases may be one or more Cas9 or Cpf1 endonucleases that produce an SSB or DSB in or near the dystrophin gene, which is a pair of single-strand breaks (SSBs) or double-strand breaks, a first break in the 5' locus and a second break in the 3' locus, facilitating the insertion of a novel sequence derived from the polynucleotide donor template into chromosomal DNA between the 5' and 3' locus, within or near the dystrophin gene, resulting in the permanent insertion or correction of a splice acceptor or donor site of one or more exons or abnormal introns within or near the dystrophin gene, resulting in the repair of the dystrophin leading frame and the repair of the activity of the dystrophin protein. 【0036】 A single gRNA can generate a pair of SSBs or DSBs. A single gRNA may contain spacer sequences complementary to the 5' locus, the 3' locus, or the segment between the 5' and 3' locus. The first gRNA may contain a spacer sequence complementary to the 5' locus segment, and the second gRNA may contain a spacer sequence complementary to the 3' locus segment. 【0037】 One or more gRNAs can be one or more single-molecule guide RNAs (sgRNAs). One or more gRNAs, or one or more sgRNAs, can be one or more modified gRNAs, or one or more modified sgRNAs. One or more DNA endonucleases can be pre-complexed with one or more gRNAs, or one or more sgRNAs. 【0038】 An insertion can exist between the 5' locus and the 3' locus. 【0039】 An insertion can be a single exon insertion. A single exon insertion can be an insertion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. A 5' locus or 3' locus can be proximal to the boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and exon 70. Proximal to an exon boundary may include splice donors and acceptors around an adjacent intron or adjacent exon. 【0040】 An insertion can be a multiexon insertion. A multiexon insertion can be an insertion of exons 45–53 or exons 45–55. A 5' locus or 3' locus can be proximal to the boundary of multiple exons selected from the group consisting of exons 45–53 or exons 45–55. Proximal to an exon boundary may include splice donors and acceptors around adjacent introns. 【0041】 At least a portion of the wild-type dystrophin gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or may include at least a portion of the dystrophin gene or the entire cDNA.At least a portion of the wild-type dystrophin gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, This may include exons 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the dystrophin gene or the entire cDNA. 【0042】 Insertion or correction may be performed by homology-guided restoration (HDR). 【0043】 The donor template can be a single-stranded or double-stranded polynucleotide. 【0044】 The mRNA, gRNA, and donor templates of Cas9 or Cpf1 can each be formulated into separate lipid nanoparticles, or they can all be co-formulated into lipid nanoparticles. 【0045】 Cas9 or Cpf1 mRNA can be formulated into lipid nanoparticles, and both the gRNA and donor template can be delivered to cells via adeno-associated virus (AAV) vectors. 【0046】 Cas9 or Cpf1 mRNA can be formulated into lipid nanoparticles, gRNA can be delivered to cells by electroporation, and donor templates can be delivered to cells by adeno-associated virus (AAV) vectors. 【0047】 The dystrophin gene can be located on the X chromosome: 31,117,228~33,344,609 (Genome Reference Consortium: GRCh38 / hg38). 【0048】 This specification also presents one or more guide ribonucleic acids (gRNAs) for editing the dystrophin gene in cells derived from patients with DMD. One or more gRNAs and / or sgRNAs may include spacer sequences selected from the group of nucleic acid sequences within sequence numbers 1 to 1,410,472 of the sequence listing. One or more gRNAs may be one or more single-molecule guide RNAs (sgRNAs). One or more gRNAs, or one or more sgRNAs, may be one or more modified gRNAs, or one or more modified sgRNAs. 【0049】 This specification presents cells modified by the preceding method to permanently delete or correct one or more exons or abnormal intron splice acceptor or donor sites within the dystrophin gene, thereby repairing the dystrophin reading frame and restoring the activity of the dystrophin protein. This specification further presents a method for improving DMD by administering cells modified by the preceding method to DMD patients. 【0050】 It should be understood that the inventions described herein are not limited to the examples summarized in this abstract. A variety of other embodiments are also described and illustrated herein. 【0051】 Various aspects of materials and methods for treating DMD disclosed and described herein can be better understood by referring to the accompanying drawings. [Brief explanation of the drawing] 【0052】 [Figure 1A] Figure 1A shows the plasmid (CTx-1) containing the codon optimization gene for S. pyogenes Cas9 endonuclease. The CTx-1 plasmid also contains a gRNA scaffold sequence, which includes a 20 bp spacer sequence from sequences listed as sequence numbers 1-467,030 in the sequence listing, or a 19 bp spacer sequence from sequences listed as sequence numbers 1,410,430-1,410,472 in the sequence listing. 【0053】 [Figure 1B] Figure 1B shows plasmid (CTx-2) containing different codon-optimizing genes for S. pyogenes Cas9 endonuclease. The CTx-2 plasmid also contains a gRNA scaffold sequence, which includes a 20 bp spacer sequence from sequences listed as sequence numbers 1-467,030 in the sequence listing, or a 19 bp spacer sequence from sequences listed as sequence numbers 1,410,430-1,410,472 in the sequence listing. 【0054】 [Figure 1C] Figure 1C shows a plasmid (CTx-3) containing yet another different codon-optimizing gene for S. pyogenes Cas9 endonuclease. The CTx-3 plasmid also contains a gRNA scaffold sequence, which includes a 20 bp spacer sequence from sequences listed in sequence numbers 1–467,030 of the sequence listing, or a 19 bp spacer sequence from sequences listed in sequence numbers 1,410,430–1,410,472 of the sequence listing. 【0055】 [Figure 2A] Figure 2A is an illustration of a Type II CRISPR / Cas system. 【0056】 [Figure 2B] Figure 2B is an illustration of a Type II CRISPR / Cas system. 【0057】 [Figure 3A] Figure 3A illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T, targeting exons 45, 51, and 53 of the dystrophin gene. 【0058】 [Figure 3B] Figure 3B illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T, targeting exons 55 and 70 of the dystrophin gene. 【0059】 [Figure 4A] Figure 4A illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T, targeting the splice acceptors in exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the dystrophin gene. 【0060】 [Figure 4B] Figure 4B illustrates the cleavage efficiency of N. meningitides, S. thermophiles, and S. aureus gRNAs in HEK293T, targeting the splice acceptors in exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the dystrophin gene. 【0061】 [Figure 4C]Figure 4C illustrates the cleavage efficiency of Cpf1 gRNA in HEK293T, targeting the splice acceptors in exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the dystrophin gene. 【0062】 [Figure 5A] Figures 5A-B illustrate the cleavage efficiency and splice acceptor knockout efficiency of S.pyogenes gRNA in HEK293T, targeting exons 51, 45, 53, 44, 46, 52, 50, 43, and 55 of the dystrophin gene. [Figure 5B] Figures 5A-B illustrate the cleavage efficiency and splice acceptor knockout efficiency of S.pyogenes gRNA in HEK293T, targeting exons 51, 45, 53, 44, 46, 52, 50, 43, and 55 of the dystrophin gene. 【0063】 [Figure 6] Figure 6 illustrates the cleavage efficiency and splice acceptor knockout efficiency of N. meningitides (NM), S. thermophiles (ST), and S. aureus (SA) gRNAs in HEK293T, targeting exons 51, 45, 53, 44, 46, 52, 50, 43, and 55 of the dystrophin gene. 【0064】 [Figure 7A] Figure 7A illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T cells, where the gRNA targets the region surrounding exon 52 of the dystrophin gene. 【0065】 [Figure 7B] Figure 7B illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T cells, showing that the gRNA targets the region surrounding exons 44, 45, and 54 of the dystrophin gene. 【0066】 [Figure 8A] Figure 8A illustrates the cleavage efficiency of S. pyogenes gRNA in iPSCs, showing how the gRNA targets the region surrounding exon 52 of the dystrophin gene. 【0067】 [Figure 8B] Figure 8B illustrates the cleavage efficiency of S. pyogenes gRNA in iPSCs, showing that the gRNA targets the region surrounding exons 44, 45, and 54 of the dystrophin gene. 【0068】 [Figure 9] Figure 9 illustrates a comparison of the cleavage efficiency of S. pyogenes gRNA in HEK293T cells and iPSCs, where the gRNA targets the region surrounding exons 44, 45, 52, and 54 of the dystrophin gene. 【0069】 [Figure 10AB] Figures 10A, 10B, and 10C illustrate the clonal analysis of clonal deletion events. [Figure 10C] Figures 10A, 10B, and 10C illustrate the clonal analysis of clonal deletion events. 【0070】 [Figure 11] Figures 11A and 11B illustrate the Sanger sequencing of the Δ52 clone. 【0071】 [Figure 12A] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcribed (IVT) gRNA screening. [Figure 12B] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcribed (IVT) gRNA screening. [Figure 12C]Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcribed (IVT) gRNA screening. [Figure 12D] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcribed (IVT) gRNA screening. [Figure 12E] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcribed (IVT) gRNA screening. 【0072】 [Figure 13A] Figure 13A illustrates homology-directed repair (HDR) between exons 45 and 55 of the dystrophin gene. 【0073】 [Figure 13B] Figure 13B shows PCR confirmation of HDR at the dystrophin gene's exon 45-55 locus. 【0074】 [Figure 14A] Figure 14A shows a 3-primer PCR assay. 【0075】 [Figure 14B] Figure 14B shows the results from the 3-primer PCR assay. 【0076】 [Figure 14C] Figure 14C illustrates the data generated from the 3-primer PCR assay. 【0077】 [Figure 15] Figure 15 illustrates five clones possessing the desired Δ45–55 deletion. 【0078】 [Figure 16]Figures 16A-B illustrate the SSEA-4 and TRA-160 staining results for five clones with the desired Δ45-55 deletions. 【0079】 [Figure 17] Figure 17 shows the expression of the internal deletion dystrophin protein for all five edited clones. 【0080】 [Figure 18] Figure 18 shows myosin heavy chain staining of differentiated clone 56. 【0081】 [Figure 19A] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19B] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19C] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19D] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19E] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19F] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19G] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19H] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19I] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19J] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19K] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19L] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19M] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19N] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19O] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19P] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19Q] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19R] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19S] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19T] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19U] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19V] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19W] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19X] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19Y]Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19Z] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19AA] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19BB] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19CC] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19DD] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19EE] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19FF] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19GG] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19HH] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19II] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19JJ] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19KK] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19LL] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19MM] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19NN] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 1900] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19PP] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19QQ] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19RR] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19SS] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19TT] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19UU] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Figure 19VV] Figures 19A–19VV illustrate the results of large-scale lentivirus screening. [Modes for carrying out the invention] 【0082】 A brief explanation of sequence listings Sequence IDs 1-467,030 are a list of 20bp gRNA spacer sequences for targeting the dystrophin gene with S. pyogenes Cas9 endonuclease. 【0083】 Sequence IDs 467,031–528,196 are a list of 20bp gRNA spacer sequences for targeting the dystrophin gene with S. aureus Cas9 endonuclease. 【0084】 Sequence IDs 528,197–553,198 are a list of 24bp gRNA spacer sequences for targeting the dystrophin gene with S. thermophilus Cas9 endonuclease. 【0085】 Sequence IDs 553,199–563,911 are a list of 24bp gRNA spacer sequences for targeting the dystrophin gene with T. denticola Cas9 endonuclease. 【0086】 Sequence IDs 563,912–627,854 are a list of 24bp gRNA spacer sequences for targeting the dystrophin gene with N. meningitides Cas9 endonuclease. 【0087】 Sequence IDs 627,855–1,410,399 are a list of 20–24 bp gRNA spacer sequences for targeting the dystrophin gene in Acidiminoccoccus, Lachnospiraceae, and Franciscella Novicida Cpf1 endonucleases. 【0088】 Sequence numbers 1,410,400 to 1,410,402 are N. meningitides. This is a list of 24bp gRNA spacer sequences for targeting the dystrophin gene with Cas9 endonuclease. 【0089】 Sequence IDs 1,410,403–1,410,429 are a list of 23bp gRNA spacer sequences for targeting the dystrophin gene in Acidiminoccoccus, Lachnospiraceae, and Franciscella Novicida Cpf1 endonucleases. 【0090】 Sequence IDs 1,410,430 to 1,410,472 are a list of 19bp gRNA spacer sequences for targeting the dystrophin gene with S. pyogenes Cas9 endonuclease. 【0091】 Detailed explanation Duchenne muscular dystrophy (DMD) 【0092】 DMD is caused by mutations in the dystrophin gene (X chromosome: 31,117,228~33,344,609 (Genome Reference Consortium: GRCh38 / hg38)). Dystrophin, with a genomic region spanning 2.2 megabases, is the second largest human gene. The dystrophin gene contains 79 exons, which are processed into 11,000 base pair mRNA, which is translated into a 427 kDa protein. Functionally, dystrophin acts as a linker between actin filaments and the extracellular matrix within muscle fibers. The N-terminus of dystrophin is an actin-binding domain, while the C-terminus interacts with the transmembrane scaffold that anchors the muscle fiber to the extracellular matrix. During muscle contraction, dystrophin provides structural support that allows muscle tissue to resist mechanical forces. DMD is caused by a variety of mutations in the dystrophin gene that result in an early stop codon, and therefore a truncated dystrophin protein. The truncated dystrophin protein lacks a C-terminus and therefore cannot provide the structural support necessary to resist the stress of muscle contraction. As a result, muscle fibers are torn apart, leading to muscle wasting. 【0093】 treatment 【0094】 This specification presents ex vivo and in vivo methods for cells to create permanent changes in the genome that can repair the dystrophin reading frame and restore the activity of the dystrophin protein, using genome manipulation tools. Such methods use endonucleases, such as CRISPR / Cas9 nucleases, to permanently delete (excavate), insert, or replace (delete and insert) exons within the genomic locus of the dystrophin gene (i.e., mutations in the coding and / or splicing sequence). In this way, the present invention mimics the product produced by exon skipping and / or repairs the reading frame with only a few treatments (rather than delivering exon skipping oligos over the patient's lifetime). Preclinical studies have been conducted on the expression of the C-terminus of dystrophin by infusing targeted changes into the genome using zinc fingers, TALE, and CRISPR / Cas9-based nucleases. In one example, a large genomic region was deleted, which is estimated to treat more than 60% of patients with DMD. 【0095】 This specification presents methods for treating patients with DMD. An example of such a method is ex vivo cell-based therapy. For example, DMD patient-specific iPS cell lines are created. Then, the chromosomal DNA of these iPS cells is corrected using the materials and methods described herein. Next, the corrected iPSCs are differentiated into Pax7+ myoprimordial cells. Finally, the myoprimordial cells are implanted into the patient. This ex vivo method has many advantages. 【0096】 One advantage of ex vivo cell therapy is the ability to perform a comprehensive analysis of the therapeutic agent before administration. All nuclease-based therapeutic agents exert some level of off-target effects. Performing ex vivo gene correction allows for the complete characterization of the corrected cell population before implantation. Aspects of this disclosure include sequencing the whole genome of the corrected cells to ensure that off-target cuts, if present, are located in genomic locations associated with minimal risk to the patient. Furthermore, the clonal population of cells can be isolated before implantation. 【0097】 Another advantage of ex vivo cell therapy lies in gene correction within iPSCs compared to other primary cell sources. iPSCs are prolific, making it easy to obtain the large number of cells required for cell-based therapies. Furthermore, iPSCs are an ideal cell type for performing clonal isolation. This allows for screening for precise genomic correction without risking reduced viability. In contrast, other potential cell types, such as primary myoblasts, are only viable for a few passages and are difficult to expand clonally. Also, patient-specific DMD myoblasts can be unhealthy due to the lack of dystrophin protein. On the other hand, patient-derived DMD iPSCs do not express dystrophin in this differentiated state and therefore do not exhibit the diseased phenotype. Thus, DMD iPSCs are much easier to manipulate and can reduce the amount of time required to perform the desired gene correction. 【0098】 A further advantage of ex vivo cell therapy lies in the implantation of myogenic Pax7+ progenitor cells, in contrast to myoblasts. Pax7+ cells are accepted as myogenic satellite cells. Pax7+ progenitor cells are mononuclear cells present at the periphery of multinucleated muscle fibers. In response to injury, the progenitor cells divide and fuse with existing fibers. In contrast, myoblasts fuse directly with muscle fibers upon implantation and have minimal proliferative capacity in vivo. Therefore, while myoblasts cannot aid in healing after repeated injuries, Pax7+ progenitor cells can function as reservoirs and aid in muscle healing throughout the patient's life. 【0099】 Another example of such methods is in vivo-based therapy, which uses the materials and methods described herein to correct the chromosomal DNA of cells in a patient. 【0100】 The advantage of in vivo gene therapy is the ease of creating and administering the therapeutic agent. The same therapeutic cocktail may have the potential to reach a subset (n>1) of the DMD patient population. In contrast, the proposed ex vivo cell therapy requires the development of a customized therapeutic agent for each patient (n=1). Developing ex vivo cell therapies is time-consuming, and certain advanced DMD patients may not have that time. 【0101】 This specification also presents cell methods for editing the dystrophin gene in human cells by genome editing. For example, cells are isolated from a patient or animal. The chromosomal DNA of the cells is then corrected using the materials and methods described herein. 【0102】 In addition to mutations within coding and splicing sequences, several types of genomic target sites may exist. 【0103】 The regulation of transcription and translation involves several different classes of sites that interact with proteins or nucleotides within cells. While DNA-binding sites of transcription factors or other proteins can often be targeted for mutations or deletions to study their role, they can also be targeted to alter gene expression. Sites can be added via direct genome editing, either through non-homologous end joining (NHEJ) or homology-guided repair (HDR). The increasing use of genome-wide studies on genome sequencing, RNA expression, and transcription factor binding is enhancing our ability to identify how sites result in developmental or transient gene regulation. These regulatory systems can be direct or involve extensive cooperative regulation requiring the integration of activity from multiple enhancers. Transcription factors typically bind to degenerate DNA sequences 6–12 bp long. The low levels of specificity mediated by individual sites suggest that complex interactions and rules are involved in binding and functional outcomes. Binding sites with low degeneracy can provide a simpler means of regulation. Artificial transcription factors can be designed to specify longer sequences with fewer similar sequences in the genome and a lower potential for off-target cleavage. Any of these types of binding sites can be mutated, deleted, or even created to enable changes in gene regulation or expression (Canver, MC et al., Nature (201) 5 years)). 【0104】 Another class of gene regulatory regions possessing these characteristics is the microRNA (miRNA) binding site. miRNAs are non-coding RNAs that play a key role in post-transcriptional gene regulation. miRNAs can regulate the expression of 30% of all mammalian protein-coding genes. Specific and potent gene silencing (RNAi) by double-stranded RNA, including further small non-coding RNAs, has been discovered (Canver, MC et al., Nature (2015)). Non-coding RNAs are crucial for gene silencing. The largest class of miRNAs is miRNA. In mammals, miRNAs are first transcribed as long RNA transcripts, which can be individual transcription units, parts of protein introns, or other transcripts. These long transcripts are called pri-miRNAs (primary miRNAs) and contain incomplete hairpin structures of base pairing. These pri-miRNAs can be cleaved into one or more short pre-miRNAs (precursor miRNAs) by microprocessors, which are nuclear protein complexes accompanied by Drosha. 【0105】 pre-miRNA is a short stem-loop approximately 70 nucleotides long with a 2-nucleotide 3' overhang that is extruded, and mature miRNA is 19-25 nucleotides long. * It becomes a double helix. A miRNA strand with low base-pairing stability (guide strand) can be loaded into the RNA-induced silencing complex (RISC). Passenger guide strand ( * (marked with ) may be functional but are usually degraded. Mature miRNAs tether RISC to a partially complementary sequence motif within the target mRNA, which is mainly found in the 3' untranslated region (UTR), inducing post-transcriptional gene silencing (Bartel, DP, Cell, vol. 136, pp. 215-233 (2009); Saj, A. and Lai, EC, Curr Opin Genet Dev, vol. 21, pp. 504-510 (2009). 11 years)). 【0106】 miRNAs can be crucial in the regulation of development, differentiation, the cell cycle, and proliferation, as well as in virtually all biological pathways in mammals and other multicellular organisms. miRNAs may also be involved in cell cycle regulation, apoptosis, stem cell differentiation, hematopoiesis, hypoxia, myogenesis, neurogenesis, insulin secretion, cholesterol metabolism, aging, viral replication, and immune responses. 【0107】 A single miRNA can target hundreds of different mRNA transcripts, while individual transcripts can be targeted by many different miRNAs. The latest version of miRBase (v.21) annotates over 28,645 microRNAs. Some miRNAs may be encoded by multiple loci, some of which can be expressed from tandem co-transcribed clusters. This characteristic enables complex regulatory networks involving multiple pathways and feedback controls. miRNAs may be integral parts of these feedback and regulatory circuits, helping to regulate gene expression by keeping protein production within limits (Herranz, H. and Cohen, SM, Gene Dev, Vol. 24, pp. 1339-1344 (2010); Posadas, DM and Carthew, RW, Curr Opin Genet Dev, Vol. 27, pp. 1-6 (2014). 【0108】 miRNAs may also be important in numerous human diseases associated with abnormal miRNA expression. This association underscores the importance of miRNA regulatory pathways. Recent miRNA deletion studies have linked miRNAs to the regulation of immune responses (Stern-Ginossar, N. et al., Science, Vol. 317, pp. 376-381 (2007)). 【0109】 miRNAs also have a strong association with cancer and can play a role in different types of cancer. miRNAs have been found to be downregulated in several tumors. They may be important in regulating key cancer-related pathways, such as cell cycle control and responses to DNA damage, and therefore can be used in diagnosis and clinical targeting. MicroRNAs can precisely modulate the equilibrium of angiogenesis, as experiments depleting all microRNAs suppress tumor angiogenesis (Chen, S. et al., Genes Dev, Vol. 28, pp. 1054-1067 (2014)). 【0110】 As has been shown with protein-coding genes, miRNA genes can also be subjected to epigenetic changes that occur with cancer. Many miRNA locus may be associated with CpG islands, which increase the opportunity for their regulation through DNA methylation (Weber, B., Stresemann, C., Brueckner, B., and Lyko, F., Cell Cycle, vol. 6, pp. 1001-1005 (2007)). Most of the research has focused on chromatin We are using treatment with remodeling drugs to elucidate the epigenetic silencing of miRNAs. 【0111】 In addition to their roles in RNA silencing, miRNAs can also activate translation (Posadas, DM and Carthew, RW, Curr Opin Genet Dev, Vol. 27, pp. 1-6 (2014)). Knockout of these sites can lead to decreased expression of target genes, while introduction of these sites can increase expression. 【0112】 Individual miRNAs can be most effectively knocked out by mutating the seed sequence (a 2-8 nucleotide microRNA) which may be important for binding specificity. Cleavage within this region, followed by misrepair by NHEJ, can effectively eliminate miRNA function by blocking binding to the target site. miRNAs may also be inhibited by specific targeting of special loop regions adjacent to palindromic sequences. Catalytically inactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. et al., Sci Rep, vol. 4, p. 3943 (2014)). miRNA targeting In addition to targeting, the binding site can also be targeted and mutated to prevent silencing by miRNA. 【0113】 human cells 【0114】 As described and illustrated herein, the primary target for gene editing to improve DMD is human cells. For example, in ex vivo methods, the human cells may be somatic cells that, after being modified using the techniques described, can result in Pax7+ myorigen cells. For example, in in vivo methods, the human cells may be muscle cells or muscle progenitor cells. 【0115】 By performing gene editing within autologous cells derived from patients who require it and therefore are already perfectly matched to the patients, it is possible to safely reintroduce these cells into the patients, thereby effectively creating a cell population that may be effective in improving one or more clinical conditions associated with the patients' disease. 【0116】 Originating cells (also referred to herein as stem cells) have the ability to generate a large number of parent cells that can either proliferate and produce more originating cells, and that can produce differentiated cells or differentiateable daughter cells. The daughter cells themselves can be induced to progeny that proliferate and then differentiate into one or more mature cell types, while also retaining one or more cells with the developmental potential of the parent. The term “stem cell” then refers to a cell that, under certain circumstances, has the ability or potential to differentiate into a more specialized or differentiated phenotype, and under certain circumstances, retains the ability to proliferate substantially without differentiation. In one aspect, the terms originating cell or stem cell refer to a general-purpose parent cell whose offspring cells (progeny) often specialize in different directions by acquiring completely distinct characteristics through differentiation, for example, in the ongoing diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can originate from pluripotent cells, and pluripotent cells themselves originate from pluripotent cells, and so on. Each of these pluripotent cells is considered a stem cell, but the range of cell types they can produce can vary considerably. Some differentiated cells also have the ability to produce cells with large developmental potential. This ability can be spontaneous or artificially induced through treatment with various factors. In many biological cases, stem cells can also be "pluripotent" because they can produce offspring of more than one distinct cell type, but this is not necessary for them to be "stem cell-like." 【0117】 Self-renewal can be another important aspect of stem cells. Theoretically, self-renewal can occur through one of two main mechanisms. Stem cells may divide asymmetrically, with one daughter cell retaining the stem cell state and the other daughter cell expressing some distinct other specific function and phenotype. Alternatively, some stem cells in a population may divide symmetrically into two stem cells, thereby maintaining some stem cells within the population as a whole, while the other cells in the population yield only differentiated progeny. Generally, “primordial cells” have a phenotype that is more primordial (i.e., at an earlier stage than fully differentiated cells along the developmental pathway or progression). Primordial cells also have significant or extremely high proliferative potential. Depending on the developmental pathway and environment in which the cell develops and differentiates, primordial cells may yield multiple distinct differentiated cell types or a single differentiated cell type. 【0118】 In the context of cell development, the adjectives "differentiated" or "to be differentiated" are relative terms. A "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell being compared. Therefore, stem cells can differentiate into lineage-limited progenitor cells (such as the progenitor cells of muscle cells), which can further differentiate into other types of progenitor cells (such as progenitor cells of muscle cells), and then differentiate into final-stage differentiated cells such as muscle cells, which may or may not retain the ability to proliferate, play a characteristic role in a particular tissue type. 【0119】 induced pluripotent stem cells 【0120】 In some cases, the genetically engineered human cells described herein may be induced pluripotent stem cells (iPSCs). The advantage of using iPSCs is that the cells can be derived from the same subject to which the progenitor cells are administered. That is, somatic cells can be obtained from a subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into progenitor cells (e.g., autologous cells) for administration to the subject. Because the progenitor cells are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells derived from another subject or group of subjects. In addition, the use of iPSCs also eliminates the need for cells obtained from embryonic sources. Thus, in one embodiment, the stem cells used in the disclosed method are not embryonic stem cells. 【0121】 While differentiation is generally irreversible in a physiological context, several methods have recently been developed for reprogramming somatic cells into iPSCs. Exemplary methods are known to those skilled in the art and are briefly described below herein. 【0122】 The term "reprogramming" refers to the process of altering or reversing the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming refers to the process of driving cell differentiation back to a more undifferentiated or more primitive cell type. It should be noted that culturing many primary cells can result in some loss of complete differentiation characteristics. Therefore, simply culturing such cells included in the term differentiated cells does not mean that these cells become undifferentiated cells (e.g., undifferentiated cells) or pluripotent cells. The transition from differentiated to pluripotent cells requires a reprogramming stimulus beyond a stimulus that results in a partial loss of differentiating characteristics in the culture. Reprogrammed cells also possess the characteristic of extended passageability without loss of proliferative capacity, compared to primary parent cells, which generally have only a limited number of mitotic counts in the culture. 【0123】 Cells to be reprogrammed may be partially differentiated or terminally differentiated before reprogramming. Reprogramming encompasses a complete reversal of the differentiated state of differentiated cells (e.g., somatic cells) to a pluripotent or multipotent state. Reprogramming may also encompass a complete or partial reversal of the differentiated state of differentiated cells (e.g., somatic cells) to an undifferentiated state (e.g., embryoid cells). Reprogramming may involve the expression of specific genes by the cell, whose expression may further contribute to the reprogramming. In certain examples described herein, reprogramming of differentiated cells (e.g., somatic cells) may cause the differentiated cells to enter an undifferentiated state (e.g., differentiated cells may become undifferentiated cells). The resulting cells are referred to as “reprogrammed cells” or “induced pluripotent stem cells (iPSCs or iPS cells).” 【0124】 Reprogramming is a genetic pattern that occurs during cell differentiation and may involve alteration of at least some of the genetic patterns, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, and genomic imprinting, e.g., retrograde reprogramming. Reprogramming is distinct from simply maintaining the existing undifferentiated state of cells that are already pluripotent, or from maintaining the existing sub-differentiated state of cells that are already multipotent (e.g., myogenic stem cells). Reprogramming is also distinct from promoting the self-regeneration or proliferation of cells that are already pluripotent or multipotent, although in some cases the compositions and methods described herein may also be useful for such purposes. 【0125】 In the art, many methods are known that can be used to produce pluripotent stem cells from somatic cells. Any such method for reprogramming somatic cells into a pluripotent phenotype would be suitable for use in the method described herein. 【0126】 Reprogramming methods for generating pluripotent cells using specified combinations of transcription factors are described. Direct transduction of Oct4, Sox2, Klf4, and c-Myc can convert mouse somatic cells into ES cell-like cells with expanded developmental potential (see, e.g., Takahashi and Yamanaka, Cell, Vol. 126 (No. 4): pp. 663-676 (2006)). iPSCs resemble ES cells because they repair most of the pluripotency-related transcriptional circuits and epigenetic landscape. In addition, mouse iPSCs satisfy all standard assays for pluripotency: specifically, in vitro differentiation into the three germ layer cell types, teratoma formation, contribution to chimeras, germline transmission [see, e.g., Maherali and Hochedlinger, Cell Stem Cell, Vol. 3 (No. 6): pp. 595-605 (2008)], and tetraploid complementation. 【0127】 Human iPSCs can be obtained using similar transduction methods, and the trio of transcription factors OCT4, SOX2, and NANOG have been established as the core set of transcription factors that govern pluripotency (e.g., Budniatzky and Gepstein, Stem Cell Transl Med., Vol. 3 (No. 4): pp. 448-457 (2014); Barrett et al., Stem Cell Trans Med. See Vol. 3: pp. 1-6, sctm.2014-0121 (2014); Focosi et al., Blood Cancer Journal, Vol. 4: e211 (2014); and the references cited therein). iPSCs can be created by introducing nucleic acid sequences encoding stem cell-related genes into adult somatic cells using a viral vector, as in the conventional method. 【0128】 iPSCs can be produced or derived from terminally differentiated somatic cells, as well as from adult stem cells or somatic stem cells. That is, non-pluripotent progenitor cells can be made pluripotent or multipotent by reprogramming. In such cases, it may not be necessary to contain as many reprogramming factors as is required to reprogram terminally differentiated cells. Furthermore, reprogramming can be induced by the non-viral introduction of reprogramming factors, for example, by introducing the protein itself, by introducing nucleic acids encoding the reprogramming factors, or by introducing messenger RNA that delivers the reprogramming factors during translation (see, for example, Warren et al., Cell Stem Cell, vol. 7(no. 5): pp. 618-630 (2010)). Reprogramming can be achieved, for example, by introducing a combination of nucleic acids encoding stem cell-related genes, including Oct-4 (also known as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox15, Sox18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. Reprogramming using the methods and compositions described herein may further include introducing one or more of Oct-3 / 4, members of the Sox family, members of the Klf family, and members of the Myc family into somatic cells. The methods and compositions described herein may further include introducing one or more of Oct-4, Sox2, Nanog, c-MYC, and Klf4 for reprogramming. As mentioned above, the exact method used for reprogramming is not necessarily essential for the methods and compositions described herein. However, when cells differentiated from reprogrammed cells are to be used, for example, in human therapy, in one embodiment, reprogramming is not performed by methods that modify the genome. Therefore, in such cases, reprogramming can be achieved, for example, without using viruses or plasmid vectors. 【0129】 The efficiency of reprogramming derived from the starting cell population (i.e., the number of reprogrammed cells) is as follows: Shi et al., Cell-Stem Cell, Vol. 2: pp. 525-528 (2008); Huangfu et al. As shown in Nature Biotechnology, Vol. 26 (No. 7): pp. 795-797 (2008); and Marson et al., Cell-Stem Cell, Vol. 3: pp. 132-135 (2008), the process can be enhanced by the addition of various drugs, such as small molecules. Therefore, drugs or combinations of drugs that enhance the efficiency or rate of induced pluripotent stem cell generation can be used in the generation of patient-specific or disease-specific iPSCs. Some non-exclusive examples of drugs that enhance reprogramming efficiency include, among others, soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacitidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA). 【0130】 Other non-limiting examples of reprogramming enhancers include suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinoline-2-yl)-N-hydroxybutanamide), phenyl butyrate (e.g., sodium phenylbutyrate and valproic acid (VPA) and other short-chain fatty acids), Scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, Apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), and Trapoxin. B, chlamydosin, depsipeptide (also known as FR901228 or FK228), benzamide (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnamate bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamfratin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)capronhydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50. Other reprogramming enhancers include, for example, dominant-negative forms of HDACs (e.g., catalytically inactive forms), HDAC inhibitors by siRNA, and antibodies that specifically bind to HDACs. Such inhibitors are available from companies such as BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich. 【0131】 To confirm the induction of pluripotent stem cells for use by the methods described herein, isolated clones can be examined for the expression of stem cell markers. Such expression in cells derived from somatic cells identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from a non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers may include, for example, RT-PCR and immunological methods for detecting the presence of encoded polypeptides, such as Western blotting or flow cytometry analysis. Detection may involve not only RT-PCR but also the detection of protein markers. Intracellular markers can best be identified via protein detection methods such as RT-PCR or immunocytochemistry, while cell surface markers can be easily identified, for example, by immunocytochemistry. 【0132】 The pluripotency of isolated cells can be confirmed by tests that assess the ability of iPSCs to differentiate into cells of each of the three germ layers. For example, teratoma formation in nude mice can be used to assess the pluripotency of isolated clones. Cells can be introduced into nude mice, and histology and / or immunohistochemistry can be performed on the tumors arising from the cells. The proliferation of tumors containing cells derived from all three germ layers further indicates, for example, that the cells are pluripotent stem cells. 【0133】 Creation of DMD patient-specific iPSCs 【0134】 One step of the ex vivo method of this disclosure may involve creating a single DMD patient-specific iPS cell, multiple DMD patient-specific iPS cells, or a DMD patient-specific iPS cell line. As described in Takahashi and Yamanaka, 2006; and Takahashi, Tanabe et al., 2007, many methods for creating patient-specific iPS cells have been established in the art. In addition, differentiation of pluripotent cells into muscle lineages can be achieved by techniques developed by Anagenesis Biotechnologies, as described in International Patent Application Publications WO2013 / 030243 and WO2012 / 101114. For example, the creation step may include a) isolating somatic cells, such as skin cells or fibroblasts, from a patient; and b) introducing a set of pluripotency-related genes into the somatic cells to induce them to become pluripotent stem cells. The set of pluripotency-related genes may be one or more genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC. 【0135】 Genome editing 【0136】 Genome editing generally refers to the process of modifying the nucleotide sequence of a genome, preferably precisely or in a predetermined manner. Examples of genome editing methods described herein include methods that use site-specific nucleases to cleave deoxyribonucleic acid (DNA) at precise target locations within the genome, thereby creating single-stranded or double-stranded DNA breaks at specific locations within the genome. Such cleavage has recently been described in Cox et al., Nature Medicine, Vol. 21 (No. 2). As reviewed in pp. 121-131 (2015), DNA repair is possible and regularly carried out by innate endogenous cellular processes, such as homology-driven repair (HDR) and non-homologous end joining (NHEJ). NHEJ directly joins DNA ends resulting from double-strand breaks, but may involve loss or addition of nucleotide sequences, which may disrupt or enhance gene expression. HDR utilizes homologous or donor sequences as templates for inserting a defined DNA sequence into the break point. Homologous sequences can be found in the endogenous genome, such as sister chromatids. Alternatively, the donor can be an exogenous nucleic acid such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, or virus, which has a region of high homology to the locus cleaved by the nuclease, but may also contain further sequences or sequence changes, including deletions that can be incorporated into the cleaved target locus. A third repair mechanism is microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ," which can result in a genetic outcome similar to NHEJ, in that small deletions and insertions may occur at the cleavage site. MMEJ can utilize a small number of base pairs of homologous sequences flanking the DNA cleavage site to drive a more favorable DNA end joining repair outcome, and recent reports have further clarified the molecular mechanism of this process (e.g., Cho and Greenberg, Nature, Vol. 518, pp. 174-176 (2015); Kent et al., Nature Structural and Molecular Biology, Adv. Online). See doi:10.1038 / nsmb.2961 (2015); Mateos-Gomez et al., Nature, Vol. 518, pp. 254-257 (2015); Ceccaldi et al., Nature, Vol. 528, pp. 258-252 (2015). In some cases, it may be possible to predict a high probability of repair outcome based on an analysis of potential microhomology at DNA break sites. 【0137】 Each of these genome editing mechanisms can be used to create a desired genomic modification. A step within the genome editing process may involve creating one or two DNA breaks, specifically two DNA breaks, either as a double-strand break or two single-strand breaks, within a target locus, close to the intended mutation site. This can be achieved through the use of site-specific polypeptides, as described and illustrated herein. 【0138】 Site-specific polypeptides, such as DNA endonucleases, can introduce double-strand or single-strand breaks into nucleic acids, for example, into genomic DNA. Double-strand breaks can stimulate the cell's endogenous DNA repair pathways (e.g., homology-dependent repair, non-homologous end joining, alternative non-homologous end joining (A-NHEJ), or microhomologous end joining). NHEJ can repair the cleaved target nucleic acid without requiring a homologous template. This may, in some cases, result in small deletions or insertions (indels) at the cleavage site within the target nucleic acid, potentially leading to disruption or alteration of gene expression. HDR can occur if a homologous repair template or donor is available. A homologous donor template may contain sequences that are homologous to the sequences flanking the target nucleic acid cleavage site. Sister chromatids may be used by cells as repair templates. However, for genome editing purposes, the repair template can be supplied as an exogenous nucleic acid such as a plasmid, double-stranded oligonucleotide, single-stranded oligonucleotide, or viral nucleic acid. Further or modified nucleic acid sequences can also be introduced between homologous flanking regions, along with the exogenous donor template, as additional nucleic acid sequences (such as transgenes) or modifications (such as single or multiple base changes or deletions) so that they are incorporated into the target locus. MMEJs can produce similar genetic outcomes to NHEJs in that small deletions and insertions may occur at the cleavage site. MMEJs may use homologous sequences of a few base pairs flanking the cleavage site to drive a favorable, end-junction DNA repair outcome. In some cases, it may be possible to predict a likely repair outcome based on an analysis of potential microhomology within the nuclease target region. 【0139】 Therefore, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. In this specification, the exogenous polynucleotide sequence is referred to as a donor polynucleotide (or donor, donor sequence, or polynucleotide donor template). A donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide may be an exogenous polynucleotide sequence, i.e., a sequence that does not exist naturally at the target nucleic acid cleavage site. 【0140】 Modification of target DNA by NHEJ and / or HDR can result in, for example, mutation, deletion, alteration, integration, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocation, and / or gene mutation. The process of deleting genomic DNA and integrating non-native nucleic acids into genomic DNA is an example of genome editing. 【0141】 CRISPR endonuclease system 【0142】 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, CRISPR locus encodes products that function as a kind of immune system, helping prokaryotes defend against foreign invaders such as viruses and phages. There are three stages of CRISPR locus function: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acids. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified. 【0143】 CRISPR locus contains several short repeat sequences called "repeats." When expressed, repeats may form secondary structures (e.g., hairpins) and / or constitute unstructured single-stranded sequences. Repeats typically occur in clusters and often differ significantly between species. Repeats are regularly separated by unique intervening sequences called "spacers," resulting in a repeat-spacer-repeat locus architecture. Spacers are either identical to or highly homologous to known foreign invader sequences. Spacer-repeat units encode crisprRNA (crRNA), which is processed into the mature form of the spacer-repeat unit. crRNA contains a "seed" or spacer sequence (in its naturally occurring form in prokaryotes, the spacer sequence targets foreign invader nucleic acids) that is involved in targeting target nucleic acids. The spacer sequence is located at the 5' or 3' end of the crRNA. 【0144】 The CRISPR locus also contains polynucleotide sequences encoding CRISPR-related (Cas) genes. Cas genes encode endonucleases involved in the biosynthesis and interference steps of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and / or tertiary structures. 【0145】 Type II CRISPR system 【0146】 The biosynthesis of crRNA in the natural type II CRISPR system requires tracrRNA (trans-activating CRISPR RNA). TracrRNA is modified by endogenous RNase III and can then hybridize with crRNA repeats in a pre-crRNA array. Endogenous RNase III can be recruited to cleave the pre-crRNA. The cleaved crRNA can be subjected to trimming with exoribonuclease (e.g., 5' trimming) to obtain a mature crRNA form. The tracrRNA can then remain hybridized with crRNA, and both tracrRNA and crRNA associate with a site-specific polypeptide (e.g., Cas9). The crRNA in the crRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleic acid that the crRNA can hybridize to. Hybridization of crRNA with the target nucleic acid can activate Cas9 for targeted nucleic acid cleavage. The target nucleic acid within the type II CRISPR system is called the protospacer-adjacent motif (PAM). In nature, PAMs are essential for facilitating the binding of site-specific polypeptides (e.g., Cas9) to the target nucleic acid. The type II system (also referred to as Nmeni or CASS4) can be further subdivided into type II-A (CASS4) and type II-B (CASS4a). Jinek et al., Science, vol. 337 (no. 6096): pp. 816-821 (2012), demonstrate the usefulness of the CRISPR / Cas9 system for RNA-programmable genome editing, and International Patent Application Publication WO2013 / 176772 presents numerous examples and applications of the CRISPR / Cas endonuclease system for site-specific gene editing. 【0147】 V-type CRISPR system 【0148】 The V-type CRISPR system has several important differences from the II-type system. For example, Cpf1, in contrast to the II-type system, is a single-RNA-guided endonuclease that lacks tracrRNA. In fact, Cpf1-associated CRISPR arrays can process to mature crRNA without the requirement of further transactivation of tracrRNA. In the V-type CRISPR array, each mature crRNA can be processed into a short mature crRNA of 42-44 nucleotides in length, beginning with a 19-nucleotide direct repeat followed by a 23-25 ​​nucleotide spacer sequence. In contrast, mature crRNAs in the II-type system may begin with a 20-24 nucleotide spacer sequence followed by approximately 22 nucleotide direct repeats. Furthermore, Cpf1 utilizes a T-rich protospacer-adjacent motif so that the Cpf1-crRNA complex efficiently cleaves target DNA preceded by a short T-rich PAM, in contrast to the type II system where the target DNA is followed by a G-rich PAM. Thus, the type V system cleaves at a point far from the PAM, while the type II system cleaves at a point adjacent to the PAM. In addition, in contrast to the type II system, Cpf1 cleaves DNA via offset double-strand breaks of DNA with 4 or 5 nucleotide 5' overhangs, while the type II system cleaves via blunt-end double-strand breaks. Similar to the type II system, Cpf1 contains a predicted RuvC-like endonuclease domain but lacks a second HNH endonuclease domain, which is in contrast to the type II system. 【0149】 Cas gene / polypeptide and protospacer adjacent motif 【0150】 An example of a CRISPR / Cas polypeptide is the Cas9 polypeptide in Figure 1 of Fonfara et al., Nucleic Acids Research, Vol. 42: pp. 2577-2590 (2014). This includes the CRISPR / Cas gene. Since the discovery of the Cas gene, systems claiming to be CRISPR / Cas genes have been extensively rewritten. Fonfara, Figure 5 above, shows Cas9 genes originating from diverse species. The PAM sequence of the polypeptide is presented. 【0151】 Site-specific polypeptides 【0152】 Site-specific polypeptides are nucleases used in genome editing to cleave DNA. Site-specific polypeptides can be administered to cells or patients as one or more polypeptides, or as one or more mRNAs encoding the polypeptides. 【0153】 In the context of CRISPR / Cas or CRISPR / Cpf1 systems, site-specific polypeptides can bind to guide RNA, which specifies the site in the target DNA to which the polypeptide is directed. In the CRISPR / Cas or CRISPR / Cpf1 systems disclosed herein, site-specific polypeptides may be endonucleases, such as DNA endonucleases. 【0154】 Site-directed polypeptides may contain multiple nucleic acid cleavage (i.e., nuclease) domains. Two or more nucleic acid cleavage domains can be linked together via a linker. For example, the linker may include a flexible linker. The linker may have an amino acid length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or more. 【0155】 Naturally occurring wild-type Cas9 enzymes contain two nuclease domains: an HNH nuclease domain and a RuvC domain. In this specification, "Cas9" refers to both naturally occurring Cas9 and recombinant Cas9. The Cas9 enzymes described herein may contain an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain. 【0156】 The HNH or HNH-like domain contains an McrA-like fold. The HNH or HNH-like domain contains two antiparallel β-chains and α-helices. The HNH or HNH-like domain contains a metal-binding site (e.g., a divalent cation-binding site). The HNH or HNH-like domain can cleave one strand of the target nucleic acid (e.g., the complementary strand of the strand targeted by crRNA). 【0157】 The RuvC or RuvC-like domain contains an RNase H or RNase H-like fold. The RuvC / RNase H domain is involved in a diverse set of nucleic acid-based functions, including actions on both RNA and DNA. The RNase H domain contains five β-chains surrounded by multiple α-helices. The RuvC / RNase H or RuvC / RNase H-like domain contains a metal-binding site (e.g., a divalent cation-binding site). The RuvC / RNase H or RuvC / RNase H-like domain can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA). 【0158】 Site-specific polypeptides can introduce double-strand or single-strand breaks into nucleic acids, such as genomic DNA. Double-strand breaks can stimulate the cell's endogenous DNA repair pathways (e.g., homology-dependent repair (HDR), non-homologous end joining (NHEJ), alternative non-homologous end joining (A-NHEJ), or microhomologous-mediated end joining (MMEJ)). NHEJ can repair the cleaved target nucleic acid without requiring a homologous template. This may, in some cases, result in small deletions or insertions (indels) at the cleavage site within the target nucleic acid, potentially leading to disruption or alteration of gene expression. HDR can occur if a homologous repair template or donor is available. A homologous donor template may contain sequences homologous to the sequences flanking the target nucleic acid cleavage site. Sister chromatids may be used by cells as repair templates. However, for genome editing purposes, the repair template can be supplied as an exogenous nucleic acid, such as a plasmid, double-stranded oligonucleotide, single-stranded oligonucleotide, or viral nucleic acid. Further or modified nucleic acid sequences can also be introduced between homologous flanking regions, along with the exogenous donor template, to be incorporated into the target locus. MMEJs can produce similar genetic outcomes to NHEJs in that small deletions and insertions may occur at the cleavage site. MMEJs may use homologous sequences of a few base pairs flanking the cleavage site to drive a favorable, end-junction DNA repair outcome. In some cases, it may be possible to predict a likely repair outcome based on an analysis of potential microhomology within the nuclease target region. 【0159】 Therefore, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. In this specification, the exogenous polynucleotide sequence is referred to as a donor polynucleotide (or donor or donor sequence). A donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide may be an exogenous polynucleotide sequence, i.e., a sequence that does not exist in nature at the target nucleic acid cleavage site. 【0160】 Modification of target DNA by NHEJ and / or HDR can result in, for example, mutation, deletion, alteration, integration, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocation, and / or gene mutation. The process of deleting genomic DNA and integrating non-native nucleic acids into genomic DNA is an example of genome editing. 【0161】 Site-specific polypeptides may include amino acid sequences having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to exemplary wild-type site-specific polypeptides [e.g., Cas9 derived from S. pyogenes; SEQ ID NO. 8 in US2014 / 0068797; or Sapranauskas et al., Nucleic Acids Res, Vol. 39 (No. 21): pp. 9275-9282 (2011)] and a variety of other site-specific polypeptides. 【0162】 Site-specific polypeptides contain at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with wild-type site-specific polypeptides (e.g., Cas9 derived from S. pyogenes, mentioned above) across 10 consecutive amino acids. Site-specific polypeptides may contain up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with wild-type site-specific polypeptides (e.g., Cas9 derived from S. pyogenes, mentioned above) across 10 consecutive amino acids. Site-specific polypeptides may contain at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with wild-type site-specific polypeptides (e.g., Cas9 derived from S. pyogenes, mentioned above) across 10 consecutive amino acids within the HNH nuclease domain of the site-specific polypeptide. Site-specific polypeptides may contain up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with wild-type site-specific polypeptides (e.g., Cas9 derived from S. pyogenes, mentioned above) across 10 consecutive amino acids within the HNH nuclease domain of the site-specific polypeptide. Site-specific polypeptides may contain at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with wild-type site-specific polypeptides (e.g., Cas9 derived from S. pyogenes, mentioned above) across 10 consecutive amino acids within the RuvC nuclease domain of the site-specific polypeptide. Site-specific polypeptides contain up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with wild-type site-specific polypeptides (e.g., Cas9 derived from S. pyogenes, mentioned above) across 10 consecutive amino acids within the RuvC nuclease domain of the site-specific polypeptide. 【0163】 Site-specific polypeptides may include modified forms of exemplary wild-type site-specific polypeptides. Exemplary modified forms of wild-type site-specific polypeptides may include mutations that reduce the nucleic acid cleavage activity of the site-specific polypeptide. Exemplary modified forms of wild-type site-specific polypeptides may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the exemplary wild-type site-specific polypeptide (e.g., Cas9 derived from S. pyogenes, mentioned above). Modified forms of site-specific polypeptides may have virtually no nucleic acid cleavage activity. When a site-specific polypeptide is a modified form with virtually no nucleic acid cleavage activity, it is referred to herein as "enzymatically inactive." 【0164】 Modifications of site-directed polypeptides may include mutations that enable them to induce single-strand breaks (SSBs) on target nucleic acids (e.g., by cleaving only one of the sugar-phosphate backbones of a double-stranded target nucleic acid). Mutations may result in a reduction of less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity in one or more of the nucleic acid cleavage domains of the wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, mentioned above). Mutations may result in one or more of the nucleic acid cleavage domains that retain the ability to cleave the complementary strand of the target nucleic acid but reduce its ability to cleave the non-complementary strand. Mutations may result in one or more of the nucleic acid cleavage domains that retain the ability to cleave the non-complementary strand of the target nucleic acid but reduce its ability to cleave the complementary strand. For example, mutations in the exemplary wild-type S.pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854, and Asn856, inactivate one or more of the nucleic acid cleavage domains (e.g., nuclease domains). The residues to be mutated may correspond to the residues Asp10, His840, Asn854, and Asn856 in the exemplary wild-type S.pyogenes Cas9 polypeptide (determined, for example, by sequence and / or structural alignment). Non-limiting examples of mutations include D10A, H840A, N854A, or N856A. Those skilled in the art will recognize that mutations other than alanine substitutions may be suitable. 【0165】 A D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to produce site-specific polypeptides that substantially lack DNA cleavage activity. A D10A mutation can be combined with one or more of the D10A, N854A, or N856A mutations to produce site-specific polypeptides that substantially lack DNA cleavage activity. A N854A mutation can be combined with one or more of the H840A, D10A, or N856A mutations to produce site-specific polypeptides that substantially lack DNA cleavage activity. A N856A mutation can be combined with one or more of the H840A, N854A, or D10A mutations to produce site-specific polypeptides that substantially lack DNA cleavage activity. A site-specific polypeptide containing one substantially inactive nuclease domain is called a "nickase". 【0166】 The specificity of CRISPR-mediated genome editing can be increased by using RNA-guided endonucleases, such as Cas9 nickase variants. Wild-type Cas9 is typically guided by a single guide RNA designed to hybridize with a specified approximately 20-nucleotide sequence within a target sequence (such as an endogenous genomic locus). However, some mismatches between the guide RNA and the target locus may be tolerated, effectively reducing the required homology length within the target site to, for example, a minimum of 13 nt. This increases the potential for binding and double-strand cleavage (also known as off-target cleavage) by the CRISPR / Cas9 complex at other locations within the target genome. Since each Cas9 nickase variant cleaves only one strand, creating a double-strand break requires a pair of nickases to bind in close proximity on the reverse strand of the target nucleic acid, thereby creating a pair of nicks, which are the equivalent of a double-strand break. This requires that two distinct guide RNAs (one for each nickase) must be in close proximity and bound on the reverse strand of the target nucleic acid. This requirement essentially doubles the minimum homology length required for a double-strand break to occur, thereby reducing the likelihood that the double-strand break event will occur elsewhere in the genome if the two guide RNA sites (if any) are unlikely to be close enough to each other to enable the formation of a double-strand break. As described in the Art, nickase can also be used to promote HDR in contrast to NHEJ. Using HDR, selected changes can be introduced into target sites in the genome through the use of specific donor sequences that effectively mediate the desired changes. Descriptions of various CRISPR / Cas systems for use in gene editing can be found, for example, in International Patent Application Publication WO2013 / 176772; and Nature Biotechnology, Vol. 32, pp. 347-355 (2014); and cited therein. It can be found in the references provided. 【0167】 Possible mutations may include substitutions, additions, and deletions, or any combination thereof. A mutation may convert the mutated amino acid to alanine. A mutation may convert the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine). A mutation may convert the mutated amino acid to a native amino acid (e.g., selenomethionine). A mutation may convert the mutated amino acid to an amino acid mimetic (e.g., a phosphorylated mimetic). Mutations can be conservative mutations. For example, a mutation can convert the mutated amino acid into an amino acid similar in size, shape, charge, polarity, and conformation, and / or a rotamer of the mutated amino acid (e.g., cysteine / serine mutation, lysine / asparagine mutation, histidine / phenylalanine mutation). Mutations can cause shifts in the leading frame and / or the creation of early stop codons. Mutations can cause changes in regulatory regions of a gene or locus that affect the expression of one or more genes. 【0168】 Site-specific polypeptides (e.g., mutant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive site-specific polypeptides) can target nucleic acids. Site-specific polypeptides (e.g., mutant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endribonucleases) can target DNA. Site-specific polypeptides (e.g., mutant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endribonucleases) can target RNA. 【0169】 Site-specific polypeptides may contain one or more non-natural sequences (for example, site-specific polypeptides are fusion proteins). 【0170】 Site-specific polypeptides may contain an amino acid sequence with at least 15% amino acid identity to Cas9, a nucleic acid-binding domain, and two nucleic acid-cleaving domains (i.e., an HNH domain and a RuvC domain) derived from bacteria (e.g., S. pyogenes). 【0171】 Site-specific polypeptides may contain an amino acid sequence that exhibits at least 15% amino acid identity with respect to Cas9 derived from bacteria (e.g., S. pyogenes) and two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain). 【0172】 Site-directed polypeptides can contain amino acid sequences with at least 15% amino acid identity to Cas9 derived from bacteria (e.g., S. pyogenes) and two nucleic acid cleavage domains, in which case one or both of the nucleic acid cleavage domains contain at least 50% amino acid identity to a nuclease domain derived from Cas9 derived from bacteria (e.g., S. pyogenes). 【0173】 Site-specific polypeptides may include Cas9 derived from bacteria (e.g., S. pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), and an amino acid sequence that has at least 15% amino acid identity with respect to a non-natural sequence (e.g., a nuclear localization signal) or linker that links the site-specific polypeptide to a non-natural sequence. 【0174】 Site-specific polypeptides can contain an amino acid sequence with at least 15% amino acid identity to Cas9 derived from bacteria (e.g., S. pyogenes), specifically to two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain), in which case the site-specific polypeptide contains a mutation in one or both of the nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%. 【0175】 Site-directed polypeptides can contain amino acid sequences with at least 15% amino acid identity to Cas9 derived from bacteria (e.g., S. pyogenes) and two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain), in which case one of the nuclease domains may contain a mutation at aspartic acid 10 and / or the other nuclease domain may contain a mutation at histidine 840, the mutation reducing the cleavage activity of the nuclease domain by at least 50%. 【0176】 One or more site-specific polypeptides, such as DNA endonucleases, may comprise two nickases that collectively produce one double-strand break in a specific locus within the genome, or four nickases that collectively produce or cause two double-strand breaks in a specific locus within the genome. Alternatively, one site-specific polypeptide, such as DNA endonucleases, may produce or cause one double-strand break in a specific locus within the genome. 【0177】 Genome-targeting nucleic acids 【0178】 This disclosure presents genome-targeting nucleic acids that can direct the activity of an associated polypeptide (e.g., a site-specific polypeptide) to a specific target sequence within a target nucleic acid. The genome-targeting nucleic acid may be RNA. Hereinafter, genome-targeting RNA is referred to as “guide RNA” or “gRNA.” The guide RNA may include at least a spacer sequence that hybridizes with the target nucleic acid sequence of interest, and a CRISPR repeat sequence. In the type II system, the gRNA also includes a second RNA called a tracrRNA sequence. In type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a double helix. In type V guide RNA (gRNA), the crRNA forms a double helix. In either system, the double helix can bind to the site-specific polypeptide so that the guide RNA and the site-specific polypeptide form a complex. The genome-targeting nucleic acid can impart target specificity to the complex through its association with the site-specific polypeptide. Therefore, genome-targeting nucleic acids can induce site-specific polypeptide activity. 【0179】 Exemplary guide RNAs include spacer sequences in the sequence listing, shown with the genomic locations of their target sequences within or near the dystrophin gene, and associated Cas9-cleaved sites, where the genomic locations are based on the GRCh38 / hg38 human genome assembly. 【0180】 Each guide RNA can be designed to include a spacer sequence complementary to the target sequence in its genome, located within or near the dystrophin gene. For example, each spacer sequence in the sequence listing can be combined into a single-chain guide RNA (sgRNA) (e.g., an RNA chimera) or crRNA (along with the corresponding tracrRNA). See Jinek et al., Science, vol. 337, pp. 816-821 (2012); and Deltcheva et al., Nature, vol. 471, pp. 602-607 (2011). 【0181】 Genome-targeting nucleic acids can be bimolecule guide RNAs. Genome-targeting nucleic acids can be single-molecule guide RNAs. 【0182】 A bimolecular guide RNA may contain two strands of RNA. The first strand may contain an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand may contain a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. 【0183】 The single-molecule guide RNA (sgRNA) within the type II system may include an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension may include elements that contribute to the further functionality (e.g., stability) of the guide RNA. The single-molecule guide linker may link the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may include one or more hairpins. 【0184】 The single-molecule guide RNA (sgRNA) within the V-type system may contain minimal CRISPR repeat sequences and spacer sequences in the 5'-3' direction. 【0185】 For illustrative purposes, guide RNAs used in the CRISPR / Cas / Cpf1 system, or other smaller RNA molecules, can be readily synthesized by chemical means, as illustrated below and as described in the Art. While chemical synthesis procedures are continuously expanding, the purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC; PAGE, etc., avoiding the use of gels) tends to become more difficult as the polynucleotide length increases well beyond about 100 nucleotides. One technique used to produce longer RNAs is to create two or more molecules that are ligated together. Much longer RNAs, such as RNA encoding Cas9 or Cpf1 endonucleases, are more readily produced enzymatically. A variety of RNA modifications, such as those described in the Art, that enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, can be introduced during or after the chemical synthesis and / or enzymatic production of RNA. 【0186】 Spacer extension arrangement 【0187】 In some examples of genome-targeting nucleic acids, spacer extension sequences can modify activity, provide stability, and / or provide a position for modifying the genome-targeting nucleic acid. Spacer extension sequences can modify on or off-target activity or specificity. In some examples, spacer extension sequences can be provided. Spacer extension sequences can have lengths exceeding 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 nucleotides or more. Spacer extension sequences may have lengths less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000, or more than these nucleotide lengths. Spacer extension sequences may be less than 10 nucleotides long. Spacer extension sequences may be between 10 and 30 nucleotides long. Spacer extension sequences may be between 30 and 70 nucleotides long. 【0188】 The spacer extension sequence may contain other parts (e.g., stability control sequences, endoribonuclease binding sequences, ribozymes). These parts may decrease or increase the stability of the nucleic acid targeting nucleic acid. These parts may be transcriptional terminator segments (i.e., transcription termination sequences). These parts may function in eukaryotic cells. These parts may function in prokaryotic cells. These parts may function in both eukaryotic and prokaryotic cells. Non-limiting examples of appropriate parts include 5' caps (e.g., 7-methylguanylate caps (m7G)), riboswitch sequences (e.g., enabling the regulation of stability and / or the accessibility of proteins and protein complexes), sequences that form dsRNA double helix (i.e., hairpins), sequences that target RNA to intracellular locations (e.g., nucleus, mitochondria, chloroplasts, etc.), modifications or sequences that result in tracking (e.g., direct conjugation to fluorescent molecules, conjugation to regions that facilitate fluorescence detection, sequences that enable fluorescence detection, etc.), and / or modifications or sequences that result in protein binding sites (e.g., proteins that act on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.). 【0189】 Spacer array 【0190】 The spacer sequence hybridizes with the sequence within the target nucleic acid. Spacers in genome-targeting nucleic acids can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid. 【0191】 In the CRISPR / Cas systems described herein, spacer sequences can be designed to hybridize with a target nucleic acid located 5' to the PAM of the Cas9 enzyme used in the system. The spacer may be a perfect match or a mismatch with the target sequence. Each Cas9 enzyme has a specific PAM sequence that it recognizes within the target DNA. For example, S. pyogenes recognizes a PAM within the target nucleic acid containing the sequence 5'-NRG-3' [wherein R is A or G, N is any nucleotide, and N is located immediately 3' to the target nucleic acid sequence targeted by the spacer sequence]. 【0192】 The target nucleic acid sequence may contain 20 nucleotides. The target nucleic acid may contain fewer than 20 nucleotides. The target nucleic acid may contain more than 20 nucleotides. The target nucleic acid may contain at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. In some examples, the target nucleic acid contains at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target nucleic acid sequence may contain 20 bases immediately 5' from the first nucleotide of the PAM. For example, 5'-NNNNNNNNNNNNNNNNNNNN NRG In sequences containing -3' (sequences 1,410,473), the target nucleic acid may contain sequences corresponding to multiple Ns [wherein N is any nucleotide, and the underlined NRG sequence is the PAM of S. pyogenes]. 【0193】 The spacer sequence that hybridizes with the target nucleic acid may have a length of at least about 6 nucleotides (nt). The spacer sequence may be at least about 6nt, at least about 10nt, at least about 15nt, at least about 18nt, at least about 19nt, at least about 20nt, at least about 25nt, at least about 30nt, at least about 35nt, or at least about 40nt, about 6nt to about 80nt, about 6nt to about 50nt, about 6nt to about 45nt, about 6nt to about 40nt, about 6nt to about 35nt, about 6nt to about 30nt, about 6nt to about 25nt, about 6nt to about 20nt, about 6nt to about 19nt, about 10nt to about 50nt, about 10nt to about 45nt, about 1 The spacer sequence can be 0nt to approximately 40nt, approximately 10nt to approximately 35nt, approximately 10nt to approximately 30nt, approximately 10nt to approximately 25nt, approximately 10nt to approximately 20nt, approximately 10nt to approximately 19nt, approximately 19nt to approximately 25nt, approximately 19nt to approximately 30nt, approximately 19nt to approximately 35nt, approximately 19nt to approximately 40nt, approximately 19nt to approximately 45nt, approximately 19nt to approximately 50nt, approximately 19nt to approximately 60nt, approximately 20nt to approximately 25nt, approximately 20nt to approximately 30nt, approximately 20nt to approximately 35nt, approximately 20nt to approximately 40nt, approximately 20nt to approximately 45nt, approximately 20nt to approximately 50nt, or approximately 20nt to approximately 60nt. In some examples, the spacer sequence may contain 20 nucleotides. The spacer sequence may contain 19 nucleotides. 【0194】 In some cases, the complementarity percentage between the spacer sequence and the target nucleic acid is at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100%. In some cases, the complementarity percentage between the spacer sequence and the target nucleic acid is at most approximately 30%, at most approximately 40%, at most approximately 50%, at most approximately 60%, at most approximately 65%, at most approximately 70%, at most approximately 75%, at most approximately 80%, at most approximately 85%, at most approximately 90%, at most approximately 95%, at most approximately 97%, at most approximately 98%, at most approximately 99%, or 100%. In some examples, the complementarity percentage between the spacer sequence and the target nucleic acid is 100% over the six most consecutive nucleotides at the 5' end of the target sequence on the complementary strand of the target nucleic acid. The complementarity percentage between the spacer sequence and the target nucleic acid can be at least 60% over approximately 20 consecutive nucleotides. The lengths of the spacer sequence and the target nucleic acid may differ by 1 to 6 nucleotides, which can be considered one or more bulges. 【0195】 Spacer sequences can be designed and selected using computer programs. These programs may use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic frequency (e.g., sequences that are identical or similar but vary in one or more spots as a result of mismatches, insertions, or deletions), methylation status, and the presence of SNPs. 【0196】 Minimal CRISPR repeat array 【0197】 A minimal CRISPR repeat sequence is a sequence that exhibits at least approximately 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with respect to a reference CRISPR repeat sequence (e.g., crRNA derived from S. pyogenes). 【0198】 Minimal CRISPR repeat sequences may contain nucleotides that can hybridize with minimal intracellular tracrRNA sequences. Minimal CRISPR repeat sequences and minimal tracrRNA sequences may form a double-stranded structure, i.e., a base-paired double-stranded structure. Together, minimal CRISPR repeat sequences and minimal tracrRNA sequences may bind to site-specific polypeptides. At least a portion of the minimal CRISPR repeat sequence may hybridize with the minimal tracrRNA sequence. At least a portion of the minimal CRISPR repeat sequence may have at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity with respect to the minimal tracrRNA sequence. At least a portion of the minimal CRISPR repeat sequence may contain complementarity of up to approximately 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% to the minimal tracrRNA sequence. 【0199】 The smallest CRISPR repeat sequence can be approximately 7 to 100 nucleotides in length. For example, the lengths of the smallest CRISPR repeat sequence are approximately 7 nucleotides (nt) to 50 nt, 7 nt to 40 nt, 7 nt to 30 nt, 7 nt to 25 nt, 7 nt to 20 nt, 7 nt to 15 nt, 8 nt to 40 nt, 8 nt to 30 nt, 8 nt to 25 nt, 8 nt to 20 nt, 8 nt to 15 nt, 15 nt to 100 nt, 15 nt to 80 nt, 15 nt to 50 nt, 15 nt to 40 nt, 15 nt to 30 nt, or 15 nt to 25 nt. In some examples, the smallest CRISPR repeat sequence is approximately 9 nucleotides in length. The smallest CRISPR repeat sequence can be approximately 12 nucleotides in length. 【0200】 The minimum CRISPR repeat sequence can be at least approximately 60% identical to a reference minimum CRISPR repeat sequence (e.g., wild-type crRNA from S. pyogenes) over a sequence of at least 6, 7, or 8 consecutive nucleotides. For example, the minimum CRISPR repeat sequence can be at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 100% identical to a reference minimum CRISPR repeat sequence over a sequence of at least 6, 7, or 8 consecutive nucleotides. 【0201】 Minimal tracrRNA sequence 【0202】 The minimal tracrRNA sequence may be a sequence with at least approximately 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., wild-type tracrRNA derived from S. pyogenes). 【0203】 Minimal tracrRNA sequences may contain nucleotides that hybridize with minimal CRISPR repeat sequences within the cell. The minimal tracrRNA sequence and the minimal CRISPR repeat sequence form a double-stranded structure, i.e., a base-paired double-stranded structure. Together, the minimal tracrRNA sequence and the minimal CRISPR repeat bind to site-specific polypeptides. At least a portion of the minimal tracrRNA sequence may hybridize with the minimal CRISPR repeat sequence. The minimal tracrRNA sequence may be at least approximately 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the minimal CRISPR repeat sequence. 【0204】 The smallest tracrRNA sequence can be approximately 7 to 100 nucleotides in length. For example, the smallest tracrRNA sequence can be approximately 7 to 50 nucleotides, 7 to 40 nucleotides, 7 to 30 nucleotides, 7 to 25 nucleotides, 7 to 20 nucleotides, 7 to 15 nucleotides, 8 to 40 nucleotides, 8 to 30 nucleotides, 8 to 25 nucleotides, 8 to 20 nucleotides, 8 to 15 nucleotides, 15 to 100 nucleotides, 15 to 80 nucleotides, 15 to 50 nucleotides, 15 to 40 nucleotides, 15 to 30 nucleotides, or 15 to 25 nucleotides in length. The smallest tracrRNA sequence can be approximately 9 nucleotides in length. The smallest tracrRNA sequence can be approximately 12 nucleotides. The smallest tracrRNA was described by Jinek et al., as mentioned above. It can consist of tracrRNA nt23-48. 【0205】 The minimum tracrRNA sequence may be at least approximately 60% identical to a reference minimum tracrRNA sequence (e.g., wild-type tracrRNA derived from S. pyogenes) over a sequence of at least 6, 7, or 8 consecutive nucleotides. For example, the minimum tracrRNA sequence may be at least approximately 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a reference minimum tracrRNA sequence over a sequence of at least 6, 7, or 8 consecutive nucleotides. 【0206】 The double helix of minimal CRISPR RNA and minimal tracrRNA may contain a double helix. The double helix of minimal CRISPR RNA and minimal tracrRNA may contain at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or more. The double helix of minimal CRISPR RNA and minimal tracrRNA may contain at most approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or more. 【0207】 A double helix can contain mismatches (i.e., the two strands of a double helix are not 100% complementary). A double helix can contain at least approximately one, two, three, four, or five mismatches, or more. A double helix can contain up to approximately one, two, three, four, or five mismatches, or more. A double helix can contain two or fewer mismatches. 【0208】 bulge 【0209】 In some cases, a "bulge" may exist within the double helix of minimal CRISPR RNA and minimal tracrRNA. A bulge is an unpaired region of nucleotides within the double helix. The bulge can contribute to the binding of the double helix to site-specific polypeptides. The bulge may contain unpaired 5'-XXXY-3' sequences [where X is any purine and Y is a nucleotide that can form a fluctuation pair with a nucleotide on the reverse strand] on one side of the double helix, and unpaired nucleotide regions on the other side of the double helix. The number of unpaired nucleotides on the two sides of the double helix may differ. 【0210】 In one example, the bulge may contain unpaired purines (e.g., adenine) on the bulge's minimal CRISPR repeat strand. In some examples, the bulge may contain unpaired 5'-AAGY-3' [in the sequence, Y contains a nucleotide that can form fluctuation pairings with a nucleotide on the minimal CRISPR repeat strand] on the bulge's minimal tracrRNA sequence strand. 【0211】 The bulge on the minimal CRISPR repeat side of a double helix may contain at least one, two, three, four, or five, or more, unpaired nucleotides. The bulge on the minimal CRISPR repeat side of a double helix may contain up to one, two, three, four, or five, or more, unpaired nucleotides. The bulge on the minimal CRISPR repeat side of a double helix may contain one unpaired nucleotide. 【0212】 The bulge on the minimal tracrRNA sequence side of the double helix may contain at least one, two, three, four, five, six, seven, eight, nine, or ten or more unpaired nucleotides. The bulge on the minimal tracrRNA sequence side of the double helix may contain up to one, two, three, four, five, six, seven, eight, nine, or ten or more unpaired nucleotides. The bulge on the second side of the double helix (for example, the minimal tracrRNA sequence side of the double helix) may contain four unpaired nucleotides. 【0213】 The bulge may include at least one wobble pair. In some examples, the bulge includes at most one wobble pair. In some examples, the bulge may include at least one purine nucleotide. The bulge may include at least 3 purine nucleotides. The bulge sequence may include at least 5 purine nucleotides. The bulge sequence may include at least one guanine nucleotide. The bulge sequence may include at least one adenine nucleotide. 【0214】 Hairpin 【0215】 In various examples, one or more hairpins may be located 3' to the minimal tracrRNA within the 3'tracrRNA sequence. 【0216】 The hairpin may begin with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20, or more nucleotides on the 3' side of the last paired nucleotide within the duplex of the minimal CRISPR repeat and the minimal tracrRNA sequence. The hairpin may begin with at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more nucleotides on the 3' side of the last paired nucleotide within the duplex of the minimal CRISPR repeat and the minimal tracrRNA sequence. 【0217】 The hairpin may include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20, or more contiguous nucleotides. The hairpin may include at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more contiguous nucleotides. 【0218】 The hairpin may include the CC dinucleotide (i.e., two contiguous cytosine nucleotides). 【0219】 The hairpin may contain double-stranded nucleotides (e.g., nucleotides that hybridize together within the hairpin). For example, the hairpin may contain CC dinucleotides that hybridize to GG dinucleotides within the hairpin duplex of the 3'tracrRNA sequence. 【0220】 One or more of the hairpins may interact with the region of the site-specific polypeptide that interacts with the guide RNA. 【0221】 In certain examples, two or more hairpins are present, and in other examples, three or more hairpins are present. 【0222】 3'tracrRNA sequence 【0223】 The 3'tracrRNA sequence may include a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., tracrRNA derived from S. pyogenes). 【0224】 The 3'tracrRNA sequence may have a length of about 6 nucleotides to about 100 nucleotides. For example, the 3'tracrRNA sequence may have a length of about6 nucleotides (nt) to about 50 nt, about 6 nt to about 40 nt, about6 nt to about 30 nt, about 6 nt to about 25 nt, about 6 nt to about 20 nt, about 6 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about25 nt, about 8 nt to about20 nt, about 8 nt to about 15 nt, about 15 nt to about100 nt, about15 nt to about 80 nt, about 15 nt to about50 nt, about 15 nt to about40 nt, about15 nt to about 30 nt, or about15 nt to about 25 nt. The 3'tracrRNA sequence may have a length of about 14 nucleotides. 【0225】 A 3' tracrRNA sequence may be at least approximately 60% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 consecutive nucleotides. For example, a 3' tracrRNA sequence may be at least approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 98%, approximately 99%, or 100% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 consecutive nucleotides. 【0226】 A 3' tracrRNA sequence may contain more than one double-stranded region (e.g., a hairpin region, a hybridization region). A 3' tracrRNA sequence may contain two double-stranded regions. 【0227】 The 3' tracrRNA sequence may contain stem-loop structures. Stem-loop structures within 3' tracrRNA may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20, or more, nucleotides. Stem-loop structures within 3' tracrRNA may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more, nucleotides. Stem-loop structures may contain functional regions. For example, stem-loop structures may contain aptamers, ribozymes, hairpins that interact with proteins, CRISPR arrays, introns, or exons. Stem-loop structures may contain at least approximately 1, 2, 3, 4, or 5, or more, functional regions. Stem-loop structures may contain up to approximately 1, 2, 3, 4, or 5, or more, functional regions. 【0228】 Hairpins within the 3' tracrRNA sequence may contain a P domain. In some cases, the P domain may contain a double-stranded region within the hairpin. 【0229】 tracrRNA elongation sequence 【0230】 Whether tracrRNA is in the context of a single-molecule guide or a double-molecule guide, it can result in a tracrRNA elongation sequence. TracrRNA elongation sequences can be approximately 1 to 400 nucleotides in length. They can also be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or longer than 400 nucleotides. TracrRNA elongation sequences can also be approximately 20 to 5000 or longer. They can also be longer than 1000 nucleotides. The tracrRNA elongation sequence may have a length of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or more than 1000 nucleotides. The tracrRNA elongation sequence may have a length of less than 1000 nucleotides. The tracrRNA elongation sequence may contain a length of less than 10 nucleotides. The tracrRNA elongation sequence may be 10 to 30 nucleotides long. The tracrRNA elongation sequence may be 30 to 70 nucleotides long. 【0231】 The tracrRNA elongation sequence may contain functional regions (e.g., stability control sequences, ribozymes, endoribonuclease binding sequences). These functional regions may contain transcriptional terminator segments (i.e., transcription termination sequences). The functional regions may have a total length of approximately 10 nucleotides (nt) to 100 nucleotides, approximately 10 nt to 20 nt, approximately 20 nt to 30 nt, approximately 30 nt to 40 nt, approximately 40 nt to 50 nt, approximately 50 nt to 60 nt, approximately 60 nt to 70 nt, approximately 70 nt to 80 nt, approximately 80 nt to 90 nt, or approximately 90 nt to 100 nt, approximately 15 nt to 80 nt, approximately 15 nt to 50 nt, approximately 15 nt to 40 nt, approximately 15 nt to 30 nt, or approximately 15 nt to 25 nt. The functional regions may function within eukaryotic cells. Functional parts can function within prokaryotic cells. Functional parts can function both within eukaryotic and prokaryotic cells. 【0232】 Non-limiting examples of functional parts of appropriate tracrRNA elongation include 3' polyadenylated tails, riboswitch sequences (e.g., enabling regulation of stability and / or accessibility by proteins and protein complexes), sequences that form dsRNA double helix (i.e., hairpins), sequences that target RNA to intracellular locations (e.g., nucleus, mitochondria, chloroplasts, etc.), modifications or sequences that provide tracking (e.g., direct conjugation to fluorescent molecules, conjugation to regions that facilitate fluorescence detection, sequences that enable fluorescence detection, etc.), and / or modifications or sequences that provide protein binding sites (e.g., proteins that act on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.). TracrRNA elongation sequences may include primer binding sites or molecular indices (e.g., barcode sequences). TracrRNA elongation sequences may include one or more affinity tags. 【0233】 Single-molecule guide linker sequence 【0234】 The linker sequence of a single-molecule guide nucleic acid can be approximately 3 to 100 nucleotides in length. Jinek et al., as mentioned above, for example, a simple 4-nucleotide "tetraloop" (-GAAA-) was used (Science, Vol. 337 (No. 6096): pp. 816-821) (2012). The exemplary linkers have lengths of approximately 3 nucleotides (nt) to 90 nt, approximately 3 nt to 80 nt, approximately 3 nt to 70 nt, approximately 3 nt to 60 nt, approximately 3 nt to 50 nt, approximately 3 nt to 40 nt, approximately 3 nt to 30 nt, approximately 3 nt to 20 nt, and approximately 3 nt to 10 nt. For example, linkers can have lengths of approximately 3nt to 5nt, 5nt to 10nt, 10nt to 15nt, 15nt to 20nt, 20nt to 25nt, 25nt to 30nt, 30nt to 35nt, 35nt to 40nt, 40nt to 50nt, 50nt to 60nt, 60nt to 70nt, 70nt to 80nt, 80nt to 90nt, or 90nt to 100nt. Linkers of single-molecule guide nucleic acids can be between 4 and 40 nucleotides. The linker can consist of at least approximately 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 nucleotides, or more. The linker can consist of at most approximately 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 nucleotides, or more. 【0235】 Linkers can contain any of a variety of sequences, but in some examples, the linker is a broad region homologous to other parts of the guide RNA and does not contain a sequence that could cause intramolecular binding that may interfere with other functional regions of the guide. (Jinek) As mentioned earlier, the simple 4-nucleotide sequence, -GAAA-, was used (Science, Vol. 337 (No. 6096): pp. 816-821 (2012)), but longer sequences are also available. Many other sequences can be used in a similar manner. 【0236】 Linker sequences may contain functional regions. For example, a linker sequence may contain one or more features, including aptamers, ribozymes, hairpins that interact with proteins, protein-binding sites, CRISPR arrays, introns, or exons. A linker sequence may contain at least about 1, 2, 3, 4, or 5, or more, functional regions. In some examples, a linker sequence may contain up to about 1, 2, 3, 4, or 5, or more, functional regions. 【0237】 A genomic manipulation strategy that corrects cells by deleting (excising), inserting, or replacing (deleting and inserting) one or more exons or abnormal intron splice acceptor or donor sites. 【0238】 The steps in the ex vivo methods of this disclosure involve editing / correcting the genome of DMD patient-specific iPS cells using genomic manipulation. Similarly, the steps in the in vivo methods of this disclosure involve editing / correcting the genome of muscle cells in DMD patients using genomic manipulation. Similarly, the steps in the cellular methods of this disclosure involve editing / correcting the dystrophin gene in human cells by genomic manipulation. 【0239】 DMD patients present extensive mutations within the dystrophin gene. Thus, different patients will generally require different correction strategies. Any CRISPR endonuclease can be used in the methods of the present disclosure, and each CRISPR endonuclease has its own associated PAM, which may or may not be disease-specific. For example, the gRNA spacer sequences for targeting the dystrophin gene with a CRISPR / Cas9 endonuclease derived from S. pyogenes are identified in SEQ ID NOs: 1-467,030 and 1,410,430-1,410,472 of the Sequence Listing. The gRNA spacer sequences for targeting the dystrophin gene with a CRISPR / Cas9 endonuclease derived from S. aureus are identified in SEQ ID NOs: 467,031-528,196 of the Sequence Listing. The gRNA spacer sequences for targeting the dystrophin gene with a CRISPR / Cas9 endonuclease derived from S. thermophilus are identified in SEQ ID NOs: 528,197-553,198 of the Sequence Listing. The gRNA spacer sequences for targeting the dystrophin gene with a CRISPR / Cas9 endonuclease derived from T. denticola are identified in SEQ ID NOs: 553,199-563,911 of the Sequence Listing. The gRNA spacer sequences for targeting the dystrophin gene with a CRISPR / Cas9 endonuclease derived from N. meningitides are identified in SEQ ID NOs: 563,912-627,854 and 1,410,400-1,410,402 of the Sequence Listing. The gRNA spacer sequences for targeting the dystrophin gene with a CRISPR / Cpf1 endonuclease derived from Acidominoccoccus, Lachnospiraceae, and Franciscella Novicida are identified in SEQ ID NOs: 627,855-1,410,399 and 1,410,403-1,410,429 of the Sequence Listing. 【0240】 One genomic manipulation strategy involves exon deletion. Targeted deletion of specific exons can be an attractive strategy for treating a large subset of patients with a single therapeutic cocktail. By repairing the dystrophin reading frame, single-exon deletions are expected to treat up to 13% of patients, while multi-exon deletions are expected to treat up to 62%. Although multi-exon deletions can reach a larger number of patients, deletion efficiency decreases significantly with increasing size for larger deletions. Therefore, preferred deletions may be in the size range of 400–350,000 base pairs (bp). For example, deletions can range in size from 400 to 1,000; 1,000 to 5,000; 5,000 to 10,000; 10,000 to 25,000; 25,000 to 50,000; 50,000 to 100,000; 100,000 to 200,000; or 200,000 to 350,000 base pairs. 【0241】 As previously stated, the DMD gene contains 79 exons. Any one or more of these 79 exons, or the splice acceptor or donor sites of the abnormal introns, can be deleted to repair the dystrophin reading frame. These methods provide gRNA pairs that can be used to delete exons 2, 8, 43, 44, 45, 46, 50, 51, 52, 53, 70, 45-53, or 45-55, as these are regions expected to reach the largest subset of patients (see Tables 1 and 2; the percentages given in Table 2 are averages reported in the literature). 【0242】 Different regions of the DMD gene can be repaired by deletion and / or by HDR. Mutations within target exons can be corrected using specific combinations of gRNAs that cleave within the target genomic region. Coordinates are based on the GRch38 / hg38 genome assembly (Table 1). [Table 1] [Table 2] 【0243】 The method provides a gRNA pair that deletes exon 2 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 2 and the other gRNA that cuts at the 3' end of exon 2. 【0244】 The method provides a gRNA pair that deletes exon 8 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 8 and the other gRNA that cuts at the 3' end of exon 8. 【0245】 The method provides a gRNA pair that deletes exon 43 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 43 and the other gRNA that cuts at the 3' end of exon 43. 【0246】 The method provides a gRNA pair that deletes exon 44 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 44 and the other gRNA that cuts at the 3' end of exon 44. 【0247】 The method provides a gRNA pair that deletes exon 45 by cutting a gene twice, comprising one gRNA that is cut at the 5' end of exon 45 and the other gRNA that is cut at the 3' end of exon 45. 【0248】 The method provides a gRNA pair that deletes exon 46 by cutting the gene twice, comprising one gRNA that cuts at the 5' end of exon 46 and the other gRNA that cuts at the 3' end of exon 46. 【0249】 The method provides a gRNA pair that deletes exon 50 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 50 and the other gRNA that cuts at the 3' end of exon 50. 【0250】 The method provides a gRNA pair that deletes exon 51 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 51 and the other gRNA that cuts at the 3' end of exon 51. 【0251】 The method provides a gRNA pair that deletes exon 52 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 52 and the other gRNA that cuts at the 3' end of exon 52. 【0252】 The method provides a gRNA pair that deletes exon 53 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 53 and the other gRNA that cuts at the 3' end of exon 53. 【0253】 The method provides a gRNA pair that deletes exon 70 by cutting a gene twice, comprising one gRNA that cuts at the 5' end of exon 70 and the other gRNA that cuts at the 3' end of exon 70. 【0254】 The method provides a gRNA pair that deletes exons 45-53 by cutting a gene twice, comprising one gRNA that is cut at the 5' end of exon 45 and the other gRNA that is cut at the 3' end of exon 53. 【0255】 The method provides a gRNA pair that deletes exons 45-55 by cutting a gene twice, comprising one gRNA that is cut at the 5' end of exon 45 and the other gRNA that is cut at the 3' end of exon 55. 【0256】 Another genomic manipulation strategy involves insertion or replacement of one or more exons or abnormal introns into splice acceptor or donor sites, accompanied by homology-guided repair (HDR), also known as homologous recombination (HR). Homology-guided repair is one strategy for treating patients with premature stop codons resulting from small insertions / deletions or point mutations. Rather than resulting in large genomic deletions that convert a DMD phenotype to a BMD phenotype, this strategy repairs the entire reading frame, completely reversing the disease state. This strategy requires a more customized approach based on the patient's premature stop location. Most dystrophin exons are small (<300 bp). This is advantageous because HDR efficiency is inversely proportional to the size of the donor molecule. It is also predicted that the donor template will be a size-constrained adeno-associated virus (AAV) molecule, which has been shown to be a viable means of donor template delivery. 【0257】 Homologous repair is a cellular mechanism for repairing double-strand breaks (DSBs). The most common form is homologous recombination. Further pathways exist within HDR, including single-strand annealing and alternative HDRs. Genomic manipulation tools allow researchers to manipulate the homologous recombination pathway of cells to create site-directed modifications in the genome. Cells have been found to be able to repair double-strand breaks using synthetic donor molecules supplied in trans. Therefore, targeted changes can be introduced into the genome by introducing a double-strand break near a specific mutation and supplying the appropriate donor. Specific breaks can be repaired by supplying a single homologous donor to cells. 6The HDR rate is increased by more than 1,000 times the rate of one occurrence per individual. Since the repair rate (HDR) derived from homology at a specific nucleotide is a function of the distance to the cleavage site, it is important to select overlapping or nearest neighbor target sites. In situ correction leaves the remaining part of the genome undisturbed, so gene editing offers advantages over gene addition. 【0258】 The donors supplied for HDR editing are sequences with small or large flanking homology arms, which vary considerably but may contain sequences intended to enable annealing with genomic DNA. The homology regions flanking the introduced gene alteration may be 30 bp or smaller, or they may be large cassettes of multi-kilobases, and these may contain promoters, cDNA, etc. Both single-stranded and double-stranded oligonucleotide donors are used. These oligonucleotides may range in size from less than 100 nt to more than 200 nt, but longer ssDNA can also be produced and used. Double-stranded donors containing PCR unit replication sequences, plasmids, and minicircles can be used. Generally, AAV vectors have been found to be a very effective means of delivering donor templates, although the packaging limit for individual donors is <5 kb. Active transcription of the donor has been shown to triple the HDR, suggesting that the inclusion of a promoter may increase the conversion rate. Conversely, methylation of donor CpG reduces gene expression and HDR. 【0259】 In addition to wild-type endonucleases such as Cas9, there are nickase variants that can result in the cleavage of only one DNA strand by inactivating one or the other nuclease domain. HDR can be driven by individual Cas nickases or by using a pair of nickases flanking the target region. The donor can be single-stranded, nicked, or dsDNA. 【0260】 Donor DNA can be supplied with a nuclease, or independently by various different methods, such as transfection, nanoparticles, microinjection, or viral transduction. A range of tethering options have been proposed to increase donor availability for HDR. Examples include conjugation of the donor to a nuclease, conjugation to a nearby DNA-binding protein, or conjugation to proteins involved in binding or repair at the DNA ends. 【0261】 The selection of repair pathways can be guided by several culture conditions, including those that affect the cell cycle, and by targeting DNA repair and related proteins. For example, key NHEJ molecules such as KU70, KU80, or DNA ligase IV can be suppressed to increase HDR. 【0262】 In the absence of a donor, several non-homologous repair pathways can be used to join DNA ends, either originating from a DNA break or from a different break, with little or no base pairing at the junction. Similar repair mechanisms exist, such as alt-NHEJ, in addition to the canonical NHEJ. When two breaks are present, the intervening segment can be deleted or inverted. The NHEJ repair pathway can result in insertions, deletions, or mutations at the junction. 【0263】 NHEJ was used to insert a 15kb inductive gene expression cassette into a defined locus within a human cell line after nuclease cleavage (Maresca, M., Lin, VG, Guo, N., and Yang, Y., Obligate ligation-gated recombination). (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining, Genome Res, vol. 23, pp. 539-546 (2013 year)). 【0264】 In addition to genome editing using NHEJ or HDR, site-directed gene insertions are being performed using both the NHEJ pathway and HR. Possibly, a combination approach may be applicable in certain situations, including intron / exon boundaries. While NHEJ has been shown to be effective for ligation within introns, error-free HDR may be better suited within coding regions. 【0265】 As already stated, the DMD gene contains 79 exons. Any one or more of these 79 exons can be corrected to correct mutations and repair the dystrophin reading frame. Since data show that the majority of early stop codons in the dystrophin gene tend to be in exon 70, some methods provide one or a pair of gRNAs that can be used to facilitate the incorporation of a new sequence derived from a polynucleotide donor template, either by inserting a sequence into exon 70 or replacing a sequence within exon 70 (Tuffery-Giraud, S. et al., Hum). Mutat, 2009, Vol. 30 (No. 6): pp. 934-945) (Flanigan, KM et al., Hum Mutat, 2009, Vol. 30 (No. 12): pp. 1657-1666). To make the method applicable to the largest number of patients, the method involves a donor template into which all exon 70 can be inserted or replaced. Alternatively, the method provides a single gRNA or a pair of gRNAs that can be used to facilitate the insertion of a new sequence derived from a polynucleotide donor template into or replace sequences within exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. See Table 1. 【0266】 After HDR, it is crucial to preserve the surrounding splicing signals to ensure proper processing of pre-mRNA. Splicing donors and acceptors are generally located within 100 base pairs of adjacent introns. Therefore, in some cases, the method may result in cleaving all gRNA within approximately ±0 to 3100 bp relative to the intron junction of the exon. 【0267】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 2, with one gRNA cleaved at the 5' end of exon 2 and the other gRNA cleaved at the 3' end of exon 2. 【0268】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 2. 【0269】 One example of the method is a gRNA pair that causes a deletion by cleaving a gene twice, and which consists of one gRNA that cleaves at the 5' end of exon 8 and the other gRNA that cleaves at the 3' end of exon 8, facilitating the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 8. 【0270】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace a sequence within exon 8. 【0271】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 43, with one gRNA cleaved at the 5' end of exon 43 and the other gRNA cleaved at the 3' end of exon 43. 【0272】 Alternatively, some methods provide a single gRNA, which, according to the preceding paragraph, yields a single double-strand break that facilitates the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 43. 【0273】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 44, with one gRNA cleaved at the 5' end of exon 44 and the other gRNA cleaved at the 3' end of exon 44. 【0274】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 44. 【0275】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 45, with one gRNA cleaved at the 5' end of exon 45 and the other gRNA cleaved at the 3' end of exon 45. 【0276】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 45. 【0277】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 46, with one gRNA cleaved at the 5' end of exon 46 and the other gRNA cleaved at the 3' end of exon 46. 【0278】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 46. 【0279】 Some methods provide a gRNA pair that causes a deletion by cutting a gene twice, and which consists of one gRNA that cuts at the 5' end of exon 50 and the other gRNA that cuts at the 3' end of exon 50, facilitating the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 50. 【0280】 Alternatively, some methods provide a single gRNA, which, according to the preceding paragraph, yields a single double-strand break that facilitates the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 50. 【0281】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 51, with one gRNA cleaved at the 5' end of exon 51 and the other gRNA cleaved at the 3' end of exon 51. 【0282】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 51. 【0283】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 52, with one gRNA cleaving at the 5' end of exon 52 and the other gRNA cleaving at the 3' end of exon 52. 【0284】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 52. 【0285】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 53, with one gRNA cleaved at the 5' end of exon 53 and the other gRNA cleaved at the 3' end of exon 53. 【0286】 Alternatively, some methods provide a single gRNA, derived from a preceding paragraph, that results in a single double-strand break, facilitating the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 53. 【0287】 Some methods provide a gRNA pair that causes a deletion by cleaving a gene twice, and which facilitates the incorporation of a new sequence derived from a polynucleotide donor template to replace the sequence in exon 70, consisting of one gRNA that cleaves at the 5' end of exon 70 and the other gRNA that cleaves at the 3' end of exon 70. 【0288】 Alternatively, some methods provide a single gRNA, which, by preceding paragraph, yields a single double-strand break that facilitates the insertion of a new sequence derived from a polynucleotide donor template to replace the sequence within exon 70. 【0289】 In addition to single-exon recombination by homology-guided repair, the inventors also describe a method for performing partial cDNA knock-in of mutation hotspots found within the DMD gene. For example, a treatment to repair exons 45–55 may treat up to 62% of patients. Another treatment option, which does not involve deleting or replacing exons 45–55 as described herein, involves replacing the entire genomic region of exons 45–55 (including introns spanning >350,000 bp) with cDNA containing only the coding region of exons 45–55, spanning approximately 1800 bp. The replacement can be performed using homology-guided repair methods. By excluding the intergene region, the cDNA of exons 45–55 can be more readily included (than the whole genomic region) in any donor vector described in the section entitled “Nucleic Acids Encoding Components of the System” of this application, along with the homology arm. This method allows for the delivery of two gRNAs and Cas9 or Cpf1, which remove the genomic region from exons 45-55, along with a donor construct that replaces the deleted region with a desired cDNA knock-in. 【0290】 The cDNA knock-in method can be used to replace any series of exons. 【0291】 The cDNA knock-in sequence can be optimized to include synthetic intron sequences. To ensure proper expression and processing of the DMD locus, smaller synthetic introns than native introns can be added between exons within the donor construct. 【0292】 Exemplary modifications within the dystrophin gene include deletions, insertions, or recombinations within or proximal to the dystrophin locus mentioned above, such as in regions less than 3kb, less than 2kb, less than 1kb, or less than 0.5kb upstream or downstream of specific exons. Given the relatively wide variation of mutations within the dystrophin gene, it can be inferred that many variations of the deletions, insertions, or recombinations mentioned above (including, without limitation, large and small deletions) are expected to result in the repair of the dystrophin leading frame and the repair of dystrophin protein activity. 【0293】 Such variants may include deletions, insertions, or recombinations that are larger in the 5' and / or 3' directions than the specific exon in question, or smaller in either direction. Thus, “neighbor” or “proximal” with respect to deletions, insertions, or recombinations of a specific exon is intended to mean that the SSB or DSB locus associated with the boundary (also referred herein as the endpoint) of the desired deletion, insertion, or replacement may be within a region of less than approximately 3 kb from the reference locus mentioned. The SSB or DSB locus may be more proximal, being within 2 kb, 1 kb, 0.5 kb, or 0.1 kb. In the case of small deletions, the desired endpoint may be in or “adjacent” the reference locus, thereby intended to mean that the endpoint may be within 100 bp, 50 bp, 25 bp, or approximately 10 bp to less than 5 bp from the reference locus. 【0294】 One advantage of replicating or mimicking the products produced by exon skipping and / or repairing the leading frame for patients with DMD is that it is already known to be safe and associated with improvement of DMD. Other examples, including large or small deletions / insertions / recombinations, can be expected to yield the same benefit insofar as they repair the leading frame of dystrophin. Therefore, many variations of deletions, insertions, and recombinations described and illustrated herein can be expected to be effective in improving DMD. 【0295】 Selection of target sequences 【0296】 By using the shifts of the 5' and / or 3' boundary positions compared to a specific reference locus, it is possible to facilitate or enhance specific applications of gene editing, which are partially dependent on the endonuclease system selected for editing, as further described and illustrated herein. 【0297】 In this first non-limiting example of target sequence selection, many endonuclease systems have rules or criteria that can guide the initial selection of potential target sites for cleavage, such as requirements for PAM sequence motifs, particularly in the case of type II or V CRISPR endonucleases, the position adjacent to the DNA cleavage site. 【0298】 In another non-limiting example of target sequence selection or optimization, the frequency of “off-target” activity (i.e., the frequency of DSBs occurring at sites other than the selected target sequence) for a particular combination of target sequence and gene-editing endonuclease can be evaluated compared to the frequency of on-target activity. In some cases, cells properly edited in a desired locus may have a selective advantage over other cells. Exemplary but non-limiting examples of selective advantages include attributes such as increased replication rate, persistence, tolerance to certain conditions, increased engraftment success or survival rate in vivo after introduction into a patient, and the acquisition of other attributes associated with the maintenance or increased number or viability of such cells. In other cases, cells properly edited in a desired locus can be positively selected by one or more screening methods used to identify, isolate, or otherwise select the properly edited cells. Both selective advantages and targeted selection methods can leverage phenotypes associated with the correction. In some cases, cells can be edited two or more times to create a second modification that generates a novel phenotype used to select or purify an intended cell population. Such a second modification may be created by adding a second gRNA for a selectable or screenable marker. In some cases, cells can be appropriately edited in the desired locus using a DNA fragment that contains both cDNA and a selectable marker. 【0299】 In any given case, whether either selective advantage is applicable or directed selection is applied, the selection of target sequences can also be guided by considering off-target frequencies to enhance the effectiveness of the application and / or reduce the potential for undesirable modifications at sites other than the desired target. As further described and illustrated herein and in the Art, the occurrence of off-target activity may be influenced by several factors, including the similarities and differences between the target site and various off-target sites, as well as the specific endonuclease used. Bioinformatics tools are available to aid in predicting off-target activity, and such tools can also be used to identify sites most likely to have off-target activity, and then evaluate these under experimental conditions to assess the relative frequency of off-targets to on-target activity, thereby enabling the selection of sequences with higher relative on-target activity. Examples of such techniques are presented herein, but other examples are known in the Art. 【0300】 Another aspect of target sequence selection relates to homologous recombination events. Sequences sharing homologous regions can be used as a focus for homologous recombination events that result in the deletion of intervening sequences. Such recombination events occur during the normal replication process of chromosomes and other DNA sequences, and regularly during the normal cell replication cycle, but can also be enhanced by the occurrence of various events (such as UV light and other inducers of DNA breaks), and can also be enhanced by the presence of certain drugs (such as various chemical inducers), and can occur at other points in time when DNA sequences are synthesized, such as in the case of double-strand break (DSB) repair. Many such inducers cause DSBs to occur indiscriminately within the genome, and DSBs can be regularly induced and repaired in normal cells. During repair, the original sequence may be reconstructed with perfect fidelity, although in some cases small insertions or deletions (referred to as "indels") may be introduced at the DSB site. 【0301】 DSBs can also be specifically induced at a particular site, as in the case of the endonuclease systems described herein, and can be used to induce a induced or preferential gene modification event at a selected chromosomal location. The tendency of homologous sequences to undergo recombination in DNA repair (and replication) can be utilized in several situations and is the basis for the application of one gene editing system, such as CRISPR, which uses homology-led repair to insert a target sequence supplied via the use of a “donor” polynucleotide into a desired chromosomal location. 【0302】 Regions of "microhomology," which may contain a small number of base pairs (10 base pairs or less), and which exhibit homology between specific sequences, can also be used to produce a desired deletion. For example, a single double-sequence branch (DSB) can be introduced into a site exhibiting microhomology with a neighboring sequence. A frequently occurring outcome during the normal course of repair of such DSBs is the deletion of the intervening sequence as a result of recombination facilitated by the DSB and the synchronous cellular repair process. 【0303】 However, in some situations, selecting a target sequence within a homologous region can also result in much larger deletions, including gene fusions (when the deletion is located within a coding region), which may or may not be desirable depending on the specific circumstances. 【0304】 The examples presented herein further illustrate the selection of diverse target regions for creating DSBs designed to induce disruption, deletion, or recombination resulting in the repair of the dystrophin leading frame, as well as the selection of specific target sequences within such regions designed to minimize off-target events compared to on-target events. 【0305】 Nucleic acid modification 【0306】 Depending on the circumstances, the polynucleotides introduced into the cells may include one or more modifications, which can be used individually or in combination, for example, to enhance activity, stability, or specificity, alter delivery, reduce the innate immune response within the host cell, or for other enhancements as further described herein and known in the art. 【0307】 In certain cases, modified polynucleotides can be used with the CRISPR / Cas9 / Cpf1 system, in which case the guide RNA (single-molecule guide or double-molecule guide) and / or the DNA or RNA encoding the Cas or Cpf1 endonuclease introduced into the cell can be modified as described and illustrated below. Such modified polynucleotides can be used with the CRISPR / Cas9 / Cpf1 system to edit any one or more genomic loci. 【0308】 The CRISPR / Cas9 / Cpf1 system may be used, for the purposes of non-limiting illustrative purposes of such use, to enhance the formation or stability of the CRISPR / Cas9 / Cpf1 genome editing complex, which may include a single-molecule guide RNA or a bimolecule guide RNA and a Cas or Cpf1 endonuclease. Guide RNA modification may also, or alternatively, be used to enhance the initiation, stability, or reaction rate of the interaction between the genome editing complex and a target sequence in the genome, for example, to enhance on-target activity. Guide RNA modification may also, or alternatively, be used to enhance specificity, for example, the relative rate of genome editing at on-target sites compared to the effect at other (off-target) sites. 【0309】 Modifications can also be used, or alternatively, to increase the stability of guide RNA by increasing its resistance to degradation by intracellular ribonucleases (RNases), thereby extending its intracellular half-life. Modifications that extend the half-life of guide RNA may be particularly useful in embodiments that introduce Cas or Cpf1 endonucleases into cells edited via RNA that needs to be translated to generate endonucleases. This is because extending the half-life of the guide RNA, introduced simultaneously with the endonuclease-encoding RNA, can extend the time that the guide RNA and the encoded Cas or Cpf1 endonuclease coexist in the cell. 【0310】 Modifications can also be used, or alternatively, to reduce the likelihood or extent to which RNA introduced into a cell triggers an innate immune response. In the context of RNA interference (RNAi), including small interfering RNAs (siRNAs), such responses, which are well-characterized and described below and in the Art, tend to be associated with a shortening of the RNA half-life and / or the induction of cytokines or other factors associated with the immune response. 【0311】 Without limitation, one or more modifications can also be applied to RNA encoding an endonucleases introduced into a cell, including modifications that enhance RNA stability (such as increasing its resistance to degradation by intracellular RNases), modifications that enhance the translation of the resulting product (i.e., endonuclease), and / or modifications that reduce the likelihood or degree to which the RNA introduced into the cell elicits an innate immune response. 【0312】 Combinations of modifications, including those mentioned above and others, can also be used. In the case of CRISPR / Cas9 / Cpf1, for example, one or more modifications can be applied to the guide RNA (including the guide RNA exemplified above), and / or one or more modifications can be applied to the RNA encoding the Cas endonuclease (including the RNA encoding the Cas endonuclease exemplified above). 【0313】 For illustrative purposes, guide RNAs used in CRISPR / Cas9 / Cpf1 systems, or other small RNAs, can be readily synthesized by chemical means, allowing for the easy incorporation of several modifications, as illustrated below and described in the Art. While chemical synthesis procedures are continuously expanding, the purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC; PAGE, etc., avoiding the use of gels) tends to become more difficult as the polynucleotide length increases well beyond about 100 nucleotides. One method that can be used to produce longer chemically modified RNAs is to create two or more molecules that are ligated together. Much longer RNAs, such as the RNA encoding Cas9 endonuclease, can be more readily produced enzymatically. While a small number of modifications are available for use in enzyme-generated RNA, as described below and further in the Art, there are still modifications that can be used, for example, to enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, and new types of modifications are regularly developed. 【0314】 To illustrate the diverse types of modifications, particularly those frequently used with chemically synthesized small RNAs, modifications may include one or more nucleotides modified at the 2' position of a sugar, and in some embodiments, nucleotides modified with 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro. In some embodiments, RNA modifications may include 2'-fluoro, 2'-amino, or 2'O-methyl modifications at the ribose, non-basic residues, or reverse base at the 3' end of the RNA. Such modifications can be incorporated into oligonucleotides in prescribed ways, and these oligonucleotides have been shown to have a larger Tm (i.e., greater binding affinity to the target) than 2'-deoxyoligonucleotides for a given target. 【0315】 Several nucleotide and nucleoside modifications have been shown to make the oligonucleotides they incorporate more resistant to nuclease digestion than natural oligonucleotides, allowing these modified oligos to remain intact for longer periods than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing modified skeletons, such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl linkages, or short-chain heteroatoms or heterocyclic linkages. Some oligonucleotides include oligonucleotides with phosphorothioate skeletons, and heteroatom skeletons, particularly CH2-NH-O-CH2, CH,~N(CH3)~O~CH2 (known as the methylene (methylimino) or MMI skeleton), CH2--O--N(CH3)-CH2 skeletons, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 skeletons [wherein the formula, the natural phosphodiester skeleton is represented as OPO-CH]; amide skeletons [see De Mesmaeker et al., Ace. Chem. Res., Vol. 28: pp. 366-374 (1995)]; and morpholino skeleton structures (see Summerton and Weller, U.S. Patent No. 5,034,506). ); These are oligonucleotides with a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of an oligonucleotide is replaced with a polyamide backbone, and the nucleotide is directly or indirectly bonded to the aza nitrogen atom of the polyamide backbone; see Nielsen et al., Science, 1991, Vol. 254, p. 1497). Phosphorus-containing linkages include methylphosphonates and other alkylphosphonates, phosphinates, phosphotriesters, aminoalkylphosphotriesters, 3'alkylenephosphonates and chiral phosphonates, phosphoamides including 3'-aminophosphoramides and aminoalkylphosphoramides, thionophosphoramides, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, which include those having the usual 3'-5' linkage, their analogues with 2'-5' linkages, and phosphorus-containing linkages having reverse polarity where adjacent pairs of nucleoside units are linked at 3'-5' for 5'-3' or 2'-5' for 5'-2'. However, this is not limited to these (US Patent Nos. 3,687,808; Nos. 4,469,863; Nos. 4,476,301; Nos. 5,023,243; Nos. 5,177,196; Nos. 5,188,897; Nos. 5,264,423; Nos. 5,276,019; Nos. 5,278,302; Nos. 5,286,717; Nos. 5,321,131; Nos. 5,399,676; Nos. 5, See Nos. 405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050). 【0316】 Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, Vol. 41 (No. 14): pp. 4503-4510 (2002); Genesis, Vol. 30, No. 3 (2001); Heasman, Dev. Biol., Vol. 243: pp. 209-214 (2002); Nasevicius et al., Nat. Genet., Vol. 26: pp. 216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci., Vol. 97: pp. 9591-9596 (2000); and in U.S. Patent No. 5,034,506, published on July 23, 1991. 【0317】 For cyclohexenyl nucleic acid oligonucleotide mimetic compounds, see Wang et al., J. Am. Chem. This is described in Soc., Vol. 122, pp. 8595-8602 (2000). 【0318】 Modified oligonucleotide skeletons that do not contain phosphorus atoms have skeletons formed by nucleoside linkages by short-chain alkyl or cycloalkyl groups, nucleoside linkages by a mixture of heteroatoms and alkyl or cycloalkyl groups, or nucleoside linkages by one or more short-chain heteroatoms or heterocycles. These include morpholino-linked skeletons (partially formed from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and other skeletons having mixtures of constituent parts of N, O, S, and CH2 (each of which is incorporated herein by reference, U.S. Patents No. 5,034,506; No. 5,166,315; No. 5,185,444; No. 5,214,134; No. 5,21 No. 6,141; No. 5,235,033; No. 5,264,562; No. 5,264,564; No. 5,405,938; No. 5,434,257; No. 5, No. 466,677; No. 5,470,967; No. 5,489,677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5 (See also Nos. 602,240; Nos. 5,610,289; Nos. 5,602,240; Nos. 5,608,046; Nos. 5,610,289; Nos. 5,618,704; Nos. 5,623,070; Nos. 5,663,312; Nos. 5,633,360; Nos. 5,677,437; and Nos. 5,677,439). 【0319】 One or more substituted sugar moieties, e.g., at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2, or O(CH2)nCH3 [wherein n is 1 to about 10]; C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaline or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaline; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; insertor; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides; and may also include one of other substituents having similar properties. In some embodiments, the modification includes the 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) modification (Martin et al., He1v. Chim. Acta, 1995, vol. 78, p. 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3), and 2'-fluoro (2'-F). Similar modifications can be applied to other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetic groups such as cyclobutyl instead of the pentofuranosyl group. 【0320】 In some cases, both the sugar and nucleoside linkages, i.e., the nucleotide unit backbone, can be replaced with novel groups. The base unit can be maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an oligonucleotide mimetic, that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide can be replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobase can be retained and directly or indirectly bonded to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262. Further teachings on PNA compounds can be found in Nielsen et al., Science, Vol. 254: pp. 1497–1500 (1991). 【0321】 Guide RNA may also, in addition or alternatively, include modifications or substitutions of nucleobases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases are nucleobases found only infrequently or transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, in particular 5-methylcytosine (also called 5-methyl-2'deoxycytosine, and often referred to as 5-Me-C in this art), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases, such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazoylalkyl)adenine, 2-(aminoalkyl(alkly)amino)adenine or other heterosubstituted alkyladenines, and 2-thioura This includes syl, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine (Kornberg, A., DNA Replication, WH Freeman & Co., San Francisco, pp. 75-77 (1980); Gebeyehu et al., Nucl. Acids Res., Vol. 15: pp. 45-13 (1997)). "Universal" bases known in the art, such as inosine, can also be incorporated. 5-Me-C substitution has been shown to increase the stability of nucleic acid double helix by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is a form of base substitution. 【0322】 Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine, and 6-azocymine, 5-uracil (Shu 8-Halouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl adenine and 8-hydroxyl guanine, as well as other 8-substituted adenines and 8-substituted guanines, 5-halouracil and 5-halocytosine, in particular, 5-bromouracil and 5-bromocytosine, 5-trifluoromethyluracil and 5-trifluoromethylcytosine, as well as other 5-substituted uracils and 5-substituted cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine This may also include other synthetic and natural nucleobases such as 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine. 【0323】 Furthermore, nucleobases are disclosed in U.S. Patent No. 3,687,808; "The Concise Encyclopedia of Polymer Science and Engineering," pp. 858-859, edited by Kroschwitz, J.I., John Wiley & Sons, 1990. Nucleo bases disclosed in; Nucleo bases disclosed in Englisch et al., "Angewandle Chemie, International Edition", 1991, Vol. 30, p. 613; This may include nucleobases disclosed by Sanghvi, YS, Chapter 15, "Antisense Research and Applications," pp. 289-302, edited by Crooke, ST and Lebleu, B., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid double helix by 0.6-1.2°C (Sanghvi, YS, Crooke, ST). (and Lebleu, B., ed., "Antisense Research and Applications", CRC Press, Boca Raton, 1993, pp. 276-278), more specifically, when combined with 2'-O-methoxyethyl sugar modification, it is a form of base substitution. For modified nucleo bases, in addition to U.S. Patent Nos. 3,687,808, see Nos. 4,845,205; Nos. 5,130,302; Nos. 5,134,066; Nos. 5,175,273; Nos. 5,367,066; Nos. 5,432,272; Nos. 5,457,187; Nos. 5,459,255; Nos. 5,484,908; Nos. 5,502,177; Nos. These patents are described in Patent Nos. 5,525,711; Nos. 5,552,540; Nos. 5,587,469; Nos. 5,596,091; Nos. 5,614,617; Nos. 5,681,941; Nos. 5,750,692; Nos. 5,763,588; Nos. 5,830,653; Nos. 6,005,096; and U.S. Patent Application Publication No. 2003 / 0158403. 【0324】 Therefore, the term "modified" refers to a non-natural sugar, phosphate, or base incorporated into the guide RNA, endonuclease, or both the guide RNA and the endonuclease. Not all positions within a given oligonucleotide need to be uniformly modified; in fact, more than one of the aforementioned modifications can be incorporated into a single oligonucleotide, or into a single nucleoside within an oligonucleotide. 【0325】 mRNA (or DNA) encoding guide RNA and / or endonucleases can be chemically linked to one or more moieties or conjugates that enhance activity, cellular distribution, or cellular uptake of oligonucleotides. Such moieties include lipid moieties such as cholesterol moieties [Letsinger et al., Proc. Natl. Acad., Sci.]. USA, Vol. 86: pp. 6553-6556 (1989); Cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., Vol. 4: pp. 1053-1060 (1994)]; Thioethers, e.g., hexyl-S-tritylthiol [Manoharan et al., Ann. NY Acad. Sci., Vol. 660: pp. 306-309 (1992); and Manoharan et al., Bioorg. Med. Chem. Let., Vol. 3: pp. 2765-2770 (1993)]; Thiocholesterol [Oberhauser et al., Nucl. Acids Res., Vol. 20: pp. 533-538 (1992)]; Aliphatic chains For example, dodecanediol or undecyl residues [Kabanov et al., FEBS Lett., Vol. 259: pp. 327-330 (1990); and Svinarchuk et al., Biochimie, Vol. 75: pp. 49] [Pages 54 (1993)]; Phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., Vol. 36: 3651-365] 4 pages (1995); and Shea et al., Nucl. Acids Res., Vol. 18: pp. 3777-3783 (1990); polyamine or polyethylene glycol chain [Mancharan et al., Nucleosides & Nucleotides, Vol. 14: pp. 969-973 (1995)]; adamantane acetate [Manoharan et al., Tetrahedron Lett., Vol. 36: pp. 3651-3654 (1995)] )]; palmityl portion [(Mishra et al., Biochim. Biophys. Acta, Vol. 1264: pp. 229-237 (1995)]; or octadecylamine or hexylamino-carbonyl-t oxycholesterol portion [Crooke et al., J. Pharmacol. Exp. This includes, but is not limited to, U.S. titles 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; and 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022 ; Same No. 5,254,469; Same No. 5,258,506; Same No. 5,262,536; Same No. 5,272,250; Same No. 5,292,873; Same No. 5,317,098; Same No. 5,371,241; Same No. 5,391,723 See also Nos. 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941. 【0326】 Using sugars and other moieties, proteins and nucleotide-containing complexes, such as cationic polysomes and liposomes, can be targeted to specific sites. For example, hepatocyte-targeted transfer can be mediated by the asialoglycoprotein receptor (ASGPR) (see, e.g., Hu et al., Protein Pept Lett., Vol. 21 (No. 10): pp. 1025-30 (2014)). Other systems known and regularly developed in the art can be used to target the biomolecules and / or complexes used in this case to the desired, specific target cells. 【0327】 These targeting moieties or conjugates may include conjugate groups covalently bonded to functional groups such as primary or secondary hydroxyl groups. The conjugate groups of the present invention include groups that enhance the pharmacodynamic properties of inserts, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In the context of this disclosure, groups that enhance pharmacodynamic properties include groups that improve uptake, groups that enhance resistance to degradation, and / or groups that enhance sequence-specific hybridization with target nucleic acids. In the context of the present invention, groups that enhance pharmacokinetic properties include groups that improve the uptake, distribution, metabolism, or efflux of the compounds of the present invention. Typical conjugate groups are disclosed in International Patent Application PCT / US92 / 09196, filed on October 23, 1992, and in U.S. Patent No. 6,287,860. The conjugate moiety includes, but is not limited to, a lipid moiety such as a cholesterol moiety, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine chain or polyethylene glycol chain, or adamantane acetate, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.For example, U.S. No. 4,828,979; No. 4,948,882; No. 5,218,105; No. 5,525,465; No. 5,541,313; No. 5,545,730; No. 5,552,538; No. 5,578,717; No. 5,580,731; No. 5,580,731; No. 5,591,584; No. 5,109,124; No. 5,118,802; No. 5,138,045; No. 5,414 ,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,2 See issues 03; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941. 【0328】 Long polynucleotides, which are not well-suited to chemical synthesis and are typically produced by enzymatic synthesis, can also be modified by a variety of means. Such modifications may include, for example, the introduction of a particular nucleotide analog, the incorporation of a particular sequence or other portion at the 5' or 3' end of the molecule, and other modifications. For illustrative purposes, the mRNA encoding Cas9 is approximately 4 kb long and can be synthesized by transcription in vitro. Modifications to mRNA can be applied, for example, to increase its translation or stability (such as by increasing its resistance to cellular degradation), or to reduce the RNA's tendency to induce an innate immune response, a tendency often observed in cells after the introduction of exogenous RNA, particularly long RNAs such as the RNA encoding Cas9. 【0329】 In the art, numerous such modifications have been described, including the use of poly(A) tails, 5' cap analogues (e.g., ARCA (Anti-Reverse Cap Analog) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTR), modified bases (such as pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP), or phosphatase treatment to remove the 5' terminal phosphate. These and other modifications are well known in the art, and new RNA modifications are regularly developed. 【0330】 For example, there are numerous commercial suppliers of modified RNA, including TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon, and many other suppliers. As described by TriLink, for example, 5-methyl-CTP can be used to confer desirable characteristics such as increased nuclease stability, increased translation, or reduced interaction of innate immune receptors with transcribed RNA in vitro. Kormann et al. and Warren et al., mentioned below, As illustrated in the publications, 5-methylcytidine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, as well as pseudo-UTP and 2-thio-UTP, have also been shown to enhance translation while reducing innate immunostimulation in culture and in vivo. 【0331】 It has been shown that improved therapeutic effects can be achieved using chemically modified mRNA delivered in vivo (see, for example, Kormann et al., Nature Biotechnology, Vol. 29, pp. 154-157 (2011)). Such modifications can be used, for example, to increase the stability of RNA molecules and / or reduce their immunogenicity. It has been found that using chemical modifications such as pseudo-U, N6-methyl-A, 2-thio-U, and 5-methyl-C to replace only one-quarter of uridine and cytidine residues with 2-thio-U and 5-methyl-C, respectively, results in a significant decrease in Toll-like receptor (TLR)-mediated mRNA recognition in mice. By reducing the activation of the innate immune system, these modifications can be used to effectively increase the stability and lifespan of mRNA in vivo (see, for example, Kormann et al., previously cited). 【0332】 Furthermore, repeated administration of synthetic messenger RNA incorporating modifications designed to circumvent the innate antiviral response has been shown to reprogram differentiated human cells into pluripotency. See, for example, Warren et al., Cell Stem Cell, vol. 7(no. 5): pp. 618-630 (2010). Such modified mRNAs, acting as primary reprogramming proteins, may be an efficient means of reprogramming multiple human cell types. These cells are called induced pluripotent stem cells (iPSCs), and it has been found that enzymatically synthesized RNA incorporating 5-methyl-CTP, pseudo-UTP, and ARCA (Anti-Reverse Cap Analog) can be used to effectively evade the cell's antiviral response (see, for example, Warren et al., cited above). 【0333】 Other modifications of polynucleotides described in the art include, for example, the use of poly(A) tails, the addition of 5' cap analogues (such as m7G(5')ppp(5')G(mCAP)), modification of the 5' or 3' untranslated region UTR, or removal of the 5' terminal phosphate, phosphatase treatment (and new methods are being developed periodically). 【0334】 Several compositions and techniques applicable to the production of modified RNA for use herein have been developed in connection with RNA interference (RNAi) modification, including small interfering RNA (siRNA). siRNAs, through mRNA interference, generally have transient effects on gene silencing, which may require repeated administration, thus presenting certain challenges in vivo. In addition, siRNAs are double-stranded RNAs (dsRNAs), and mammalian cells have an immune response that has evolved to detect and neutralize dsRNAs, which are often byproducts of viral infection. Thus, mammalian enzymes such as PKR (dsRNA-responsive kinase) can mediate cellular responses to dsRNA, and potentially, Toll-like receptors (TLR3, TLR7, and TLR8, etc.) can induce cytokines in response to such molecules, as well as retinoic acid-inducible gene I (RIG-I) (e.g., Angart et al., Pharmaceuticals (Basel), Vol. 6 (No. 4): pp. 440-468 (2013); Kanasty et al., Molecular Therapy, Vol. 20 (No. 3): pp. 513-524 (2012); Burnett et al., Biotechnol J., Vol. 6 (No. 9): pp. 1130-1146 (2011); Judge and MacLachlan Review article in Hum Gene Ther, Vol. 19 (No. 2): pp. 111-124 (2008); (Please also refer to the references cited in these documents.) 【0335】 A wide variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other benefits that may be useful in connection with the introduction of polynucleotides into human cells as described herein (e.g., Whitehead KA et al., Annual Review of Chemical and Biomolecular Engineering, Vol. 2:7). Pages 7-96 (2011); Gaglione and Messere, Mini Rev Med Chem, Vol. 10 ( Issue 7): pp. 578-595 (2010); Chernolovskaya et al., Curr Opin Mol Ther., Vol. 12 (Issue 2): pp. 158-167 (2010); Deleavey et al., Curr Protoc Nucleic Acids Chem, Chapter 16: Part 16.3 (2009); Behlke, Oligonucleotides, 18 Volume (No. 4): pp. 305-319 (2008); Fucini et al., Nucleic Acid Ther, Vol. 22 (Issue 3): pp. 205-210 (2012); Bremsen et al., Front Genet, Vol. 3: 154 See the review article by [page number] (2012). 【0336】 As mentioned above, there are several commercial suppliers of modified RNAs, many of which are specifically designed to improve the efficacy of siRNA. Based on the diverse findings reported in the literature, various methods are offered. For example, Dharmacon is described in Kole, Nature Reviews Drug Discovery, Vol. 11: pp. 125-140 (2012). As previously reported, the replacement of non-crosslinked oxygen with sulfur (phosphorothioate, PS) has been used to improve the nuclease resistance of siRNA. Modification at the 2' position of ribose has been shown to improve the nuclease resistance of the phosphate bond between nucleotides while increasing the double-strand stability (Tm), which also provides protection from immune activation. (Soutschek et al., Nature, 43) As reported in Vol. 2: pp. 173-178 (2004), the combination of moderate PS backbone modification with small, well-tolerated 2'-substitutions (2'-O-methyl, 2'-fluoro, 2'-hydro) is associated with highly stable siRNA for in vivo application, and as reported by Volkov, Oligonucleotides, Vol. 19: pp. 191-202 (2009), 2'-O-methyl modification has been reported to be effective in improving stability. In relation to reducing the induction of innate immune responses, modification of specific sequences with 2'-O-methyl, 2'-fluoro, and 2'-hydro has been reported to reduce TLR7 / TLR8 interactions while generally preserving silencing activity (see, e.g., Judge et al., Mol. Ther., Vol. 13: pp. 494-505 (2006); and Cekaite et al., J. Mol. Biol., Vol. 365: pp. 90-108 (2007)). Further modifications such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize immune activity mediated by TLR3, TLR7, and TLR8 (see, e.g., Kariko, K. et al., Immunity, Vol. 23: pp. 165-175 (2005)). stomach). 【0337】 Furthermore, several conjugates known and commercially available in the art, for example, comprising cholesterol, tocopherol, and folic acid, lipids, peptides, polymers, linkers, and aptamers, which can enhance their delivery and / or uptake by cells, can be applied to polynucleotides such as RNA for use herein (see, for example, the review by Winkler, Ther. Deliv., Vol. 4: pp. 791-809 (2013), and the references cited herein). 【0338】 Codon optimization 【0339】 Polynucleotides encoding site-specific polypeptides can be codon-optimized for expression in cells containing the target DNA of interest, following methods standard in the art. For example, if the intended target nucleic acid is in a human cell, a human polynucleotide with optimized codons encoding Cas9 is envisioned for use in producing the Cas9 polypeptide. 【0340】 Complex of genome-targeting nucleic acid and site-specific polypeptide 【0341】 Genome-targeting nucleic acids interact with site-specific polypeptides (e.g., nucleic acid-guided nucleases such as Cas9) to form a complex. The genome-targeting nucleic acid guides the site-specific polypeptide to the target nucleic acid. 【0342】 RNP 【0343】 Site-specific polypeptides and genome-targeting nucleic acids can each be administered individually to cells or patients. On the other hand, site-specific polypeptides can be pre-complexed with one or more guide RNAs, or one or more crRNAs combined with tracrRNA. The pre-complexed material can then be administered to cells or patients. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs). 【0344】 Nucleic acids that code for the components of a system 【0345】 This disclosure presents nucleic acids comprising nucleotide sequences encoding any nucleic acid or proteinaceous molecule necessary to carry out the genome-targeting nucleic acids of this disclosure, the site-specific polypeptides of this disclosure, and / or embodiments of the methods of this disclosure. 【0346】 The genome-targeting nucleic acids of this disclosure, the site-directed polypeptides of this disclosure, and / or nucleic acids encoding any nucleic acid or proteinaceous molecule necessary to carry out embodiments of the methods of this disclosure may include vectors (e.g., recombinant expression vectors). 【0347】 The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop that can ligate further nucleic acid segments. Another type of vector is a viral vector, which can ligate further nucleic acid segments into a viral genome. Certain vectors are capable of self-replication within the host cell into which they are introduced (e.g., bacterial vectors with origins of replication in bacteria, and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) are integrated into the host cell's genome upon introduction into the host cell, thereby replicating alongside the host genome. 【0348】 In some cases, vectors may be capable of inducing the expression of nucleic acids to which they are operatively linked. Hereinafter, such vectors will be referred to as “recombinant expression vectors,” or more simply, “expression vectors,” but they perform equivalent functions. 【0349】 The term "operably linked" means that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that enables the expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art, for example, Goeddel, Gene Expression Technology, Methods in This was described in Enzymology, Vol. 185, Academic Press, San Diego, CA (1990). It is stated that regulatory sequences include regulatory sequences that lead to the constitutive expression of nucleotide sequences in many types of host cells, and regulatory sequences that lead to the expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will realize that the design of expression vectors may depend on factors such as the selection of target cells and the desired expression level. 【0350】 The proposed expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., mouse leukemia virus, splenic necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. Other proposed vectors for eukaryotic target cells include, but are not limited to, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Further proposed vectors for eukaryotic target cells include, but are not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3, which are shown in Figures 1A-1C. Other vectors may be used insofar as they are compatible with the host cell. 【0351】 In some cases, a vector may contain one or more transcriptional and / or translational regulatory elements. Depending on the host / vector system used, any of several suitable transcriptional and translational regulatory elements, including constitutive and inducible promoters, transcriptional enhancer elements, and transcriptional terminators, may be used within the expression vector. The vector may be a self-inactivating vector that inactivates the viral sequence or components of the CRISPR mechanism or other elements. 【0352】 A non-limiting example of a suitable eukaryotic promoter (i.e., a promoter that functions within a eukaryotic cell) includes cytomegalovirus (CMV) i-initial, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retrovirus-derived LTRs (long terminal repeats), human elongation factor 1 promoter (EF1), hybrid constructs including a cytomegalovirus (CMV) enhancer fused to a chicken beta-actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and a promoter derived from mouse metallothionein I. 【0353】 To express small RNAs, including guide RNAs used in association with Cas endonucleases, a variety of promoters, such as the RNA polymerase III promoter including U6 and H1, may be advantageous. In the art, descriptions of enhancing the use of such promoters and the parameters for this are publicly known, and further information and methods are regularly described (see, for example, Ma, H. et al., Molecular Therapy - Nucleic Acids, Vol. 3, p. e161 (2014), doi:10.1038 / mtna.2014.12). 【0354】 Expression vectors may also contain ribosome-binding sites and transcriptional terminators for translation initiation. Expression vectors may also contain sequences suitable for amplifying expression. Expression vectors may also contain nucleotide sequences encoding non-natural tags (e.g., histidine tags, hemagglutinin tags, green fluorescent protein, etc.) that are fused to site-specific polypeptides, thus resulting in a fusion protein. 【0355】 The promoter may be an inductive promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). The promoter may be a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some cases, the promoter may be a spatially and / or temporally limited promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.). 【0356】 Nucleic acids encoding genome-targeting nucleic acids and / or site-specific polypeptides of this disclosure can be packaged in or on the surface of a delivery medium for delivery to cells. Conceived delivery mediums include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. Various targeting moieties can be used to enhance the preferential interaction of such mediums with desired cell types or locations. 【0357】 The complexes, polypeptides, and nucleic acids of this disclosure can be introduced into cells by means of viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, gene gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. 【0358】 Delivery 【0359】 Guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotides (RNA or DNA) may be delivered by a virus or by non-viral delivery media known in the art. Alternatively, endonuclease polypeptides may be delivered by non-viral delivery media known in the art, such as electroporation or lipid nanoparticles. In further alternative embodiments, DNA endonucleases may be delivered as one or more polypeptides alone, or as one or more polypeptides pre-complexed with one or more guide RNAs or tracrRNAs, or with one or more crRNAs. 【0360】 Polynucleotides can be delivered by nonviral delivery media, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Some exemplary nonviral delivery media are described in Peer and Lieberman, Gene Therapy, Vol. 18: pp. 1127–1133 (2011) (which focuses on nonviral delivery media for siRNA, which are also useful for the delivery of other polynucleotides). 【0361】 Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding endonucleases, can be delivered to cells or patients via lipid nanoparticles (LNPs). 【0362】 LNPs refer to any particles with a diameter of 1000nm, 500nm, 250nm, 200nm, 150nm, 100nm, 75nm, 50nm, or less than 25nm. Alternatively, nanoparticles can be in the size range of 1-1000nm, 1-500nm, 1-250nm, 25-200nm, 25-100nm, 35-75nm, or 25-60nm. 【0363】 LNPs can be prepared from cationic lipids, anionic lipids, or neutral lipids. Neutral lipids such as DOPE (a fusion phospholipid) or cholesterol (a membrane component) can be incorporated into LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to low stability and rapid clearance, as well as the potential for inflammatory or anti-inflammatory responses. 【0364】 LNPs may also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. 【0365】 LNPs can be prepared using any lipid or combination of lipids known in the art. Examples of lipids used to prepare LNPs are DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are PEG-DMG, PEG-CerC14, and PEG-CerC20. 【0366】 LNPs can be synthesized by combining lipids in any molar ratio. In addition, LNPs can be synthesized by combining polynucleotides with lipids in a wide range of molar ratios. 【0367】 As already stated, site-specific polypeptides and genome-targeting nucleic acids can be administered individually to cells or patients. On the other hand, site-specific polypeptides can be pre-complexed with one or more guide RNAs, or one or more crRNAs combined with tracrRNA. The pre-complexed material can then be administered to cells or patients. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs). 【0368】 RNA can form specific interactions with other RNA or DNA. While this property is utilized in many biological processes, it also carries the risk of indiscriminate interactions in the nucleic acid-rich intracellular environment. One solution to this problem is the formation of ribonucleoprotein particles (RNPs) that pre-complex RNA with endonucleases. Another benefit of RNPs is the protection of RNA from degradation. 【0369】 Endonucleases within RNPs can be modified or left unmodified. Similarly, gRNAs, crRNAs, tracrRNAs, or sgRNAs can be modified or left unmodified. Numerous modifications are known and usable in the art. 【0370】 Endonucleases and sgRNAs can be combined in a 1:1 molar ratio. Alternatively, endonucleases, crRNAs, and tracrRNAs can generally be combined in a 1:1:1 molar ratio. However, a wide range of molar ratios can be used to construct RNPs. 【0371】 Recombinant adeno-associated virus (AAV) vectors can be used for delivery. In the art, it is standard practice to produce rAAV particles that deliver a packaged AAV genome, including the polynucleotides, rep and cap genes, and helper virus function to be delivered, into a cell. The production of rAAV typically requires that the following components—the rAAV genome, the AAV rep and cap genes separate from the rAAV genome (i.e., not present within the rAAV genome), and the helper virus function—are present in a single cell (described herein as a packaging cell). The AAV rep and cap genes can be derived from any AAV serotype from which recombinant viruses can be derived, and may be derived from AAV serotypes distinct from the rAAV genome ITR, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. For example, the construction of pseudotyped rAAV is disclosed in International Patent Application Publication WO01 / 83692. See Table 3. [Table 3] 【0372】 The method for creating packaging cells involves the step of creating a cell line that stably expresses all the components necessary for producing AAV particles. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and selectable markers such as neomycin resistance genes is incorporated into the cell genome. The AAV genome is incorporated by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, Vol. 79: pp. 2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, Vol. 23: pp. 65-73), or direct blunt-end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 2 (Volume 59: pp. 4661-4666) The rAAV genome is introduced into a bacterial plasmid. Subsequently, the packaging cell line can be infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to introduce the rAAV genome and / or rep and cap genes into packaging cells using adenovirus or baculovirus instead of plasmid. 【0373】 The general principles of rAAV production are reviewed, for example, in Carter, 1992, *Current Opinions in Biotechnology*, pp. 1533-539; and Muzyczka, 1992, *Curr. Topics in Microbial. and Immunol.*, Vol. 158: pp. 97-129. Various methods are described in Ratschin et al., Mol. Cell. Biol., Vol. 4: 2072 p. (1984); Hermonat et al., Proc. Natl. Acad., Sci. USA, Vol. 81: 6466 p. (1984); Tratschin et al., Mol. Cell. Biol., Vol. 5: 3251 p. (1985); McLaughlin et al., J. Virol., Vol. 62: 1963 p. (1988); and Lebkowski et al., 1988, Mol. Cell. Biol., Vol. 7: 349 p. (1988). Samulski et al. (1989, J. Virol, Vol. 63: pp. 3822-3828); US Patent No. 5,173,414; WO95 / 13365 and corresponding US Patent No. 5,658,776; WO95 / 13392; WO96 / 17947; PCT / US98 / 18600; WO97 / 09441 (PCT / US96 / 14423); WO97 / 08298 (PCT / US96 / 13872); WO97 / 21825 (PCT / US96 / 20777); WO97 / 06243 (PCT / FR96 / 01064); WO99 / 11764; Perrin et al. (1995), Vaccine, Vol. 13: pp. 1244-1250; Paul et al. (1993), Human Gene Therapy, Vol. 4: pp. 609-615; Clark et al. (1996), Gene Therapy, Vol. 3: pp. 1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,8 U.S. Patent No. 71,982; and U.S. Patent No. 6,258,595. 【0374】 The serotype of the AAV vector can be matched to the target cell type. For example, the following exemplary cell types can be transduced with the indicated AAV serotype. See Table 4. [Table 4] 【0375】 Genetically modified cells 【0376】 The term "genetically modified cell" refers to a cell containing at least one genetic modification introduced by genome editing (e.g., using the CRISPR / Cas system). In some ex vivo examples herein, genetically modified cells may be genetically modified origin cells. In some in vivo examples herein, genetically modified cells may be genetically modified muscle cells or genetically modified muscle progenitor cells. Hereinafter, genetically modified cells containing exogenous genome-targeting nucleic acids and / or exogenous nucleic acids encoding genome-targeting nucleic acids are assumed. 【0377】 The term "control-treated population" refers to a population of cells treated with the same medium, viral induction, nucleic acid sequence, temperature, confluence, flask size, pH, etc., except for the addition of genome editing components. Repair of the dystrophin reading frame can be measured using any method known in the art, such as Western blotting analysis of the dystrophin protein or quantification of dystrophin mRNA. 【0378】 The term "isolated cells" refers to cells taken from the organism in which they are originally found, or the offspring of such cells. Optionally, cells can be cultured in vitro, for example, under specified conditions or in the presence of other cells. Optionally, cells can later be introduced into a second organism, and they can be reintroduced into the isolated organism (or cells descended from it). 【0379】 The term “isolated population” in relation to an isolated population of cells refers to a population of cells that has been taken out and separated from a mixed or heterogeneous population of cells. In some cases, an isolated population may be substantially purer than the heterogeneous population from which the cells are isolated or enriched. In some cases, an isolated population may be substantially purer than an isolated population of human primordial cells, for example, a heterogeneous population containing human primordial cells and cells from which human primordial cells are derived. 【0380】 The term "substantially enhanced" in reference to a particular cell population refers to a cell population in which the occurrence of a particular type of cell increases by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least fifty, at least one hundred, at least four hundred, at least one thousand, at least five thousand, at least two hundred thousand, at least one hundred thousand, at least one hundred thousand, at least one hundred thousand, at least two hundred thousand, at least one hundred thousand thousand, at least one hundred thousand thousand, at least one hundred thousand thousand, at least one hundred thousand thousand, at least one hundred thousand thousand, or more, compared to the existing or reference level, depending on the desired level of such cells, for example, to improve DMD. 【0381】 The term "substantially enriched" in reference to a particular cell population refers to a population that represents at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or more of the cells that make up the entire cell population. 【0382】 The term “substantially pure” with respect to a particular cell population refers to a cell population that is at least about 75%, at least about 85%, at least about 90%, or at least about 95% pure with respect to the cells that make up the entire cell population. That is, with respect to a primordial cell population, the terms “substantially pure” or “essentially purified” refer to a cell population that contains fewer than 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than 1% of cells that are not primordial cells as defined herein. 【0383】 Differentiation of corrected iPSCs into Pax7+ myoprimordial cells 【0384】 Another step of the ex vivo method of this disclosure involves differentiating the corrected iPSCs into Pax7+ myoprimordial cells. The differentiation step can be carried out according to any method known in the art. For example, the differentiation step is described in Chal, Oginuma et al., 201 As shown in 5 years, this may involve contacting genome-edited iPSCs with a specific culture medium formulation containing small molecule drugs to differentiate them into Pax7+ myogenic cells. Alternatively, iPSCs, myogenic cells, and other cell lineages are used, as described by Tapscott, Davis et al., 1988. Langen, Schols et al., 2003; Fujita, Endo et al., 2010; Xu, Tabebordbar et al. As demonstrated in 2013; and in the method of Shoji, Woltjen et al., 2015, differentiation into muscle can be achieved using any one of several established methods involving transgene overexpression, serum depletion, and / or small molecule drugs. 【0385】 Implantation of Pax7+ myoprimordial cells into patients 【0386】 Another step of the ex vivo method of this disclosure involves implanting Pax7+ myoprimordial cells into a patient. This implantation step can be achieved using any implantation method known in the art. For example, genetically modified cells can be injected directly into the patient's muscle. 【0387】 Pharmacologically acceptable carriers 【0388】 The ex vivo methods for administering progenitor cells to a subject as envisioned herein involve the use of a therapeutic composition containing progenitor cells. 【0389】 The therapeutic composition may contain a physiologically tolerable carrier in combination with the cell composition, and optionally, at least one further bioactive agent as described herein, in which the bioactive agent is dissolved or dispersed as an active ingredient. In some cases, the therapeutic composition may not be substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless otherwise desired. 【0390】 In general, the progenitor cells described herein can be administered as a suspension with a pharmaceutically acceptable carrier. Those skilled in the art will recognize that the pharmaceutically acceptable carrier used in the cell composition does not contain buffers, compounds, cryopreservatives, preservatives, or other agents in amounts that substantially interfere with the viability of the cells delivered to the subject. The cell formulation may, for example, include an osmotic buffer that allows for the maintenance of cell membrane integrity, and optionally, nutrients that maintain cell viability or enhance engraftment at the time of administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the progenitor cells described herein using prescribed experiments. 【0391】 The cell composition may also be emulsified and presented as a liposome composition, provided that the emulsification procedure does not adversely affect the viability of the cells. The cells and any other active ingredients may be mixed with excipients in amounts suitable for use in the therapeutics described herein, provided that the excipients are pharmaceutically acceptable, compatible with the active ingredients. 【0392】 Further agents contained in the cell composition may include pharmaceutically acceptable salts of its components. These pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups of polypeptides) formed by inorganic acids such as hydrochloric acid or phosphoric acid, or by organic acids such as acetic acid, tartaric acid, or mandelic acid. Salts formed by free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine. 【0393】 In the art, physiologically tolerable carriers are well known. Exemplary liquid carriers are sterile aqueous solutions containing the active ingredient and water, plus buffers such as sodium phosphate, physiological saline, or phosphate-buffered physiological saline, or both, at a physiological pH value. Furthermore, aqueous carriers may contain one or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases with and without water. Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in a cell composition effective for treating a particular disorder or condition may depend on the nature of the disorder or condition and can be determined by standard clinical methods. 【0394】 Administration and efficacy 【0395】 In the context of transferring cells, such as progenitor cells, into a subject by a method or route that results in at least partial localization of the introduced cells to a desired site, such as a site of injury or repair, to produce the desired effect, the terms “administering,” “introducing,” and “implanting” are used interchangeably. Cells, such as progenitor cells, or their differentiated offspring, can be administered by any suitable route that results in delivery to a desired site in the subject, in which case at least some of the implanted cells or components of the cells remain viable. The cell survival period after administration to the subject can be as short as a few hours, e.g., 24 hours, to several days, to several years, or even longer, i.e., the patient’s lifetime, i.e., long-term engraftment. For example, in some embodiments described herein, an effective amount of myogenic progenitor cells is administered via a systemic route of administration, such as an intraperitoneal or intravenous route. 【0396】 In this specification, the terms “individual,” “subject,” “host,” and “patient” are used interchangeably to refer to any subject for which diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. 【0397】 When administered prophylactically, the myoprimordial cells described herein can be administered to a subject prior to any symptom of DMD, for example, before the onset of muscle loss. Therefore, prophylactic administration of myoprimordial cell populations can be used to prevent DMD. 【0398】 When administered therapeutically, myoprimordial cells can be administered at the onset (or after the onset) of symptoms or signs of DMD, for example, at the onset of muscle loss. 【0399】 The myoprimordial cell population administered according to the methods described herein may include allogeneic myoprimordial cells obtained from one or more donors. "Allogeneic" means myoprimordial cells or myoprimordial cell-containing biological specimens obtained from one or more different donors of the same species, wherein the genes in one or more loci are not identical. For example, the myoprimordial cell population administered to a subject may originate from one or more unrelated donor subjects, or from one or more non-identical siblings. Depending on the case, syngeneic myoprimordial cell populations, such as populations obtained from genetically identical animals or identical twins, may also be used. The myoprimordial cells may be autologous cells, that is, myoprimordial cells obtained from or isolated from a subject and administered to the same subject, i.e., the donor and recipient are the same. 【0400】 The term “effective dose” refers to the amount of progenitor cells or a population of their offspring required to prevent or alleviate at least one sign or symptom of DMD, and relates to the amount of a composition sufficient to treat, for example, a subject having DMD, to produce the desired effect. Thus, the term “therapeutic effective dose” refers to the amount of progenitor cells or a composition containing progenitor cells sufficient to promote a particular effect when administered to a typical subject, such as a subject having or at risk of having DMD. An effective dose would also include an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., slow the progression of disease symptoms), or reverse disease symptoms. It is understood that, in any given case, a suitable “effective dose” can be determined by a person skilled in the art using prescribed experiments. 【0401】 For use in the various embodiments described herein, an effective amount of progenitor cells is provided, with at least 10 progenitor cells. 2 Individual cells, at least 5 × 10¹⁶ primordial cells 2 individual, at least 10 primordial cells 3 Individual cells, at least 5 × 10¹⁶ primordial cells 3cells, at least 10 progenitor cells 4 cells, at least 5×10 progenitor cells 4 cells, at least 10 progenitor cells 5 cells, at least 2×10 progenitor cells 5 cells, at least 3×10 progenitor cells 5 cells, at least 4×10 progenitor cells 5 cells, at least 5×10 progenitor cells 5 cells, at least 6×10 progenitor cells 5 cells, at least 7×10 progenitor cells 5 cells, at least 8×10 progenitor cells 5 cells, at least 9×10 progenitor cells 5 cells, at least 1×10 progenitor cells 6 cells, at least 2×10 progenitor cells 6 cells, at least 3×10 progenitor cells 6 cells, at least 4×10 progenitor cells 6 cells, at least 5×10 progenitor cells 6 cells, at least 6×10 progenitor cells 6 cells, at least 7×10 progenitor cells 6 cells, at least 8×10 progenitor cells 6 cells, at least 9×10 progenitor cells 6 cells, or include multiples of these. Progenitor cells can also be derived from one or more donors and can be obtained from autologous sources. In some examples described herein, progenitor cells can be expanded in culture prior to administration to a subject that requires them. 【0402】 Gradually increasing the level of functional dystrophin expressed in cells of patients with DMD may be beneficial in improving one or more symptoms of the disease, increasing long-term survival, and / or reducing side effects associated with other treatments. When such cells are administered to human patients, the presence of myoprimordial cells that result in elevated functional dystrophin levels is beneficial. In some cases, effective treatment of a target results in at least approximately 3%, 5%, or 7% functional dystrophin compared to total dystrophin in the treated subject. In some cases, functional dystrophin may account for at least approximately 10% of total dystrophin. In other cases, functional dystrophin may account for at least approximately 20%–30% of total dystrophin. Similarly, in some situations, normalized cells have a selective advantage over affected cells, so even the introduction of a relatively limited subpopulation of cells with significantly elevated functional dystrophin levels may still be beneficial in a variety of patients. However, even small levels of myoprimordial cells that increase functional dystrophin levels can be beneficial in improving one or more aspects of DMD in patients. In some cases, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more myoprimordial cells in patients treated with such cells produce high levels of functional dystrophin. 【0403】 "Administered" means the delivery of the progenitor cell composition to the subject by a method or route that results in at least partial localization of the cell composition at the desired site. The administration of the cell composition by any suitable route that results in an effective treatment at the subject, i.e., delivery to the desired site at the subject, includes at least a portion of the composition to be delivered, i.e., at least 1 × 10 cells 4Cells can be administered by delivery to a desired site over a period of time. Administration methods include injection, infusion, intravenous infusion, or oral administration. “Injection” includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In some cases, the route is intravenous. To deliver cells, administration may be carried out by injection or infusion. 【0404】 Cells are administered systemically. The terms “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” refer to administration of a population of progenitor cells other than direct administration to a target site, tissue, or organ, where instead they enter the target circulatory system and are therefore subjected to metabolism and other similar processes. 【0405】 A person skilled in the art can determine the efficacy of a treatment comprising a composition for treating DMD. However, to give only a few examples, a treatment is considered “effective” if any or all of the signs or symptoms of functional dystrophin levels are modified in a beneficial manner (e.g., increased by at least 10%), or if other clinically acceptable symptoms or markers of the disease are improved or alleviated. Efficacy can also be measured by the non-exacerbation of the individual (e.g., reduction of muscle loss or cessation or at least slowing of disease progression), which is assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to a person skilled in the art and / or are described herein. A treatment includes any treatment of the disease in an individual or animal (some non-limiting examples include humans or mammals), and includes (1) inhibiting the disease, e.g., cessation or slowing of symptom progression; or (2) alleviating the disease, e.g., causing a reduction in symptoms; and (3) preventing or reducing the likelihood of symptom onset. 【0406】 Treatments according to this disclosure may alleviate one or more symptoms associated with DMD by increasing the amount of functional dystrophin in the individual. Early signs typically associated with DMD include, for example, delayed gait, calf muscle hypertrophy (due to scar tissue), and frequent falls. As the disease progresses, children become wheelchair-bound due to muscle loss and pain. The disease becomes fatal due to cardiac and / or respiratory complications. 【0407】 kit 【0408】 This disclosure presents a kit for carrying out the methods described herein. The kit may include one or more, or any combination thereof, of genome-targeting nucleic acids, polynucleotides encoding genome-targeting nucleic acids, site-specific polypeptides, polynucleotides encoding site-specific polypeptides, and / or any nucleic acid or proteinaceous molecules necessary to carry out embodiments of the methods described herein. 【0409】 The kit may include (1) a vector containing a nucleotide sequence encoding a genome-targeting nucleic acid, (2) a site-specific polypeptide or a vector containing a nucleotide sequence encoding a site-specific polypeptide, and (3) reagents for repairing and / or diluting the vector and / or polypeptide. 【0410】 The kit may include (1) a vector comprising (i) a nucleotide sequence encoding a genome-targeting nucleic acid and (ii) a nucleotide sequence encoding a site-specific polypeptide, and (2) reagents for repairing and / or diluting the vector. 【0411】 Some kits may include a single-molecule genome targeting guide nucleic acid. Any of the above kits may include a dual-molecule genome targeting nucleic acid. Any of the kits may include two or more dual-molecule or single-molecule guides. The kits may include vectors encoding nucleic acid targeting nucleic acids. 【0412】 In some of the kits, the kit may further contain polynucleotides that are inserted to produce the desired genetic modification. 【0413】 The components of the kit may be in separate containers, or they may be combined within a single container. 【0414】 Any kit may further include one or more additional reagents, in which case such additional reagents are selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, wash buffers, control reagents, control vectors, control RNA polynucleotides, reagents for in vitro preparation of polypeptides from DNA, adapters for sequencing, etc. Buffers may be stabilizing buffers, repair buffers, dilution buffers, etc. The kit may also include one or more components that can be used to facilitate or enhance binding to the on-target or cleavage of DNA by endonucleases, or to improve targeting specificity. 【0415】 In addition to the components mentioned above, the kit may further include instructions for performing the method using the components of the kit. Instructions for performing the method may be recorded on a suitable recording medium. For example, instructions may be printed on a substrate such as paper or plastic. Instructions may also be present within the kit as package documentation, or on the labeling of the kit or its components (i.e., associated with packaging or partial packaging). Instructions may also exist as electronically stored data files on a suitable computer-readable storage medium, such as a CD-ROM, diskette, or flash drive. In some cases, the actual instructions may not be present within the kit, but a means of obtaining the instructions from a remote source (e.g., via the internet) may be provided. An example of this is a kit that includes a web address from which the instructions can be viewed and / or downloaded. Similar to the instructions themselves, this means of obtaining the instructions may be recorded on a suitable substrate. 【0416】 Guide RNA preparations 【0417】 The guide RNAs of this disclosure can be formulated with pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, and diluents, depending on the specific administration method and dosage form. The guide RNA compositions can be formulated to achieve a physiologically compatible pH, depending on the formulation and route of administration, ranging from approximately 3 to approximately 11, and from approximately 3 to approximately 7. Optionally, the pH can be adjusted to a range of approximately 5.0 to approximately 8. Optionally, the compositions may contain at least one therapeutically effective amount of one of the compounds described herein, in combination with one or more pharmaceutically acceptable excipients. Optionally, the compositions may include combinations of the compounds described herein, a second active ingredient (e.g., an antimicrobial agent or antimicrobial agent, to the effect of, and without limitation, an antimicrobial agent) useful in treating or preventing bacterial growth, or combinations of the reagents of this disclosure. 【0418】 Suitable excipients include carrier molecules containing large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients may include antioxidants (e.g., ascorbic acid), chelating agents (e.g., EDTA), carbohydrates (e.g., dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (e.g., oil, water, physiological saline, glycerol, and ethanol), humectants or emulsifiers, pH buffers, and the like. 【0419】 Other possible treatments 【0420】 Gene editing can be performed using nucleases engineered to target specific sequences. Currently, there are four main types of nucleases: meganucleases and their derivatives, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 nuclease system. In particular, the specificity of ZFNs and TALENs is mediated through protein-DNA interactions, while RNA-DNA interactions primarily guide Cas9, so nuclease platforms vary in design difficulty, targeting density, and mode of action. Cas9 cleavage also requires PAMs, which are different flanking motifs between different CRISPR systems. Cas9 from Streptococcus pyogenes cleaves using the PAM NGG, while CRISPR from Neisseria meningitidis can cleave at sites with PAMs including NNNNGATT, NNNNGTTTT, and NNNNGCTT. Some other Cas9 orthologues target alternative PAMs and adjacent protospacers. 【0421】 The methods of this disclosure may use CRISPR endonucleases such as Cas9. However, the teachings described herein, including the target site for treatment, may also be applied to other forms of endonucleases such as ZFNs, TALENs, HEs, or MegaTALs, and may also be applied to the use of nuclease combinations. However, applying the teachings of this disclosure to such endonucleases will, in particular, require the manipulation of proteins that are directed to specific target sites. 【0422】 Further binding domains can be fused to the Cas9 protein to increase specificity. The target sites of these constructs map to identified gRNA designation sites, but would require additional binding motifs, such as zinc finger domains. In the case of Mega-TAL, a meganuclease can be fused to the TALE DNA-binding domain. The meganuclease domain can increase specificity and lead to cleavage. Similarly, inactivated Cas9 or dead Cas9 (dCas9) can be fused to a cleavage domain, which requires an sgRNA / Cas9 target site and an adjacent binding site for the fused DNA-binding domain. This would likely require some protein manipulation of dCas9 in addition to catalytic inactivation to reduce binding if no further binding sites are present. 【0423】 Zinc finger nuclease 【0424】 Zinc finger nucleases (ZFNs) are modular proteins composed of a modified zinc finger DNA-binding domain linked to the catalytic domain of the type II endonuclease FokI. Since FokI functions only as a dimer, a pair of ZFNs must be manipulated to bind to a target "half-site" sequence of the cognitive on the reverse DNA strand, with a precise spacing between them, thereby enabling the formation of a catalytically active FokI dimer. When the FokI domain, which does not inherently possess sequence specificity, dimerizes, a DNA double-strand break is created between the ZFN half-sites, serving as the initiation step in genome editing. 【0425】 The DNA-binding domain of each ZFN consists of 3 to 6 zinc fingers in a complex Cys2-His2 architecture. Typically, each finger is composed of zinc fingers that primarily recognize a nucleotide triplet on one strand of the target DNA sequence, although cross-interaction with a fourth nucleotide can also be important. Modifications to the amino acids of the fingers at the key contact points with DNA alter the sequence specificity of a given finger. Thus, a 4-finger zinc finger protein selectively recognizes a 12 bp target sequence, although the triplet priority is variable and can be influenced by adjacent fingers; the target sequence is a complex of triplet priorities to which each finger contributes. A key aspect of ZFNs is that they can be easily retargeted to almost any genomic address by modifying individual fingers, but doing so effectively requires considerable skill. Most ZFN applications use 4 to 6-finger proteins, each recognizing 12 to 18 bp. Therefore, a pair of ZFNs would typically recognize a combination of target sequences of 24–36 bp, without a typical 5–7 bp spacer between half-sites. The binding sites can be further separated by a larger spacer containing 15–17 bp. Target sequences of this length are likely unique within the human genome, assuming that repeat sequences or gene homologs are excluded during the design process. However, since ZFN protein-DNA interactions are not absolute in their specificity, off-target binding and cleavage events can occur as heterodimers between two ZFNs, or as homodimers by one or the other ZFN. The latter possibility is effectively eliminated by manipulating the dimerization interface of the FokI domain to create “plus” and “minus” variants, also known as obligate heterodimer variants, which can only dimerize with each other and cannot dimerize by themselves. By forcing obligate heterodimerization, homodimerization is prevented. This greatly enhances the specificity of ZFNs, as well as any other nucleases employing these FokI variants. 【0426】 In this technical field, various ZFN-based systems have been described, their modifications are reported regularly, and numerous references describe the rules and parameters used to guide ZFN design (e.g., Segal et al., Proc Natl Acad Sci USA, Vol. 96). (No. 6): pp. 2758-2763 (1999); Dreier B et al., J Mol Biol., Vol. 303 (No. 4): pp. 489-502 (2000); Liu Q et al., J Biol Chem., Vol. 277 (No. 6): pp. 3850-386 (2002); Dreier et al., J Biol Chem, Vol. 280 (No. 42): See pp. 35588-97 (2005); and Dreier et al., J Biol Chem., Vol. 276 (No. 31): pp. 29466-78 (2001). 【0427】 Transcription activator-like effector nucleases (TALENs) 【0428】 TALEN is another format of modular nuclease, similar to ZFN, in which a manipulated DNA-binding domain is ligated to a FokI nuclease domain, and a pair of TALENs operate in tandem to achieve targeted DNA cleavage. The main difference from ZFN lies in the nature of the DNA-binding domain and its associated target DNA sequence recognition properties. The DNA-binding domain of TALEN originates from the TALE protein, originally described in the plant bacterial pathogen Xanthomonas sp. TALE is a tandem array of 33-35 amino acid repeats, typically a target DNA sequence up to 20 bp in length, with the total target sequence length up to 40 bp, where each repeat recognizes a single base pair within the target DNA sequence. The nucleotide specificity of each repeat is determined by the RVD (repeat variable diresidue), which contains only two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine, and thymine are primarily recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. This constitutes a much simpler recognition code than that of zinc fingers and therefore represents an advantage over the latter for nuclease design. However, like ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs also benefit from the use of obligate heterodimer variants of the FokI domain to reduce off-target activity. 【0429】 Further variants of the FokI domain have been created, in which their catalytic functions are inactivated. If one of the TALEN or ZFN pair contains an inactive FokI domain, only single-strand DNA breaks (nicking) will occur at the target site, rather than double-strand breaks (DSBs). The results are equivalent to using a CRISPR / Cas9 / Cpf1 "nickase" mutant in which one of the Cas9 cleavage domains is inactivated. DNA nicks can be used to drive HDR-mediated genome editing, but the efficiency is lower than that of DSBs. The main benefit is that off-target nicks are repaired rapidly and accurately, unlike DSBs, which are susceptible to misrepair mediated by NHEJs. 【0430】 In this field, various TALEN-based systems have been described, and modifications to them are regularly reported (e.g., Boch, Science, Vol. 326 (No. 5959): 1509~). 12 pages (2009); Mak et al., Science, Vol. 335 (No. 6069): pp. 716-719 (2012); and Moscou et al., Science, Vol. 326 (No. 5959): p. 1501 (2009) See (year). The use of TALENs based on the "GoldenGate" platform or cloning scheme has been described by several groups (e.g., Cermak et al., Nucleic Acids Res., Vol. 39 (No. 12): e82 (2011); Li et al., Nucleic Acids Res., Vol. 39 (No. 14): e6315-6325 (2011); Weber et al., PLoS One., Vol. 6 (No. 2): e16765 (2011); Wang et al., J Genet Genomics, Vol. 41 (No. 6): e339-347, Epub, May 17, 2014 (201 See also Cermak T et al., Methods Mol Biol., Vol. 1239: pp. 133-159 (2015). 【0431】 Homing Endonuclease Homing endonucleases (HEs) are sequence-specific endonucleases that possess long recognition sequences (14–44 base pairs) and cleave DNA with high specificity (often to specific sites within the genome). There are at least six known families of HEs, classified by their structure, derived from a wide range of hosts including eukaryotes, protists, bacteria, archaea, cyanobacteria, and phages, including LAGLIDADG (SEQ ID NOs. 1,410,474), GIY-YIG, His-Cis box, HNH, PD-(D / E)xK, and Vsr-like enzymes. Similar to ZFNs and TALENs, HEs can be used as a first step in genome editing to create double-strand breaks (DSBs) at a target locus. In addition, some native and engineered HEs cleave only single strands of DNA, thereby functioning as site-specific nickases. Due to the large target sequences of HEs and the specificity they provide, HEs are attractive candidates for creating site-specific DSBs. 【0432】 In this technical field, various HE-based systems have been described, and modifications to them are reported regularly (e.g., Steentoft et al., Glycobiology, vol. 24(no. 8):663-80). See the review articles by Belfort and Bonocora, Methods Mol Biol., Vol. 1123: pp. 1-26 (2014); and Hafez and Hausner, Genome, Vol. 55 (No. 8): pp. 553-69 (2012), as well as the references cited therein. 【0433】 MegaTAL / Tev-mTALEN / MegaTev 【0434】 As further examples of hybrid nucleases, the MegaTAL and Tev-mTALEN platforms utilize a fusion of the TALE DNA-binding domain and catalytically active HE, leveraging both the tunable DNA-binding and TALE specificity, as well as the cleavage sequence specificity of HE (e.g., Boissel et al., NAR, Vol. 42: pp. 2591-2601 (2014); Kleinstiver et al., G3, Vol. 4: pp. 1155-1165). (2014); see also Boissel and Scharenberg, Methods Mol. Biol., Vol. 1239: pp. 171–96 (2015). 【0435】 In a further variation, the MegaTev architecture is a fusion of the meganuclease (Mega) with I-TevI(Tev), a nuclease domain derived from the GIY-YIG homing endonuclease. The two active sites are located approximately 30 bp apart on the DNA substrate and generate two double-segment breaks (DSBs) with incompatible sticky ends (see, e.g., Wolfs et al., NAR, vol. 42, pp. 8816-8829 (2014)). It is expected that other combinations of existing nuclease-based methods will evolve and prove useful in achieving targeted genome modifications as described herein. 【0436】 dCas9-FokI or dCpf1-Fok1 and other nucleases 【0437】 By combining the structural and functional properties of the nuclease platform described above, we can provide further methods for genome editing that potentially overcome some of the inherent shortcomings. For example, CRISPR genome editing systems typically use a single Cas9 endonuclease to create double-side blocks (DSBs). Targeting specificity is driven by a 20 or 24-nucleotide sequence in the guide RNA, which undergoes Watson-Crick base pairing with the target DNA (in the case of Cas9 from S. pyogenes, this includes two additional bases in the PAM sequence, which are adjacent NAG or NGG). While such sequences are long enough to be unique within the human genome, the specificity of the RNA / DNA interaction is not absolute, and in some cases, significant indifferentiation is tolerated, particularly at the 5' end of the target sequence, effectively reducing the number of bases that drive specificity. One solution to this was to fuse the FokI domain to the inactivated Cas9, instead of completely deactivating the catalytic function of Cas9 or Cpf1 (while retaining only the RNA-guided DNA binding function) (see, for example, Tsai et al., Nature Biotech, Vol. 32: pp. 569-576 (2014); and Guilinger et al., Nature Biotech, Vol. 32: pp. 577-572 (2014)). Since FokI must dimerize to become catalytically active, two guide RNAs are required to tether the two FokI fusions in close proximity to form a dimer and cleave the DNA. This essentially doubles the number of bases in the target site combination, thereby increasing the precision of targeting by CRISPR-based systems. 【0438】 As a further example, the fusion of the TALE DNA-binding domain to catalytically active HEs such as I-TevI ​​is expected to further reduce off-target cleavage, utilizing both the tunable DNA-binding and TALE specificity, as well as the cleavage sequence specificity of I-TevI. 【0439】 Methods and compositions of the present invention 【0440】 Accordingly, this disclosure relates in particular to the following non-limiting inventions. In Method 1, the first method, the disclosure presents a method for editing the dystrophin gene in a human cell by genome editing, comprising the step of introducing one or more deoxyribonucleic acid (DNA) endonucleases into a human cell to produce one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the dystrophin gene, which result in the permanent deletion, insertion, or replacement of one or more exons or abnormal intron splice acceptor or donor sites in or near the dystrophin gene, thereby repairing the dystrophin reading frame and repairing the activity of the dystrophin protein. 【0441】 In another method, Method 2, the present disclosure presents a method for editing the dystrophin gene in human cells by genome editing, wherein the human cells are muscle cells or muscle progenitor cells. 【0442】 In another method, Method 3, the present disclosure presents an ex vivo method for treating patients with Duchenne muscular dystrophy (DMD), comprising the steps of: i) creating DMD patient-specific induced pluripotent stem cells (iPSCs); ii) editing the dystrophin gene of the iPSCs in or near it; iii) differentiating the genome-edited iPSCs into Pax7+ myoprimordial cells; and iv) implanting the Pax7+ myoprimordial cells into the patient. 【0443】 In another method, Method 4, the present disclosure presents an ex vivo method for treating a patient with DMD, as presented in Method 3, the method comprising the steps of a) isolating somatic cells from the patient; and b) introducing a set of pluripotency-related genes into the somatic cells to induce the somatic cells to become pluripotent stem cells. 【0444】 In another method, Method 5, the present disclosure presents an ex vivo method for treating a patient with DMD, as presented in Method 4, wherein the somatic cells are fibroblasts. 【0445】 In an alternative method, Method 6, the present disclosure presents an ex vivo method for treating patients with DMD, as presented in Methods 4 and 5, wherein the set of pluripotency-related genes is one or more genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC. 【0446】 In an alternative method, Method 7, the present disclosure presents an ex vivo method for treating patients with DMD, as presented in any one of Methods 3 to 6, wherein the editing step involves introducing one or more deoxyribonucleic acid (DNA) endonucleases into an iPSC to produce one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the dystrophin gene, resulting in the permanent deletion, insertion, or replacement of one or more exons or abnormal intron splice acceptor or donor sites in or near the dystrophin gene, thereby resulting in the repair of the dystrophin reading frame and the repair of the activity of the dystrophin protein. 【0447】 In another method, Method 8, the present disclosure presents an ex vivo method for treating patients with DMD, as presented in any one of Methods 3 to 7, wherein the differentiation step comprises one or more of the following: contacting genome-edited iPSCs with a specific culture medium formulation containing a small molecule drug; trans gene overexpression; or serum depletion, to differentiate the genome-edited iPSCs into Pax7+ myoprimordial cells. 【0448】 In another method, Method 9, the present disclosure presents an ex vivo method for treating a patient with DMD, as presented in any one of Methods 3 to 8, wherein the implantation step includes implanting Pax7+ myoprimordial cells into the patient by local injection into a desired muscle. 【0449】 In another method, Method 10, the present disclosure presents an in vivo method for treating a patient with DMD, which includes the step of editing the dystrophin gene in the patient's cells. 【0450】 In another method, Method 11, the present disclosure presents an in vivo method for treating a patient with DMD, as presented in Method 10, wherein the editing step involves introducing one or more deoxyribonucleic acid (DNA) endonucleases into the patient's cells to produce one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in or near the dystrophin gene, resulting in the permanent deletion, insertion, or replacement of one or more exons or abnormal intron splice acceptor sites or donor sites in or near the dystrophin gene, thereby resulting in the repair of the dystrophin leading frame and the repair of the activity of the dystrophin protein. 【0451】 In another method, Method 12, the present disclosure presents an in vivo method for treating a patient with DMD, as presented in Method 11, wherein the cells are muscle cells or muscle progenitor cells. 【0452】 In another method, Method 13, the present disclosure provides an in vivo method for treating a patient with DMD, as presented in any one of Methods 1, 7, and 11, wherein one or more DNA endonucleases are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6 We present in vivo methods for Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonucleases; their homologs, recombinants of these naturally occurring molecules, codon-optimized versions of these, modified forms of these, and combinations thereof. 【0453】 In another method, Method 14, the present disclosure presents a method presented in Method 13, which includes the step of introducing one or more polynucleotides encoding one or more DNA endonucleases into a cell. 【0454】 In another method, Method 15, the present disclosure presents a method presented in Method 13, which includes the step of introducing one or more ribonucleic acids (RNAs) encoding one or more DNA endonucleases into a cell. 【0455】 In another method, Method 16, the Disclosure presents a method presented in Methods 14 and 15, wherein one or more polynucleotides or one or more RNAs are one or more modified polynucleotides or one or more modified RNAs. 【0456】 In another method, Method 17, the Disclosure presents a method presented in Method 13, wherein one or more DNA endonucleases are one or more proteins or polypeptides. 【0457】 In another method, Method 18, the present disclosure presents a method presented in any one of Methods 1 to 17, further comprising the step of introducing one or more guide ribonucleic acids (gRNAs) into cells. 【0458】 In another method, Method 19, the Disclosure presents a method presented in Method 18, wherein one or more gRNAs are single-molecule guide RNAs (sgRNAs). 【0459】 In another method, Method 20, the Disclosure presents a method presented in Methods 18 and 19, wherein one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs. 【0460】 In another method, Method 21, the Disclosure presents a method, as presented in any one of Methods 18-20, in which one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs. 【0461】 In another method, Method 22, the present disclosure presents a method presented in any one of Methods 1 to 21, further comprising the step of introducing a polynucleotide donor template containing at least a portion of the wild-type dystrophin gene or cDNA into a cell. 【0462】 In another method, Method 23, the present disclosure relates to at least one portion of the gene or cDNA of wild-type dystrophin, specifically exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 42, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 The present invention provides a method as presented in Method 22, comprising exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or at least a portion of the dystrophin gene or the entire cDNA. 【0463】 In another method, Method 24, the present disclosure relates to at least one portion of the gene or cDNA of wild-type dystrophin, specifically exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42. Method 22 presents a method comprising exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the dystrophin gene or the entire cDNA. 【0464】 In another method, Method 25, the Disclosure presents a method presented in any one of Methods 22-24, wherein the donor template is a single-stranded or double-stranded polynucleotide. 【0465】 In an alternative method, Method 26, the present disclosure further comprises the step of introducing one or more guide ribonucleic acids (gRNAs) into a cell, wherein the one or more DNA endonucleases are one or more Cas9 or Cpf1 endonucleases that produce an SSB or DSB which is a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs) which are a first SSB or DSB break in the 5' locus and a second SSB or DSB break in the 3' locus, resulting in a permanent deletion or replacement of one or more exons or abnormal intron splice acceptor or donor sites between the 5' and 3' locus in or near the dystrophin gene, resulting in repair of the dystrophin leading frame and repair of the activity of the dystrophin protein, as presented in any one of Methods 1, 7, and 11. 【0466】 In another method, Method 27, the Disclosure presents a method, as presented in Method 26, in which one gRNA generates a pair of SSBs or DSBs. 【0467】 In another method, Method 28, the present disclosure presents a method presented in Method 26, wherein a single gRNA includes a spacer sequence that is complementary to the 5' locus, the 3' locus, or the segment between the 5' locus and the 3' locus. 【0468】 In an alternative method, Method 29, the present disclosure presents a method presented in Method 26, comprising a first gRNA and a second gRNA, wherein the first gRNA comprises a spacer sequence complementary to the 5' locus segment, and the second gRNA comprises a spacer sequence complementary to the 3' locus segment. 【0469】 In another method, Method 30, the present disclosure presents a method presented in Methods 26-29 in which one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs). 【0470】 In another method, Method 31, the Disclosure presents a method, as presented in Methods 26-30, in which one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs. 【0471】 In another method, Method 32, the Disclosure presents a method, as presented in any one of Methods 26-31, in which one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs. 【0472】 In another method, Method 33, the present disclosure presents a method presented in any one of Methods 26-32, wherein a deletion of chromosomal DNA exists between the 5' locus and the 3' locus. 【0473】 In another method, Method 34, the Disclosure presents a method presented in any one of Methods 26-33, wherein the deletion is a single exon deletion. 【0474】 In another method, Method 35, the Disclosure presents a method presented in Method 34 in which the deletion of a single exon is a deletion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, or exon 53. 【0475】 In another method, Method 36, the Disclosure presents a method presented in Method 34 or 35, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53. 【0476】 In another method, Method 37, the Disclosure presents a method presented in any one of Methods 34 to 36, wherein the 3' locus is proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53. 【0477】 In another method, Method 38, the Disclosure presents a method presented in any one of Methods 34-37, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53, and the 3' locus is proximal to the 3' boundaries of these exons. 【0478】 In another method, Method 39, the Disclosure presents a method, as presented in any one of Methods 36-38, in which the proximal portion of an exon to its boundary includes splice donors and acceptors around an adjacent intron. 【0479】 In another method, Method 40, the Disclosure presents a method presented in any one of Methods 26-33, wherein the deletion is a multi-exon deletion. 【0480】 In another method, Method 41, the Disclosure presents a method presented in Method 40 in which the multi-exon deletion is a deletion of exons 45-53 or exons 45-55. 【0481】 In another method, Method 42, the Disclosure presents a method presented in any one of Methods 40-41, wherein the 5' locus is proximal to the 5' boundary of a plurality of exons selected from the group consisting of exons 45-53 and exons 45-55. 【0482】 In another method, Method 43, the Disclosure presents a method presented in any one of Methods 40 to 42, wherein the 3' locus is proximal to the 3' boundary of a plurality of exons selected from the group consisting of exons 45 to 53 and exons 45 to 55. 【0483】 In another method, Method 44, the Disclosure presents a method presented in any one of Methods 40 to 43, wherein the 5' locus is proximal to the 5' boundaries of a plurality of exons selected from the group consisting of exons 45 to 53 and exons 45 to 55, and the 3' locus is proximal to these 3' boundaries. 【0484】 In another method, Method 45, the Disclosure presents a method, as presented in any one of Methods 42-44, in which the proximal portion of an exon to its boundary includes splice donors and acceptors around an adjacent intron. 【0485】 In another method, Method 46, the present disclosure presents a method presented in any one of Methods 26–32, wherein a substitution of chromosomal DNA exists between the 5' locus and the 3' locus. 【0486】 In another method, Method 47, the Disclosure presents a method presented in any one of Methods 26-32 and 46, wherein the replacement is a single exon replacement. 【0487】 In another method, Method 48, the Disclosure presents a method presented in any one of Methods 26-32 and 46-47, wherein the single exon replacement is a replacement of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. 【0488】 In another method, Method 49, the Disclosure presents a method presented in any one of Methods 47-48, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. 【0489】 In another method, Method 50, the Disclosure presents a method presented in any one of Methods 47-49, wherein the 3' locus is proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. 【0490】 In another method, Method 51, the Disclosure presents a method presented in any one of Methods 47-50, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70, and the 3' locus is proximal to these 3' boundaries. 【0491】 In another method, Method 52, the Disclosure presents a method presented in any one of Methods 49-51, wherein the proximal part of the exon boundary includes splice donors and acceptors around the adjacent intron or adjacent exon. 【0492】 In another method, Method 53, the Disclosure presents a method presented in any one of Methods 26-32 or 46, wherein the replacement is a multiexon replacement. 【0493】 In another method, Method 54, the Disclosure presents a method presented in Method 53, wherein the replacement of a multiexon is a replacement of exons 45-53 or exons 45-55. 【0494】 In another method, Method 55, the Disclosure presents a method presented in any one of Methods 53 to 54, wherein the 5' locus is proximal to the 5' boundary of a plurality of exons selected from the group consisting of exons 45 to 53 and exons 45 to 55. 【0495】 In another method, Method 56, the Disclosure presents a method presented in any one of Methods 53 to 55, wherein the 3' locus is proximal to the 3' boundary of a plurality of exons selected from the group consisting of exons 45 to 53 and exons 45 to 55. 【0496】 In another method, Method 57, the Disclosure presents a method presented in any one of Methods 53 to 56, wherein the 5' locus is proximal to the 5' boundaries of a plurality of exons selected from the group consisting of exons 45 to 53 and exons 45 to 55, and the 3' locus is proximal to these 3' boundaries. 【0497】 In another method, Method 58, the Disclosure presents a method, as presented in any one of Methods 55-57, in which the proximal portion of an exon to its boundary includes splice donors and acceptors around an adjacent intron. 【0498】 In another method, Method 59, the present disclosure further includes the step of introducing a polynucleotide donor template containing at least a portion of the wild-type dystrophin gene or cDNA into a cell, wherein the replacement is by homology-guided repair (HDR), as presented in any one of Methods 46–58. 【0499】 In another method, Method 60, the present disclosure relates to at least one portion of the gene or cDNA of wild-type dystrophin, specifically exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 42, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41 Method 59 presents a method comprising exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or at least a portion of the dystrophin gene or the entire cDNA. 【0500】 In another method, Method 61, the present disclosure relates to at least one portion of the gene or cDNA of wild-type dystrophin, specifically exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42. Method 59 presents a method comprising exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA. 【0501】 In another method, Method 62, the present disclosure further comprises the step of introducing into a cell one guide ribonucleic acid (gRNA) and a polynucleotide donor template containing at least a portion of the wild-type dystrophin gene, wherein one or more DNA endonucleases produce one single-strand break (SSB) or double-strand break (DSB) in or near the dystrophin gene locus, which facilitates the insertion of a novel sequence derived from the polynucleotide donor template into the chromosomal DNA in the locus, resulting in the permanent insertion or correction of one or more exons or abnormal intron splice acceptor or donor sites in or near the dystrophin gene, resulting in the repair of the dystrophin leading frame and the repair of the dystrophin protein activity, and the gRNA includes a spacer sequence complementary to the locus segment, as presented in any one of Methods 1, 7, or 11. 【0502】 In another method, Method 63, the present disclosure further comprises the step of introducing into a cell one or more guide ribonucleic acids (gRNAs) and a polynucleotide donor template comprising at least a portion of the wild-type dystrophin gene, wherein one or more DNA endonucleases facilitate the insertion of a novel sequence derived from the polynucleotide donor template into chromosomal DNA between the 5' and 3' locuses, by creating a pair of single-strand breaks (S) which are a first break at the 5' locus and a second break at the 3' locus within or near the dystrophin gene. The present invention provides a method, as presented in any one of methods 1, 7, or 11, that produces an SSB or DSB, which is a double-strand break (SB) resulting in the permanent insertion or correction of one or more exons or abnormal intron splice acceptor or donor sites between the 5' locus and 3' locus within or near the dystrophin gene, thereby repairing the dystrophin leading frame and restoring the activity of the dystrophin protein, comprising one or more Cas9 or Cpf1 endonucleases. 【0503】 In another method, Method 64, the Disclosure presents a method, as presented in Method 63, in which one gRNA generates one pair of SSBs or DSBs. 【0504】 In another method, Method 65, the present disclosure presents a method presented in Method 63, wherein one gRNA includes a spacer sequence that is complementary to the 5' locus, the 3' locus, or the segment between the 5' locus and the 3' locus. 【0505】 In an alternative method, Method 66, the Disclosure presents a method presented in Method 63, comprising a first gRNA and a second gRNA, wherein the first gRNA comprises a spacer sequence complementary to a 5' locus segment, and the second gRNA comprises a spacer sequence complementary to a 3' locus segment. 【0506】 In another method, Method 67, the Disclosure presents a method presented in Method 62 or 63, in which one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs). 【0507】 In another method, Method 68, the Disclosure presents a method, as presented in Methods 62-63 or 67, in which one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs. 【0508】 In another method, Method 69, the Disclosure presents a method, as presented in any one of Methods 62-63 or 67-68, in which one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs. 【0509】 In another method, Method 70, the Disclosure presents a method presented in any one of Methods 62-69, wherein the insertion is a single exon insertion. 【0510】 In another method, Method 71, the Disclosure presents a method presented in Method 70 in which the insertion of a single exon is the insertion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. 【0511】 In another method, Method 72, the Disclosure presents a method presented in any one of Methods 70 to 71, wherein the locus, 5'-locus, or 3'-locus is proximal to the boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and exon 70. 【0512】 In another method, Method 73, the Disclosure presents a method presented in Method 72 in which the proximal part of the exon boundary includes splice donors and acceptors around the adjacent intron or adjacent exon. 【0513】 In another method, Method 74, the Disclosure presents a method presented in any one of Methods 62-69, wherein the insertion is a multi-exon insertion. 【0514】 In another method, Method 75, the Disclosure presents a method presented in Method 74 in which the multi-exon insertion is an insertion of exons 45-53 or exons 45-55. 【0515】 In another method, Method 76, the Disclosure presents a method presented in any one of Methods 74-75, wherein the locus, 5'-locus, or 3'-locus is proximal to the boundary of a multiexon selected from the group consisting of exons 45-53 or exons 45-55. 【0516】 In another method, Method 77, the Disclosure presents a method presented in Method 76 in which the proximal portion of the exon boundary includes splice donors and acceptors around the adjacent intron. 【0517】 In another method, Method 78, the present disclosure relates to at least one portion of the gene or cDNA of wild-type dystrophin, specifically exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 Methods presented in Method 62 or 63 are presented, which include exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or at least a portion of the dystrophin gene or the entire cDNA. 【0518】 In another method, Method 79, the present disclosure relates to at least a portion of the gene or cDNA of wild-type dystrophin, specifically exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, 【0519】 Exxon 7, Exxon 8, Exxon 9, Exxon 10, Exxon 11, Exxon 12, Exxon 13, Exxon 14, Exxon 15, Exxon 16, Exxon 17, Exxon 18, Exxon 19, Exxon 20, Exxon 21, Exxon 22, Exxon 23, Exxon 24, Exxon 25, Exxon 26, Exxon 27, Exxon 28, Exxon 29, Exxon 30, Exxon 31, Exxon 32, Exxon 33, Exxon 34, Exxon 35, Exxon 36, Exxon 37, Exxon 38, Exxon 39, Exxon 40, Exxon 41, Exxon 42, Exxon 43, Exxon 44, Exxon 45, Exxon 46, Exxon 47, Exxon 48, Exxon Methods presented in Method 62 or 63 are presented, including 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA. 【0520】 In another method, Method 80, the Disclosure presents a method presented in any one of Methods 62-79, wherein the insertion is by homology-guided restoration (HDR). 【0521】 In another method, Method 81, the Disclosure presents a method presented in any one of Methods 62-80, wherein the donor template is a single-stranded or double-stranded polynucleotide. 【0522】 In another method, Method 82, the Disclosure presents a method, as presented in any one of Methods 26 to 81, for formulating the mRNA, gRNA, and donor template of Cas9 or Cpf1 into separate lipid nanoparticles, or for co-formulating all of these into lipid nanoparticles. 【0523】 In another method, Method 83, the Disclosure presents a method presented in any one of Methods 26-81, which involves formulating Cas9 or Cpf1 mRNA into lipid nanoparticles and delivering both the gRNA and donor template to cells via an adeno-associated virus (AAV) vector. 【0524】 In another method, Method 84, the present disclosure presents a method presented in any one of Methods 26 to 81, wherein Cas9 or Cpf1 mRNA is formulated into lipid nanoparticles, the gRNA is delivered to cells by electroporation, and the donor template is delivered to cells by an adeno-associated virus (AAV) vector. 【0525】 In an alternative method, Method 85, the present disclosure presents a method, as presented in any one of Methods 1 to 84, in which the dystrophin gene is located on the X chromosome: 31,117,228~33,344,609 (Genome Reference Consortium: GRCh38 / hg38). 【0526】 In the first composition, Composition 1, the present disclosure presents one or more guide ribonucleic acid (gRNAs) for editing the dystrophin gene in cells derived from a patient with Duchenne muscular dystrophy (DMD), the gRNAs comprising a spacer sequence selected from the group consisting of nucleic acid sequences within sequence numbers 1 to 1,410,472 of the sequence listing. 【0527】 In a different composition, Composition 2, the Disclosure presents one or more gRNAs of Composition 1, wherein one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs). 【0528】 In another composition, composition 3, the disclosure presents one or more gRNAs or sgRNAs of composition 1 or 2, wherein one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs. 【0529】 definition 【0530】 The terms "including" or "containing" are used to refer to compositions, methods, and each of their components, which are essential to the present invention but are open to the inclusion of unspecified elements, whether essential or essential. 【0531】 The term "essentially derived from" refers to an element required for a given embodiment. The term allows for the presence of further elements that do not substantially affect the basic, novel, or functional features of that embodiment of the invention. 【0532】 The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not enumerated in the description of the embodiments. 【0533】 The singular forms "a," "an," and "sono" refer to multiple objects unless explicitly indicated otherwise by the context. 【0534】 Any numerical ranges enumerated herein describe all subranges that are included within the enumerated range, having the same numerical precision (i.e., having the same number of digits as specified). For example, the enumerated range "1.0 to 10.0" describes all subranges between (and including) the enumerated minimum value of 1.0 and the enumerated maximum value of 10.0, such as "2.4 to 7.6," even if the range "2.4 to 7.6" is not explicitly enumerated in the text of this specification. Accordingly, the applicant reserves the right to amend this specification, including the claims, to explicitly enumerate any subranges of the same numerical precision that are included within the ranges explicitly enumerated herein. All such ranges are described herein in such a way that any amendment to explicitly enumerate any such subranges complies with the requirements of description, sufficiency of description, and novel matter, including the requirements under 35 U.S.C § 112(a) and EPC § 123(2). Unless explicitly specified or required otherwise by the context, all numerical parameters described herein (numerical parameters representing values, ranges, quantities, percentages, etc.) can also be read as if the word "approximately" is preceding the number, even if the word does not explicitly appear before the number. In addition, numerical parameters described herein shall be interpreted by applying the usual rounding method in light of the numerical precision, which is the number of significant figures reported. It is also understood that numerical parameters described herein will inevitably have inherent variability characteristic of the underlying measurement method used to determine the numerical value of the parameter. [Examples] 【0535】 The present invention will be more fully understood by referring to the following embodiments which provide exemplary and non-limiting aspects of the present invention. 【0536】 This embodiment describes the use of the CRISPR system as an exemplary genome editing technique for creating defined therapeutic genomic deletions, insertions, or replacements, collectively referred to herein as “genomic modifications,” in the dystrophin gene (DMD gene), resulting in permanent deletion or correction of a problematic exon from a genomic locus (gene locus) that repairs the dystrophin reading frame and restores dystrophin protein activity. 【0537】 Single-chain gRNAs spanning various regions of the DMD gene were selected and tested for cleavage efficiency (Table 5). The gRNAs targeted exons, introns, and splice acceptors in numerous target regions of the DMD gene. The nomenclature convention for all gRNAs discussed in the examples is #(corresponding to gRNA)-NN(Cas protein: SP-S.pyogenes, SA-S.aureus, NM-N.meningitides, ST-S.thermophiles, TD-T.denticola, Cpf1)-NN##(SA-splice acceptor, E-exon, I-intron). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] 【0538】 All tested gRNAs can be used for HDR / correction-based editing techniques. Single-chain gRNAs targeting splice acceptors can be used to induce exon skipping and repair the reading frame of the DMD gene. Selected gRNA pairs can be used to create deletions in the DMD gene that repair the reading frame. Selected gRNA pairs can be used to create deletions that simulate patient mutations and can be used to generate model DMD mutant strains. 【0539】 Various Cas orthologues were evaluated for cleavage. SP, NM, ST, SA, and Cpf1 gRNAs were delivered as RNA expressed from the U6 promoter in plasmids or lentiviruses. The corresponding Cas proteins were delivered either constitutively by knock-in into target cell lines, as mRNA, or as proteins. The activity of all gRNAs in the above forms was evaluated in HEK293T cells, K562 cells, or induced pluripotent stem cells (iPSCs) using TIDE analysis or next-generation sequencing. 【0540】 Overall, it was determined that most of the gRNAs tested induced cleavage. However, the amount of cleavage was highly dependent on the Cas protein tested. Generally, SP Cas9 gRNA was found to induce the highest level of cleavage, followed by SA Cas9 gRNA. Generally, it is beneficial to select the gRNA with the highest possible cleavage efficiency for therapeutic application. However, for iPSC-based therapies, cleavage efficiency is less critical. iPSCs are highly proliferative and simplify the isolation of clonal populations of cells with the desired edits, even if the editing efficiency is less than 10%. 【0541】 The introduction of the defined therapeutic modifications described above represents a novel therapeutic strategy for the potential improvement of DMD, as will be further described and illustrated herein. 【0542】 (Example 1) CRISPR / SPCas9 target sites of the dystrophin gene The dystrophin gene region was scanned for target sites. Each region was scanned for protospacer-adjacent motifs (PAMs) containing the sequence NRG. 20bp gRNA spacer sequences corresponding to PAMs were identified, as shown in sequence numbers 1-467,030. 19bp gRNA spacer sequences corresponding to PAMs were identified, as shown in sequence numbers 1,410,430-1,410,472 in the sequence listing. 【0543】 (Example 2) CRISPR / SACas9 target sites of the dystrophin gene The dystrophin gene region was scanned for target sites. Each region was scanned for protospacer-adjacent motifs (PAMs) containing the sequence NNGRRT. The gRNA 20bp spacer sequences corresponding to the PAMs were identified, as shown in sequence numbers 467,031–528,196 of the sequence listing. 【0544】 (Example 3) CRISPR / STCas9 target sites of the dystrophin gene The dystrophin gene region was scanned for target sites. Each region was scanned for protospacer-adjacent motifs (PAMs) containing the sequence NNAGAAW. The gRNA 24bp spacer sequences corresponding to the PAMs were identified, as shown in sequence numbers 528,197–553,198 of the sequence listing. 【0545】 (Example 4) CRISPR / TDCas9 target sites of the dystrophin gene The dystrophin gene region was scanned for target sites. Each region was scanned for protospacer-adjacent motifs (PAMs) containing the sequence NAAAAC. The gRNA 24bp spacer sequences corresponding to the PAMs were identified, as shown in sequence numbers 553,199–563,911 of the sequence listing. 【0546】 (Example 5) CRISPR / NMCas9 target sites of the dystrophin gene The dystrophin gene region was scanned for target sites. Each region was scanned for protospacer-adjacent motifs (PAMs) containing the sequence NNNNGHTT. The gRNA 24bp spacer sequences corresponding to the PAMs were identified, as shown in sequence numbers 563,912–627,854 and 1,410,400–1,410,402 of the sequence listing. 【0547】 (Example 6) CRISPR / Cpf1 target sites of the dystrophin gene The dystrophin gene region was scanned for target sites. Each region was scanned for protospacer-adjacent motifs (PAMs) containing the sequence YTN. As shown in sequence numbers 627,855–1,410,399 and 1,410,403–1,410,429 of the sequence listing, gRNA 20–24 bp spacer sequences corresponding to the PAMs were identified. 【0548】 (Example 7) Exemplary genome editing strategies that target exon 2 Several methods provide a pair of gRNAs that delete exon 2 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 2 and the other gRNA cutting at the 3' end of exon 2. 【0549】 (Example 8) Exemplary genome editing strategies targeting exon 8 Several methods provide a pair of gRNAs that delete exon 8 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 8 and the other gRNA cutting at the 3' end of exon 8. 【0550】 (Example 9) Exemplary genome editing strategies targeting exon 43 Several methods provide a pair of gRNAs that delete exon 43 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 43 and the other gRNA cutting at the 3' end of exon 43. 【0551】 (Example 10) An exemplary genome editing method that targets exon 44 Several methods provide a pair of gRNAs that delete exon 44 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 44 and the other gRNA cutting at the 3' end of exon 44. 【0552】 (Example 11) Exemplary genome editing strategies targeting exon 45 Several methods provide a pair of gRNAs that delete exon 45 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 45 and the other gRNA cutting at the 3' end of exon 45. 【0553】 (Example 12) Exemplary genome editing strategies targeting exon 46 Several methods provide a pair of gRNAs that delete exon 46 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 46 and the other gRNA cutting at the 3' end of exon 46. 【0554】 (Example 13) Exemplary genome editing strategies targeting exon 50 Several methods provide a pair of gRNAs that delete exon 50 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 50 and the other gRNA cutting at the 3' end of exon 50. 【0555】 (Example 14) Exemplary genome editing strategies targeting exon 51 Several methods provide a pair of gRNAs that delete exon 51 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 51 and the other gRNA cutting at the 3' end of exon 51. 【0556】 (Example 15) Exemplary genome editing strategies targeting exon 52 Several methods provide a pair of gRNAs that delete exon 52 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 52 and the other gRNA cutting at the 3' end of exon 52. 【0557】 (Example 16) Exemplary genome editing strategies targeting exon 53 Several methods provide a pair of gRNAs that delete exon 53 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 53 and the other gRNA cutting at the 3' end of exon 53. 【0558】 (Example 17) Exemplary genome editing strategies targeting exon 70 Several methods provide a pair of gRNAs that delete exon 70 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 70 and the other gRNA cutting at the 3' end of exon 70. 【0559】 (Example 18) Exemplary genome editing strategies targeting exons 45-53 Several methods provide a pair of gRNAs that delete exons 45-53 by cutting the gene twice, with one gRNA being cut at the 5' end of exon 45 and the other at the 3' end of exon 53. 【0560】 (Example 19) Exemplary genome editing strategies targeting exons 45-55 Several methods provide a pair of gRNAs that delete exons 45-55 by cutting the gene twice, with one gRNA being cut at the 5' end of exon 45 and the other at the 3' end of exon 55. 【0561】 (Example 20) Bioinformatics analysis of guide chains Candidate guides were screened and selected through a multi-step process involving both theoretical binding and experimentally assessed activity. For example, to assess the potential for effects at chromosomal locations other than the intended chromosomal location, one or more of the various bioinformatics tools available for assessing off-target binding can be used, as described and illustrated in more detail below, to assess the potential for candidate guides with sequences that fit specific on-target sites, such as sites within or near the dystrophin gene with adjacent PAMs, to cleave at off-target sites with similar sequences. Candidates predicted to have a relatively low potential for off-target activity can then be experimentally assessed to measure their on-target activity, and subsequently their off-target activity at various sites. A preferred guide has sufficiently high on-target activity to achieve the desired level of gene editing at the selected locus and relatively low off-target activity to reduce the likelihood of modification at other chromosomal loci. The on-target to off-target activity ratio is often referred to as the guide's "specificity." 【0562】 For initial screening of predicted off-target activity, several known and generally available bioinformatics tools can be used to predict the most likely off-target sites. Since binding to target sites in the CRISPR / Cas9 nuclease system is driven by Watson-Crick base pairing between complementary sequences, the degree of difference (and therefore reduction of the potential for off-target binding) is essentially related to primary sequence differences, mismatches, and bulges, i.e., bases replaced with non-complementary bases, as well as base insertions or deletions, at the target site and at the potential off-target site. An exemplary bioinformatics tool called COSMID (CRISPR Off-target Sites with Mismatches, Insertions and Deletions) (available on the web at crispr.bme.gatech.edu) aggregates such similarities. Other bioinformatics tools include, but are not limited to, GUIDO, autoCOSMID, and CCtop. 【0563】 Bioinformatics was used to minimize off-target cleavage and reduce the adverse effects of mutations and chromosomal rearrangements. Studies on the CRISPR / Cas9 system suggested the possibility of high off-target activity, particularly distal to the PAM region, due to nonspecific hybridization of the guide strand to DNA sequences with base pair mismatches and / or bulges. Therefore, it is important to have bioinformatics tools that can identify potentially off-target sites with insertions and / or deletions between the RNA guide strand and the genomic sequence, in addition to base pair mismatches. Bioinformatics-based tools, such as COSMID (CRISPR Off-target Sites with Mismatches, Insertions), are used. Therefore, COSMID (and Deletions) was used to search the genome for potentially CRISPR 30D5 target sites (available on the web at crispr.bme.gatech.edu). COSMID output ranks a list of potentially off-target sites based on the number and location of mismatches, enabling a more informed selection of target sites and avoiding the use of sites with more likely off-target cuts. 【0564】 An additional bioinformatics pipeline was used to compare the estimated on-target and / or off-target activity of gRNA targeting sites in a given region. Other features that can be used to predict activity include information about the cell type in question, DNA accessibility, chromatin state, transcription factor binding sites, transcription factor binding data, and other CHIP-seq data. Additional factors that predict editing efficiency, such as the relative position and orientation of gRNA pairs, local sequence features, and microhomology, were also compared. 【0565】 (Example 21) Testing of preferred guides in cells for on-target activity. Subsequently, the gRNA predicted to have the lowest off-target activity is tested for on-target activity in human embryonic kidney epithelial cells, HEK293T, by transient transfection, and indel frequency is evaluated using TIDE or next-generation sequencing. TIDE is a web-based tool for rapidly assessing CRISPR-Cas9 genome editing at target loci determined by guide RNA (gRNA or sgRNA). Based on quantitative sequence trace data from two standard capillary sequencing reactions, the TIDE software quantifies editing effectiveness and identifies the dominant types of insertions and deletions (indels) in the DNA of the target cell pool. For detailed explanations and examples, see Brinkman et al., Nucl. Acids Res. (2014). Next-generation sequencing (NGS), also known as high-throughput sequencing, is a broad term used to describe several different modern sequencing techniques, including Illumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent:Proton / PGM sequencing, and SOLiD sequencing. These recent techniques have revolutionized genomics and molecular biology research by enabling the sequencing of DNA and RNA considerably faster and cheaper than previously used Sanger sequencing. Since both HEK293T and iPSC cell types are known to have loose chromatin structures, HEK293T is an excellent model system for gene correction in iPSCs. 【0566】 Chromatin is organized by coiling, forming individual structures called nucleosomes. This coiling affects the accessibility of genomic material to the transcription mechanism. Open genomic regions are called euchromatin, while tightly coiled regions are called heterochromatin. It is a well-accepted paradigm that stem cells generally have loose chromatin conformation, and as cells differentiate into more specialized cell types, certain regions of the genome close to form heterochromatin (Sims, RJ and D. Reinberg (2009), "Stem cells: Escaping fates with open states," Nature 460 (No. 7257): pp. 802-803). 【0567】 (Example 22) Tests in related model cell lines All guide RNAs are individually evaluated to identify effective gRNAs, and all permutations of gRNA pairs are tested in relevant model cell lines for their ability to modify the DNA sequence of the dystrophin gene, which is predicted to repair the dystrophin reading frame. Myoblast and iPSC cell lines with modifications similar to or identical to those found in patient samples were generated. These cells were treated with various individual and paired combinations of gRNAs and donor DNA templates, where applicable and when applicable. The samples can then be evaluated for repair of dystrophin expression using one or more biological methods known to those skilled in the art, e.g., enzyme-linked immunosorbent assay (ELISA) that specifically recognizes the C-terminus of the dystrophin protein (note that truncated proteins do not contain an intact C-terminus). The gRNA pairs that repair dystrophin expression can then be further evaluated by additional biological techniques, such as Western blotting, to confirm the expression of the dystrophin protein at an appropriate size. 【0568】 (Example 23) Testing various methods for HDR gene editing After testing gRNAs for both on-target and off-target activity, exon correction and knock-in strategies are tested for HDR gene editing. 【0569】 For exon correction techniques, donor DNA templates are provided as short single-stranded oligonucleotides, short double-stranded oligonucleotides (PAM sequence intact / PAM sequence mutants), long single-stranded DNA molecules (PAM sequence intact / PAM sequence mutants), or long double-stranded DNA molecules (PAM sequence intact / PAM sequence mutants). In addition, the donor DNA templates are delivered by AAV. 【0570】 For DNA knock-in techniques, single-stranded or double-stranded DNA with a homology arm to the Xp21.2 locus may contain 40 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, the 3'UTR of the dystrophin gene, and at least 40 nt of subsequent introns. Single-stranded or double-stranded DNA with a homology arm to the Xp21.2 locus may contain 80 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, the 3'UTR of the dystrophin gene, and at least 80 nt of subsequent introns. Single-stranded or double-stranded DNA having a homology arm to the Xp21.2 locus may contain 100 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, the 3'UTR of the dystrophin gene, and at least 100 nt of subsequent introns. Single-stranded or double-stranded DNA having a homology arm to the Xp21.2 locus may contain 150 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, the 3'UTR of the dystrophin gene, and at least 150 nt of subsequent introns. Single-stranded or double-stranded DNA having a homology arm to the Xp21.2 locus may contain 300 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, and at least 300 nt of the 3'UTR of the dystrophin gene, followed by introns.A single-stranded or double-stranded DNA having a homology arm to the Xp21.2 locus may contain 400 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete CDS of the dystrophin gene, and at least 400 nt of the 3'UTR of the dystrophin gene, followed by introns. Alternatively, the DNA template is delivered by AAV. 【0571】 For cDNA knock-in techniques, a single-stranded or double-stranded cDNA may contain 40 nt or more of a single exon target of the dystrophin gene. A single-stranded or double-stranded cDNA may contain 80 nt or more of a single exon target of the dystrophin gene. A single-stranded or double-stranded cDNA may contain 100 nt or more of a single exon target of the dystrophin gene. A single-stranded or double-stranded cDNA may contain 150 nt or more of a single exon target of the dystrophin gene. A single-stranded or double-stranded cDNA may contain 300 nt or more of a single exon target of the dystrophin gene. A single-stranded or double-stranded cDNA may contain 400 nt or more of a single exon target of the dystrophin gene. Alternatively, the DNA template is delivered by AAV. 【0572】 For cDNA knock-in techniques, single-stranded or double-stranded cDNA may contain 40 nt or more of multiple exon targets of the dystrophin gene. Single-stranded or double-stranded cDNA may contain 80 nt or more of multiple exon targets of the dystrophin gene. Single-stranded or double-stranded cDNA may contain 100 nt or more of multiple exon targets of the dystrophin gene. Single-stranded or double-stranded cDNA may contain 150 nt or more of multiple exon targets of the dystrophin gene. Single-stranded or double-stranded cDNA may contain 300 nt or more of multiple exon targets of the dystrophin gene. Single-stranded or double-stranded cDNA may contain 400 nt or more of multiple exon targets of the dystrophin gene. Alternatively, the DNA template is delivered by AAV. 【0573】 (Example 24) Reassessment of lead CRISPR-Cas9 / DNA donor combinations After testing various strategies for HDR gene editing, lead CRISPR-Cas9 / DNA donor combinations are reassessed in therapeutically relevant cells for deletion efficiency, recombination, and off-target specificity. Cas9 mRNA or RNP is formulated into lipid nanoparticles for delivery, sgRNA is formulated into nanoparticles or delivered as AAN, and donor DNA is formulated into nanoparticles or delivered as AAV. 【0574】 (Example 25) In vivo studies in related animal models After reassessing the CRISPR-Cas9 / DNA donor combinations, lead formulations will be tested in vivo in therapeutically relevant mouse models. 【0575】 Culturing in human cells allows for direct testing of human targets and the background human genome, as described above. 【0576】 Preclinical efficacy and safety evaluations can be observed through the engraftment of modified mice or human cells in therapeutically relevant mouse models. Modified cells can be observed several months after engraftment. 【0577】 (Example 26) Cleavage efficiency of S. pyogenes gRNA targeting exons 45, 51, 53, 55, and 70 of the DMD gene. S. pyogenes (SP) gRNAs targeting exons 45, 51, 53, 55, and 70 of the DMD gene were tested (Figures 3A-3B). Each of exons 45, 51, 53, 55, and 70 may be edited using HDR / correction-based techniques. 【0578】 SP gRNA was cloned into plasmids co-expressing SP Cas protein. These plasmids were transfected into HEK293T cells using lipofectamine 2000. Cells were harvested 48 hours after transfection, genomic DNA was isolated, and cleavage efficiency was evaluated using TIDE analysis. Data were aggregated from a single experiment containing 3-4 replicates (N=3-4). Data were plotted as mean and SEM. 【0579】 The data from Figures 3A-3B show that most gRNAs are cleaved with more than 50% efficiency in HEK293T cells. 【0580】 (Example 27) gRNA cleavage efficiency targeting splice acceptors in exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the DMD gene. A viable option for treating DMD is to induce exon skipping to repair the reading frame of the DMD gene. To induce exon skipping, gene editing techniques must remove the AG sequence immediately upstream of the exon recognized by the endogenous splicing mechanism. When single-stranded gRNA induces a double-strand break, the cell repairs the break. Part of this time, the endogenous repair mechanism makes an error and inserts or deletes bases adjacent to the break site. A gRNA that mutates the AG sequence can induce exon skipping at this site because the splicing mechanism can no longer recognize this site as a splice acceptor site and skips to the next splice acceptor in the adjacent exon. 【0581】 We designed and tested S.pyogenes (SP), S.aureus (SA), S.thermophiles (ST), N.Meningitides (NM), and Cpf1 gRNAs that target the splice acceptors in exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the DMD gene (Figures 4A, 4B, and 4C). 【0582】 SP gRNA was designed to target the splice acceptors of nine exons of the DMD gene. The gRNA was ordered as split RNA gRNA from Integrated DNA Technologies (IDT). The split gRNA was annealed to tracRNA as instructed by the manufacturer. Subsequently, the annealed split gRNA was transfected into HEK293T cells stably expressing SP Ca...

Claims

[Claim 1] A composition comprising a guide RNA (gRNA) for use in the treatment of patients with Duchenne muscular dystrophy, wherein the gRNA comprises a) a sequence having a length of 19 to 25 nucleotides and b) a spacer sequence comprising an RNA version of a sequence shown in any one of sequence numbers 186215, 1410444, 1410464, 1410433, 1410472, 1410460, 1410441, 181240, 185602 or 236915. [Claim 2] The composition according to claim 1, wherein the spacer sequence consists of an RNA version of the sequence shown in any one of sequence numbers 186215, 1410444, 1410464, 1410433, 1410472, 1410460, 1410441, 181240, 185602, or 236915. [Claim 3] The composition according to any one of claims 1 to 2, wherein the gRNA is a single molecule gRNA (sgRNA) and / or the gRNA is a modified gRNA. [Claim 4] The composition according to any one of claims 1 to 2, wherein the gRNA further comprises a spacer extension sequence having a length greater than 1 and less than 15 nucleotides. [Claim 5] The composition according to any one of claims 1, 2, or 4, wherein the gRNA further comprises a minimal CRISPR repeat sequence. [Claim 6] The composition according to any one of claims 1, 2, 4, or 5, wherein the gRNA further comprises a minimal tracrRNA sequence. [Claim 7] The composition according to any one of claims 1, 2, or 4 to 6, wherein the gRNA further comprises a 3' tracrRNA sequence. [Claim 8] The composition according to any one of claims 1, 2, or 4 to 7, wherein the gRNA further comprises a tracrRNA elongation sequence. [Claim 9] A composition according to any one of claims 1 to 8, which is pre-complexed with a Cas9 endonuclease, wherein the Cas9 endonuclease optionally contains a nuclear localization signal. [Claim 10] A composition comprising nucleic acid for use in the treatment of patients with Duchenne muscular dystrophy, wherein the nucleic acid encodes the gRNA described in any one of claims 1 to 8. [Claim 11] The composition according to claim 10, further comprising a second nucleic acid encoding a Cas9 endonuclease, wherein the Cas9 endonuclease optionally comprises a nuclear localization signal, and optionally the Cas9 endonuclease is Streptococcus pyogenes Cas9 endonuclease. [Claim 12] A composition for use according to any one of claims 10 or 11, which is delivered to cells by a viral vector. [Claim 13] The composition for use according to claim 12, wherein the viral vector is an adeno-associated virus (AAV) vector, and optionally the AAV vector is an AAV9 vector. [Claim 14] A composition for use according to any one of claims 1 to 13, wherein treatment of the patient having Duchenne muscular dystrophy comprises editing the dystrophin gene in the patient's cells. [Claim 15] The composition for use according to claim 14, wherein the cells are muscle cells or muscle progenitor cells.

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