CN110760511B

CN110760511B - gRNA, expression vector and CRISPR-Cas9 system for treating duchenne muscular dystrophy

Info

Publication number: CN110760511B
Application number: CN201810851087.1A
Authority: CN
Inventors: 祝海宝; 喻小鲁; 黄雨亭; 陶米林; 罗思施
Original assignee: Guangdong Chimeng Medical Technology Co ltd
Current assignee: Guangdong Chimeng Medical Technology Co ltd
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2021-09-07
Anticipated expiration: 2038-07-27
Also published as: CN110760511A

Abstract

The invention provides a gRNA for treating Duchenne muscular dystrophy, which comprises a gRNA for knocking out an intron 43 of a Dys gene and a gRNA for knocking out an intron 54 of the Dys gene. The invention also provides a gRNA, an expression vector, a CRISPR-Cas9 system for treating Duchenne muscular dystrophy, and uses thereof. Compared with the prior art, the invention can cover not less than 60% of DMD patients; the utilization of the CRISPR-SaCas9 system is beneficial to using AAV as a therapeutic vector, has higher pertinence and higher safety; the gRNA screened by the invention has higher targeting efficiency, so that the correction efficiency of the Dys gene reading frame is higher.

Description

gRNA, expression vector and CRISPR-Cas9 system for treating duchenne muscular dystrophy

Technical Field

The invention relates to a gRNA for treating duchenne muscular dystrophy, an expression vector, a CRISPR-Cas9 system and application thereof.

Background

Rare diseases refer primarily to chronic, degenerative diseases with very low incidence. More than 7000 known rare diseases are inherited, 80% of which are caused by genetic defects. The rare diseases are not rare, and according to statistics, about 3.5 hundred million rare patients exist in the world, China accounts for over 2000 ten thousand, and more than 20 ten thousand new born patients are born each year. China mainly depends on means such as prenatal diagnosis and labor induction to control the expansion of rare disease groups. However, the rare diseases relate to complex and various genes, the pathogenesis is unclear, the conventional items of marriage detection and production detection are not listed, the cognition of the national and basic medical and health institutions to the diseases is to be enhanced, the diagnosis technology is not complete, and the like, so that the birth rate of the children is high. Patients with rare diseases face the dilemma of ' no medical treatment and ' no medical treatment ' once birth, because the rare diseases which can be treated currently only account for 5 percent, and in addition, 95 percent of patients are not available with medical treatment. Mainly because the research and development difficulty of rare medicines is high, the cost is high, the audiences are few, and few pharmaceutical enterprises are willing to invest in research and development. In the face of 7000 rare diseases around the world, the medicines with specific therapeutic action are only 400-500, while the medicines on the market in China are less than 20%, and the majority are imported medicines.

Duchenne muscular dystrophy, also known as pseudohypertrophic muscular dystrophy (DMD), is caused by mutations in the Dystrophin gene (Dys), which has a total length of about 2220kb, contains 79 exons, and has a cDNA length of 14kb, which is the largest human gene known at present. DMD is recessive inheritance of the X chromosome, mainly occurs in boys, women are carriers of pathogenic genes, the prevalence rate is about 3.3/10 ten thousand, and accounts for 20-30/10 ten thousand of born boys and boys, wherein about 1/3 is sporadic cases and has no family history.

For a long time, there are no effective clinical intervention measures or drugs for DMD at home and abroad. In 2016, 9 months, the U.S. FDA approved a new drug, Exondys 51(Eteplirsen), an injection by Sarepta Therapeutics, the first approved drug in the world for the treatment of DMD, by an expedited approval process (only 12 clinical trials). Eteplirsen injection can only be used for patients with deletion of exon 51 of the Dys gene (about 13% of patients with DMD), and there is currently no sufficient evidence for the therapeutic effect of Eteplirsen on DMD. Eteplirsen is a phosphorylmorpholine oligonucleotide that targets exon 51, and promotes dystrophin production by inducing skipping of exon 51 of the Dys gene, thereby converting lethal DMD to Becker Muscular Dystrophy (BMD), which is a less clinical phenotype. Except that No. 51 exon of the Dys gene was deleted, mutations of the Dys gene were mostly concentrated in the No. 2-20 exon regions and the No. 45-53 exon regions. Therefore Sarepta Therapeutics is also developing 7 additional exon skipping products that treat DMD patients with other gene mutations by skipping

exons

53, 45, 50, 44, 52, 55 and 8.

Exon skipping products developed by Sarepta Therapeutics require regular injections. With the development of science, genetic defects are corrected directly at the genome level by a gene editing technology, so that the method is considered to be an effective means for treating monogenic genetic diseases, and the treatment is effective for a lifetime without repeated administration. CRISPR-Cas9 is known as a third-generation gene editing technology, its use and design are simpler, mutation of a target gene is also more efficient, as long as a specific gRNA sequence is designed according to the target gene, Cas9 can be guided to be cut near a target site to generate Double Strand Break (DSB), and the broken DNA double strand can be re-linked in a non-homologous end joining (NHEJ) manner (as shown in fig. 1).

In the prior art, Sp.CRISPR-Cas9 gene editing technology is adopted to respectively design gRNAs aiming at an intron 44 and an intron 55 of a Dys gene, DNA double-strand cutting is induced and connected through NHEJ, exons 45-55 which are most easy to mutate are cut off, and a reading frame of the Dys gene is corrected to express dystrophin with certain function.

However, sp.crispr-gRNA designed for intron 44 and intron 55 of the Dys gene is less efficient, resulting directly in less efficient NHEJ after DNA double strand cleavage.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a gRNA, an expression vector and a CRISPR-Cas9 system for treating Duchenne muscular dystrophy, and also provides application of the gRNA, the expression vector and the CRISPR-Cas9 system. The gRNA screened by the invention has higher targeting efficiency, so that the correction efficiency of the Dys gene reading frame is higher. In addition, the Sa.Cas9 nucleic acid sequence adopted by the invention has the full length (including a nuclear localization signal) of 3258bp, can be used for effectively packaging AAV (adeno-associated virus), and has more advantages for local intramuscular injection in the treatment process. AAV is one of the most commonly used gene therapy vectors in the world, has the advantages of wide host range, high safety, low immunogenicity, stable expression, stable physical properties and the like, and has been widely applied to basic research and clinical trials. AAV has multiple serotypes, each directed against different tissues and cells, and can be selected according to clinical needs. The AAV has genome of about 4700bp and can load maximum fragment of about 4500bp of exogenous nucleic acid sequence. The total length of the Sp.Cas9 nucleic acid sequence (containing a nuclear localization signal) is 4200bp, and the total length is more than 4500bp after a promoter and polyA are added, so that the AAV cannot be effectively packaged.

In order to realize the purpose, the technical scheme is as follows: a gRNA for treating Duchenne muscular dystrophy, comprising a gRNA for knockout of intron 43 of the Dys gene and a gRNA for knockout of intron 54 of the Dys gene.

Preferably, the target sequence of the gRNA for knocking out intron 43 of the Dys gene is shown in at least one of SEQ ID NO. 1-SEQ ID NO. 50; preferably, the target sequence of the gRNA for knocking out the No. 43 intron of the Dys gene is shown as at least one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 28, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 44 and SEQ ID NO. 50.

Preferably, the target sequence of the gRNA for knocking out intron 54 of the Dys gene is shown in at least one of SEQ ID NO. 51-SEQ ID NO. 65; preferably, the target sequence of the gRNA for knocking out intron 54 of the Dys gene is shown in at least one of SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 63 and SEQ ID NO 64.

Preferably, the gRNA consists of a gRNA with a target sequence shown in one of SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 16, SEQ ID NO 22 and SEQ ID NO 39 and a gRNA with a target sequence shown in one of SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 58, SEQ ID NO 59 and SEQ ID NO 63, or consists of a gRNA with a target sequence shown in SEQ ID NO 55 and a gRNA with a target sequence shown in SEQ ID NO 53.

The invention provides an expression vector for treating duchenne muscular dystrophy, wherein the expression vector expresses the gRNA; preferably, the base vector of the expression vector is an AAV vector.

The invention provides a CRISPR-Cas9 system for treating duchenne muscular dystrophy, which comprises the gRNA and the Cas9 protein; preferably, the Cas9 protein is a SaCas9 protein.

The invention provides an expression vector for treating duchenne muscular dystrophy, which expresses Cas9 protein and the gRNA.

Preferably, the cas9 protein is a SaCas9 protein, and the basic vector of the expression vector is an AAV vector.

The invention provides application of the gRNA or the expression vector in preparing a medicament for preventing or treating Duchenne muscular dystrophy.

The invention provides application of the CRISPR-Cas9 system or the expression vector in preparing a medicament for preventing or treating Duchenne muscular dystrophy.

The present invention provides a method for treating duchenne muscular dystrophy, comprising administering the expression vector described above to a patient suffering from duchenne muscular dystrophy.

The invention adopts a CRISPR-SacAS9 gene editing system derived from Staphylococcus aureus (Staphylococcus aureus), and designs a pair of gRNAs to guide Sa.Cas9 to respectively perform double-strand cleavage at the upstream and the downstream of No. 45-53 exon in a Dys gene mutation hotspot region, so that large fragment deletion is generated between the upstream and the downstream of No. 45-53 exon, thereby correcting a reading frame of a Dys gene (see figure 2) and converting a DMD patient to a BMD with lighter symptoms. The treatment method is a broad-spectrum and universal treatment technology, is suitable for all mutation types (about 60 percent of DMD patients) such as deletion, duplication, insertion and the like of No. 45-53 exon regions, corrects the reading frame error of (3N +1) or (3N +2) into (3N), and converts DMD into non-lethal BMD.

The invention has the beneficial effects that: compared with the prior art, the invention can cover not less than 60% of DMD patients; the utilization of the CRISPR-SaCas9 system is beneficial to using AAV as a therapeutic vector, has higher pertinence and higher safety; the gRNA screened by the invention has higher targeting efficiency, so that the correction efficiency of the Dys gene reading frame is higher.

Drawings

Fig. 1 is a schematic diagram of CRISPR-Cas 9-mediated non-homologous end joining repair;

FIG. 2 is a CRISPR-Sa.Cas9 mediated large fragment deletion between the upstream and downstream of No. 45-53 exon of Dys gene;

FIG. 3 is a diagram of plasmid pX 601;

FIG. 4 shows the results of the detection of SEQ ID NO. 1 to SEQ ID NO. 5 in example 1 of the present invention;

FIG. 5 shows the results of the detection of SEQ ID NO. 6 to SEQ ID NO. 15 in example 1 of the present invention;

FIG. 6 shows the result of detection of SEQ ID NO. 16 in example 1 of the present invention;

FIG. 7 shows the results of the detection of SEQ ID NO 17 to SEQ ID NO 23 in example 1 of the present invention;

FIG. 8 shows the results of the detection of SEQ ID NO. 24 to SEQ ID NO. 26 in example 1 of the present invention;

FIG. 9 shows the result of detection of SEQ ID NO. 27 in example 1 of the present invention;

FIG. 10 shows the results of the detection of SEQ ID NO 28 to SEQ ID NO 31 in example 1 of the present invention;

FIG. 11 shows the results of the detection of SEQ ID NO 32 to SEQ ID NO 37 in example 1 of the present invention;

FIG. 12 shows the results of the detection of SEQ ID NO 38 to SEQ ID NO 41 in example 1 of the present invention;

FIG. 13 shows the results of the detection of SEQ ID NO 42 to SEQ ID NO 50 in example 1 of the present invention;

FIG. 14 shows the results of the detection of SEQ ID NO 51 to SEQ ID NO 53 in example 2 of the present invention;

FIG. 15 shows the result of detection of SEQ ID NO. 54 in example 2 of the present invention;

FIG. 16 shows the results of the detection of SEQ ID NO:55 to SEQ ID NO:60 in example 2 of the present invention;

FIG. 17 shows the results of the detection of SEQ ID NOS 61 to 65 in example 2 of the present invention;

FIG. 18 shows the results of PCR detection of Group NO:3-Group NO:24 in example 3 of the present invention;

FIG. 19 shows the results of PCR detection of Group NO:1-Group NO:2 in example 3 of the present invention,

FIG. 20 shows the results of PCR detection of Group NO:25-Group NO:26 in example 3 of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1 Dys Gene intron 43 Sa-gRNA screening

1.1 gRNA preparation

(1) Designing a 21nt gRNA sequence according to a Dys gene sequence, wherein a target sequence of the gRNA is shown in Table 1 and is shown in one of SEQ ID NO 1-SEQ ID NO 55;

(2) synthesizing a sense strand and an antisense strand of the DNA sequence of the gRNA respectively (the 5 '-end of the sense strand is added with cacc, if the first nucleotide at the 5' -end of the sense strand is not guanine G, caccG is added at the 5 '-end of the sense strand; aaac is added at the 5' -end of the antisense strand, and if the first nucleotide at the 5 '-end of the sense strand is not guanine G, C is added at the 3' -end of the antisense strand);

(3) mixing the sense strand and the antisense strand of the gRNA, treating at 90 ℃, naturally cooling to room temperature for annealing treatment, and synthesizing double-stranded gRNA with a sticky end.

TABLE 1 sense strand of Sa-gRNA DNA sequence designed for intron No. 43 of Dys gene and detection primer thereof

1.2 vector preparation

(1) The pX601 plasmid (fig. 3) was amplified and extracted, and the plasmid concentration was determined;

(2) the pX601 is cut by restriction enzyme Bsa I, and the reaction is terminated by adding loading buffer after 1h of enzyme cutting at 37 ℃.

(3) After agarose gel electrophoresis, cutting gel and recovering linearized plasmid pX601, and determining the concentration of the recovered product, and storing at-20 ℃ for later use.

1.3 ligation transformation

(1) Performing a connection reaction on the linearized pX601 vector recovered from the gel cutting and the annealed gRNA double strand;

(2) and (3) transforming the escherichia coli competent cell TOP10 by a ligation product heat shock method, adding a sterile LB liquid culture medium (without antibiotics) into each centrifuge tube after transformation, uniformly mixing, and placing in a constant temperature shaking table at 37 ℃ and 200rpm for shaking culture for 45min to recover the thalli.

(3) Recovered TOP10 cells were plated on LB solid plates (Amp)⁺) And inversely placing the mixture in a constant temperature incubator at 37 ℃ for static culture for 12-16 h.

(4) Single colonies were picked from the above plates and inoculated into LB liquid medium (Amp)⁺) Medium-scale culture.

(5) The above-mentioned bacterial solutions were sequenced using primer 601SaF-F (5'-TTCCTTgACCCTggAAggTg-3') (SEQ ID NO:66), respectively;

(6) extracting plasmids from the bacterial liquid with correct sequencing, measuring the concentration of the plasmids, and storing at-20 ℃ for later use.

1.4 transfection of cells

(1) HEK293T cell plating;

(2) respectively transfecting HEK293T cells with the plasmids extracted in the step 1.3(6) by using a Lipofectamine 3000 kit;

(3) the transfected cells were cultured for 48 hours and harvested by centrifugation.

1.5 enzyme digestion analysis of mutation efficiency of T7E1

(1) Extracting cell genome from the cells collected in the step 1.4(3), and detecting the genome concentration;

(2) designing PCR primers respectively upstream and downstream of the gRNA binding site, as shown in Table 1;

(3) amplifying target fragments with target sites by using a PCR method respectively;

(4) purifying and recovering PCR products, and determining the product concentration;

(5) annealing the purified PCR product, namely heating to 95 ℃, preserving heat for 10min, and then cooling to room temperature at the speed of reducing the temperature by 2-3 ℃ every 30 s;

(6) t7 endonuclease 1(T7E1) was added to each tube of the annealed product, and mock (untransformed cells) and blank (ddH without T7E1) were set₂O), digesting for 1h at 37 ℃.

(7) The cleavage effect was examined by 2% agarose gel electrophoresis using DL2,000 DNArker from Takara as a reference (2000 bp, 1000bp, 750bp, 500bp, 250bp, and 100bp bands in this order from top to bottom (TAKARA DL2000 Marker is used for all the electrophoretograms described below).

The results are shown in the following figure (lane numbers in the figure correspond to those of SEQ ID NO), FIG. 4 is the sequence of SEQ ID NO:1-SEQ ID NO:5, detecting the result of the detection, and determining the result, FIG. 5 is the sequence of SEQ ID NO:6-SEQ ID NO:15, the result of the detection is that, FIG. 6 is the sequence of SEQ ID NO:16, fig. 7 shows the results of SEQ ID NO:17-SEQ ID NO:23, as a result of the detection, a, FIG. 8 is the amino acid sequence of SEQ ID NO:24-SEQ ID NO: as a result of the detection 26, it is, FIG. 9 is SEQ ID NO:27, fig. 10 shows the results of SEQ ID NO:28-SEQ ID NO:31 the result of the detection is detected by the detector, FIG. 11 is the amino acid sequence of SEQ ID NO:32-SEQ ID NO:37, the result of the detection is that, FIG. 12 is SEQ ID NO:38-SEQ ID NO:41, the result of the detection is detected, FIG. 13 is SEQ ID NO:42-SEQ ID NO: and (5) detecting results by 50.

According to the principle of T7E1 detection, the brighter the cleavage band and the higher the cleavage efficiency, the higher the mutation efficiency of the gRNA. In the figure, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 44 and SEQ ID NO 50 have high cleavage efficiency.

Example 2 Dys Gene intron 54 Sa-gRNA screening

2.1 gRNA preparation

(1) Designing a gRNA sequence of 21nt according to the sequence of a Dys gene, wherein the target sequence of the gRNA is shown in one of SEQ ID NO 56-SEQ ID NO 69 as shown in Table 2;

TABLE 2 sense strand of Sa-gRNA DNA sequence designed for intron No. 54 of Dys gene and detection primer thereof

2.2 vector preparation

2.3 ligation transformation

2.4 transfection of cells

(1) HEK293T cell plating;

(2) respectively transfecting HEK293T cells with the plasmids extracted in the step 2.3(6) by using a Lipofectamine 3000 kit;

2.5 enzyme digestion analysis of mutation efficiency of T7E1

(1) Extracting cell genome from the cells collected in the step 2.4(3), and detecting the genome concentration;

(2) designing PCR primers respectively upstream and downstream of the gRNA binding site, as shown in Table 2;

(7) The cleavage effect was examined by 2% agarose gel electrophoresis using DL2,000 DNA Marker from Takara as a reference.

The results are shown in the following figures (the numbers of lanes in the figure correspond to those of SEQ ID NO), FIG. 14 shows the results of detection of SEQ ID NO:51 to SEQ ID NO:53, FIG. 15 shows the results of detection of SEQ ID NO:54, FIG. 16 shows the results of detection of SEQ ID NO:55 to SEQ ID NO:60, and FIG. 17 shows the results of detection of SEQ ID NO:61 to SEQ ID NO: 65.

According to the principle of T7E1 detection, the brighter the enzyme digestion band, the higher the enzyme digestion efficiency, indicating that the mutation efficiency of the gRNA is higher. In the figure, the cleavage efficiency of

SEQ ID NO

51, 53, 54, 58, 59, 60, 63 and 64 is high.

Example 3 Combined screening of upstream and downstream Sa-gRNA of exons 43-55 of the Dys Gene

3.1 Sa-gRNA combinations

Combining the target point of example 1 with the target point of example 2, as shown in table 3;

TABLE 3 combination of upstream and downstream Sa-gRNAs for exons 43-55 of the Dys gene

3.2 transfection of cells

(1) HEK293T cell plating;

(2) the plasmids extracted in 1.3(6) and 2.3(6) were transfected into HEK293T cells according to the combination in 3.1, respectively, using a Lipofectamine 3000 kit;

3.3 PCR detection of mutation Effect and sequencing analysis

(1) Extracting cell genome from the cells collected in the step 3.2(3), and detecting the genome concentration;

(2) PCR primers were designed upstream and downstream of the gRNA binding sites, respectively, as shown in Table 4;

TABLE 4 PCR assay primer List

(4) the targeting knockout effect was examined by 1% agarose gel electrophoresis using DL2,000 DNA Marker of Takara as a reference.

The results are shown in the following graph (lane numbers correspond to Group NO numbers in the figure), in which FIG. 18 shows the results of PCR detection of Group NO:3-Group NO:24, in which FIG. 19 shows the results of PCR detection of Group NO:1-Group NO:2, and in which FIG. 20 shows the results of PCR detection of Group NO:25-Group NO: 26.

Following genome disruption, the cell will tend to link the two DNA breaks to each other according to the principle of nonhomologous end-joining repair. After co-transformation of the vectors in 1.3(6) and 2.3(6) into cells according to Table 3, the DNA fragment between the two Sa-gRNAs was excised, and the DNA ends were ligated to each other to repair them. Primers are designed at the 5 'end of the Sa-gRNA of the intron 43 of the Dys gene and the 3' end of the Sa-gRNA of the intron 54 of the Dys gene for PCR, and whether non-homologous end connection occurs or not can be judged according to the size of a PCR product. In FIG. 18, the numbers below the lanes indicate the sizes of PCR products obtained after repair of non-homologous end joining between two Sa-gRNAs, and the arrows indicate the positions of the aforementioned PCR products.

The PCR product was recovered by cutting gel, and the band indicated by the arrow was subjected to sanger generation sequencing and analyzed by comparison with the Dys genome. PCR forward primers were used for all PCR product sequencing. The results are shown in Table 5: all Sa-gRNA combinations are subjected to sequence excision between two targets, the 3 rd base position at the 5 'end of the upstream target PAM sequence is subjected to non-homologous end connection with the 3 rd base position at the 5' end of the downstream target PAM sequence, exons 44 to 54 of the Dys gene are excised, and the frame reading error of (3N +1) or (3N +2) is corrected to be (3N), so that the DMD is converted into non-lethal BMD. Group NO 1(SEQ ID NO:105)392bp position is a non-homologous end connecting site; 399bp of Group NO 3(SEQ ID NO:107) is a non-homologous end connecting site; group NO:4(SEQ ID NO:108)394bp is a non-homologous end connection site; group NO:5(SEQ ID NO:109)401bp is a non-homologous end connection site; group NO:6(SEQ ID NO:110)410bp is a non-homologous end connection site; 417bp of Group NO:7(SEQ ID NO:11) is a non-homologous end connection site; 418bp of Group NO 8(SEQ ID NO:112) is a non-homologous end connection site; 421bp of Group NO:9(SEQ ID NO:113) is a non-homologous end connection site; group NO:10(SEQ ID NO:114)424bp is a non-homologous end connection site; 290bp of Group NO:11(SEQ ID NO:115) is a non-homologous end connection site; 12(SEQ ID NO:116)289bp of Group NO: a non-homologous end connection site; 13(SEQ ID NO:117)289bp of GroupNO is a non-homologous end connecting site; 292bp of Group NO:14(SEQ ID NO:118) is a non-homologous end connection site; group NO:15(SEQ ID NO:119)289bp is a non-homologous end connection site; 486bp of Group NO:16(SEQ ID NO:120) is a non-homologous end connecting site; the 488bp position of Group NO:17(SEQ ID NO:121) is a non-homologous end connecting site; is a non-homologous end connection site; group NO 19(SEQ ID NO:123)487bp is a non-homologous end connection site; 486bp of Group NO:20(SEQ ID NO:124) is a non-homologous end connecting site; 286bp of Group NO:21(SEQ ID NO:125) is a non-homologous end connection site; 286bp of Group NO:22(SEQ ID NO:126) is a non-homologous end connection site; group NO:23(SEQ ID NO:127)289bp is a non-homologous end connection site; 288bp of Group NO:24(SEQ ID NO:128) is a non-homologous end connection site; the position 298bp of Group NO:25(SEQ ID NO:129) is a non-homologous end connection site; group NO:26(SEQ ID NO:130)528bp is a non-homologous end joining site.

TABLE 5 sequencing results of upstream and downstream Sa-gRNA combined Sanger generation of exons 43-55 of Dys gene

NG-012232.1, the Dys gene NCBI Sequence ID, the Dys gene intron 43 Sequence range from 1057082 to 1127546, and the Dys gene intron 54 Sequence range from 1686618 to 1716744.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

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<213> Artificial sequence

<400> 29

gagagagagt tggtggtggg g 21

<210> 30

<211> 21

<212> DNA

<213> Artificial sequence

<400> 30

gtggggcctg gtgagaggtg a 21

<210> 31

<211> 21

<212> DNA

<213> Artificial sequence

<400> 31

ccaggcccca cctccaacat t 21

<210> 32

<211> 21

<212> DNA

<213> Artificial sequence

<400> 32

gacttcccag cctccagaac t 21

<210> 33

<211> 21

<212> DNA

<213> Artificial sequence

<400> 33

tcccagcctc cagaactgtg a 21

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence

<400> 34

ttggtctggg tgatggtcac c 21

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence

<400> 35

catggttggc catgcctatc c 21

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence

<400> 36

cagggatagg catggccaac c 21

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence

<400> 37

catgctagtg tctctctggt g 21

<210> 38

<211> 21

<212> DNA

<213> Artificial sequence

<400> 38

acacaaaggc agacagagtg g 21

<210> 39

<211> 21

<212> DNA

<213> Artificial sequence

<400> 39

taccttccca ctctgcccct t 21

<210> 40

<211> 21

<212> DNA

<213> Artificial sequence

<400> 40

gggcagagtg ggaaggtagt g 21

<210> 41

<211> 21

<212> DNA

<213> Artificial sequence

<400> 41

gctccattaa ttgggctgcc c 21

<210> 42

<211> 21

<212> DNA

<213> Artificial sequence

<400> 42

tccaggccca caagagggag g 21

<210> 43

<211> 21

<212> DNA

<213> Artificial sequence

<400> 43

ccagctctgt cttacagagc t 21

<210> 44

<211> 21

<212> DNA

<213> Artificial sequence

<400> 44

ctcagactgt ggtagcagga g 21

<210> 45

<211> 21

<212> DNA

<213> Artificial sequence

<400> 45

ctgtggtagc aggaggaggg t 21

<210> 46

<211> 21

<212> DNA

<213> Artificial sequence

<400> 46

actgatgtcc tgggcagaga a 21

<210> 47

<211> 21

<212> DNA

<213> Artificial sequence

<400> 47

tatggtgctg cctcagagta c 21

<210> 48

<211> 21

<212> DNA

<213> Artificial sequence

<400> 48

ttaccggcta tggtgctgcc t 21

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<400> 49

agcaccatag ccggtaatct g 21

<210> 50

<211> 21

<212> DNA

<213> Artificial sequence

<400> 50

agttggtctg gaatggagcc c 21

<210> 51

<211> 21

<212> DNA

<213> Artificial sequence

<400> 51

ccctcccaag ctccagttta g 21

<210> 52

<211> 21

<212> DNA

<213> Artificial sequence

<400> 52

tccagctaaa ctggagcttg g 21

<210> 53

<211> 21

<212> DNA

<213> Artificial sequence

<400> 53

tgggtgtgtg gagtgagata c 21

<210> 54

<211> 21

<212> DNA

<213> Artificial sequence

<400> 54

gcttgggcca tcaaggagca t 21

<210> 55

<211> 21

<212> DNA

<213> Artificial sequence

<400> 55

gctgggaagg gtagggggtg a 21

<210> 56

<211> 21

<212> DNA

<213> Artificial sequence

<400> 56

tggtggttac ctgatgctgg g 21

<210> 57

<211> 21

<212> DNA

<213> Artificial sequence

<400> 57

tgtggagggt gggaggagcg a 21

<210> 58

<211> 21

<212> DNA

<213> Artificial sequence

<400> 58

tgctggggaa gtgagactgg a 21

<210> 59

<211> 21

<212> DNA

<213> Artificial sequence

<400> 59

gtggtgctgg ggaagtgaga c 21

<210> 60

<211> 21

<212> DNA

<213> Artificial sequence

<400> 60

agagagagag ggaggtggaa g 21

<210> 61

<211> 21

<212> DNA

<213> Artificial sequence

<400> 61

agcactttgg gaggccgagg t 21

<210> 62

<211> 21

<212> DNA

<213> Artificial sequence

<400> 62

gggaggccga ggtgggggtg g 21

<210> 63

<211> 21

<212> DNA

<213> Artificial sequence

<400> 63

gtgggaggat cactagaggc c 21

<210> 64

<211> 21

<212> DNA

<213> Artificial sequence

<400> 64

gtctcactac gtcacccagg c 21

<210> 65

<211> 21

<212> DNA

<213> Artificial sequence

<400> 65

tacctcggcc tcccaaagtg c 21

<210> 66

<211> 20

<212> DNA

<213> Artificial sequence

<400> 66

ttccttgacc ctggaaggtg 20

<210> 67

<211> 24

<212> DNA

<213> Artificial sequence

<400> 67

ccaaaattgt aatgggtaga cttc 24

<210> 68

<211> 22

<212> DNA

<213> Artificial sequence

<400> 68

aatccagctt tcacttatcc ca 22

<210> 69

<211> 23

<212> DNA

<213> Artificial sequence

<400> 69

ccaactctac cttttacttc cca 23

<210> 70

<211> 23

<212> DNA

<213> Artificial sequence

<400> 70

accatgtttt ccattaaccc tac 23

<210> 71

<211> 23

<212> DNA

<213> Artificial sequence

<400> 71

cttctagctt ctcctatgct ctc 23

<210> 72

<211> 22

<212> DNA

<213> Artificial sequence

<400> 72

ctagttgggc aagatgtact ag 22

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<400> 73

agtacatctt gcccaactag c 21

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence

<400> 74

gcacgaggga aatgttcttg 20

<210> 75

<211> 21

<212> DNA

<213> Artificial sequence

<400> 75

caggtttaga catgtgcctt c 21

<210> 76

<211> 22

<212> DNA

<213> Artificial sequence

<400> 76

tgaaaccaat ctcaaaggtt gc 22

<210> 77

<211> 20

<212> DNA

<213> Artificial sequence

<400> 77

tgccaccatt gagcacttac 20

<210> 78

<211> 21

<212> DNA

<213> Artificial sequence

<400> 78

ccacactaaa tgctccctaa g 21

<210> 79

<211> 21

<212> DNA

<213> Artificial sequence

<400> 79

ttaggatggt gttgaacttg c 21

<210> 80

<211> 21

<212> DNA

<213> Artificial sequence

<400> 80

acctcacttc ctactttggt c 21

<210> 81

<211> 22

<212> DNA

<213> Artificial sequence

<400> 81

tctagcttgg aaatcattga ct 22

<210> 82

<211> 23

<212> DNA

<213> Artificial sequence

<400> 82

cttcaactaa catctacagc agg 23

<210> 83

<211> 20

<212> DNA

<213> Artificial sequence

<400> 83

tcagtcagct tgctctcttg 20

<210> 84

<211> 21

<212> DNA

<213> Artificial sequence

<400> 84

catctggcag tgttccacaa g 21

<210> 85

<211> 20

<212> DNA

<213> Artificial sequence

<400> 85

agcagcacaa ttcacagttg 20

<210> 86

<211> 22

<212> DNA

<213> Artificial sequence

<400> 86

ctcatggtcc aaactgtatg tg 22

<210> 87

<211> 22

<212> DNA

<213> Artificial sequence

<400> 87

gcacataaat gaagcagcag tg 22

<210> 88

<211> 22

<212> DNA

<213> Artificial sequence

<400> 88

tgctactgtc ttaggacttg tg 22

<210> 89

<211> 20

<212> DNA

<213> Artificial sequence

<400> 89

gtttagcagt tgcatgctgt 20

<210> 90

<211> 22

<212> DNA

<213> Artificial sequence

<400> 90

cattgttcca tactggtgag ac 22

<210> 91

<211> 20

<212> DNA

<213> Artificial sequence

<400> 91

acttggcctc tgttttctcc 20

<210> 92

<211> 21

<212> DNA

<213> Artificial sequence

<400> 92

gaaccagggc tgtatgttta c 21

<210> 93

<211> 20

<212> DNA

<213> Artificial sequence

<400> 93

acttggccct gaaacttctc 20

<210> 94

<211> 21

<212> DNA

<213> Artificial sequence

<400> 94

acaaggctta gtttgaggag a 21

<210> 95

<211> 22

<212> DNA

<213> Artificial sequence

<400> 95

ggcaaacttg aatatggagc tg 22

<210> 96

<211> 20

<212> DNA

<213> Artificial sequence

<400> 96

agcagcagga tctacatgtc 20

<210> 97

<211> 21

<212> DNA

<213> Artificial sequence

<400> 97

agatatgggt gagactcaag g 21

<210> 98

<211> 22

<212> DNA

<213> Artificial sequence

<400> 98

tgccatcctg tcatagttgt ag 22

<210> 99

<211> 22

<212> DNA

<213> Artificial sequence

<400> 99

acatactcaa gtctccctca tc 22

<210> 100

<211> 20

<212> DNA

<213> Artificial sequence

<400> 100

tcagatgcca gttacccctc 20

<210> 101

<211> 20

<212> DNA

<213> Artificial sequence

<400> 101

tcatcagatg ttgggcactg 20

<210> 102

<211> 20

<212> DNA

<213> Artificial sequence

<400> 102

cctgaagagc cgaaagacac 20

<210> 103

<211> 22

<212> DNA

<213> Artificial sequence

<400> 103

gaattgaatg accacagaga cg 22

<210> 104

<211> 21

<212> DNA

<213> Artificial sequence

<400> 104

gacaactact cccttgctgt c 21

<210> 105

<211> 526

<212> DNA

<213> Artificial sequence

<400> 105

attaaagatg acttatgtct ctgatctttt tttccttcta agtaataact accttgcaga 60

gttattactt gaattggcca taaggtaaag aggacaccaa atacagtaaa taataacata 120

ataataaaga aaccttatga attctattca gtgatatcca tattgaggta tttaaggtga 180

atgatactga tgtcagcagt ttacctggat atgcatcaaa aatatgattg attgatggat 240

ggatcggagg aaggaaaaga tatgtgatca aaacgagtat agtaaaatgt tcatggtgga 300

atctagatat atagatatca cttgtaattt ttaaaaatgt tattgcattt ttgaaatttg 360

ttatgttaaa atattgggtg aaaattctga tgtctcactc cacacaccca atgggaaaat 420

ttgtggaggg caatatgact cgtcacttca tttcccatta tatatgaatg gaaattaaca 480

gcgcttatag acagtatctc ctcaaactaa agccatatca aaagat 526

<210> 106

<211> 816

<212> DNA

<213> Artificial sequence

<400> 106

ccaactctac cttttacttc ccatgtgaac attaaaagat gacttaatgt ctctgaatct 60

ttttttcctt ctaagtaata actaccttgc agagttatta cttgaattgg ccataaggta 120

aagaggacac caaatacagt aaataataac ataataataa agaaacctta tgaattctat 180

tcagtgatat ccatattgag gtatttaagg tgaatgatac tgatgtcagc agtttacctg 240

gatatgcatc aaaaatatga ttgattgatg gatggatcgg aggaaggaaa agatatgtga 300

tcaaaacgag tatagtaaaa tgttcatggt ggaatctaga tatatagata tcacttgtaa 360

tttttaaaaa tgttattgca tttttgaaat ttgttatgtt aaaatattgg gtgaaaattc 420

tgatgctcct tgatggccca agccactctc tcagtttttt aaaaaattgt tttatcaagg 480

tctctggatt cttcatggga atgacttcca gtttatattt tttggcttgg ttccaaaaag 540

ctatcagcta aggaatgcat atacttactt cccctatggg taaagtaaat gagaatttta 600

gaagccaact cacattttta gcctgtacag aatctgcaat tcaccaagct acttctgact 660

catgtctata aagttcttcc ctgttctttt ctcacttcac atgtactctt tgcaagaatt 720

catccacttg tgtagtttca gtctgttgat gactacccat ctataattcc agctgagaat 780

gatcttttga gttttagaca tgtagatcct gctgct 816

<210> 107

<211> 989

<212> DNA

<213> Artificial sequence

<400> 107

gggaaattaa agatggactt aatgtctctg atcttttttt ccttctaagt aataactacc 60

ttgcagagtt attacttgaa ttggccataa ggtaaagagg acaccaaata cagtaaataa 120

taacataata ataaagaaac cttatgaatt ctattcagtg atatccatat tgaggtattt 180

aaggtgaatg atactgatgt cagcagttta cctggatatg catcaaaaat atgattgatt 240

gatggatgga tcggaggaag gaaaagatat gtgatcaaaa cgagtatagt aaaatgttca 300

tggtggaatc tagatatata gatatcactt gtaattttta aaaatgttat tgcatttttg 360

aaatttgtta tgttaaaata ttgggtgaaa attctgatga gtctcacttc cccagcacca 420

ccactttatg aaaacaagga cttactaaga tcatcagtga ctttgtaata gctaattagt 480

gtattttaat tcgtccatct tcttgactat attttaacat tgatcctgtt ggtcaactct 540

gctaatcaaa actttatcct ccttggttcc cagaacaata ttatcttgaa tatctcattt 600

ctctaatcat ataataattg tgaggtgctt ggcacaatgc ctagtgcgta gtaagaactc 660

agtaaaatat catctgccat cgacaccata aaaattaatt tacttactca acaaatactt 720

ttgtatgaag tttgtgctag gtaggcccag taattggtac ttggtataga gcaatgaaaa 780

gccctaaccc tcataaagct tatattcttg gaagcagaag ttggaagaca gacattgaca 840

aataaaaatt aaatacatga tgtgtcagat ggtcatacac acagtgtgga agaacaaaga 900

ggaaaacaag tggagagaga gagggaggtg ggaagaggag tgctgccatg aaaatgtggt 960

aattcaaaaa aggtctttac tgaaaaggt 989

<210> 108

<211> 1000

<212> DNA

<213> Artificial sequence

<400> 108

attaaaagga tgacttatgt ctctgatctt tttttccttc taagtaataa ctaccttgca 60

gagttattac ttgaattggc cataaggtaa agaggacacc aaatacagta aataataaca 120

taataataaa gaaaccttat gaattctatt cagtgatatc catattgagg tatttaaggt 180

gaatgatact gatgtcagca gtttacctgg atatgcatca aaaatatgat tgattgatgg 240

atggatcgga ggaaggaaaa gatatgtgat caaaacgagt atagtaaaat gttcatggtg 300

gaatctagat atatagatat cacttgtaat ttttaaaaat gttattgcat ttttgaaatt 360

tgttatgtta aaatattggg tgaaaattct gatgtcactt ccccagcacc accactttat 420

gaaaacaagg acttactaag atcatcagtg actttgtaat agctaattag tgtattttaa 480

ttcgtccatc ttcttgacta tattttaaca ttgatcctgt tggtcaactc tgctaatcaa 540

aactttatcc tccttggttc ccagaacaat attatcttga atatctcatt tctctaatca 600

tataataatt gtgaggtgct tggcacaatg cctagtgcgt agtaagaact cagtaaaata 660

tcatctgcca tcgacaccat aaaaattaat ttacttactc aacaaatact tttgtatgaa 720

gtttgtgcta ggtaggccca gtaattggta cttggtatag agcaatgaaa agccctaccc 780

tcataaagct tatattcttg gaagcagaag ttggaagaca gacattgaca aataaaaatt 840

aaatacatga tgtgtcagat ggtcatacac acagtgtgaa gacaagagga aaacaagtga 900

gagagagagg gagtggagag agtgctgcca tgaaattgtg taatcaaaaa gtcttactga 960

aagtggcatt aagcaattct aaagactgag atgtggcata 1000

<210> 109

<211> 562

<212> DNA

<213> Artificial sequence

<400> 109

aggggactta aagaatgact ttaatgtctc tgatcttttt ttccttctaa gtaataacta 60

ccttgcagag ttattacttg aattggccat aaggtaaaga ggacaccaaa tacagtaaat 120

aataacataa taataaagaa accttatgaa ttctattcag tgatatccat attgaggtat 180

ttaaggtgaa tgatactgat gtcagcagtt tacctggata tgcatcaaaa atatgattga 240

ttgatggatg gatcggagga aggaaaagat atgtgatcaa aacgagtata gtaaaatgtt 300

catggtggaa tctagatata tagatatcac ttgtaatttt taaaaatgtt attgcatttt 360

tgaaatttgt tatgttaaaa tattgggtga aaattctgat ggccaagagt ttgagaccag 420

cctgggcaac atagtgagat accatttcta caaaacataa aaaaaaaaaa aaaaaggcca 480

gttttttgtc cccccctctt tgggaagggg aggcaaagga tttttttgaa ccaaggattt 540

catttcaggg ggccgggggc ta 562

<210> 110

<211> 540

<212> DNA

<213> Artificial sequence

<400> 110

tttaagatga cttatgtctc tgatcttttt ttccttctaa gtaataacta ccttgcagag 60

ttattacttg aattggccat aaggtaaaga ggacaccaaa tacagtaaat aataacataa 120

taataaagaa accttatgaa ttctattcag tgatatccat attgaggtat ttaaggtgaa 180

tgatactgat gtcagcagtt tacctggata tgcatcaaaa atatgattga ttgatggatg 240

gatcggagga aggaaaagat atgtgatcaa aacgagtata gtaaaatgtt catggtggaa 300

tctagatata tagatatcac ttgtaatttt taaaaatgtt attgcatttt tgaaatttgt 360

tatgttaaaa tattgggtga aaattctgat gccctggcca tgtcccaaat tctcactcca 420

cacacccaat gggaaaattt gtggagggca atatgactcg tcacttcatt tcccattata 480

tatgaatgga aattaacagc gcttatagac agtatctcct caaactaagc cttgtgggag 540

<210> 111

<211> 813

<212> DNA

<213> Artificial sequence

<400> 111

aggaatttta agatgactta atgtctctga tctttttttc cttctaagta ataactacct 60

tgcagagtta ttacttgaat tggccataag gtaaagagga caccaaatac agtaaataat 120

aacataataa taaagaaacc ttatgaattc tattcagtga tatccatatt gaggtattta 180

aggtgaatga tactgatgtc agcagtttac ctggatatgc atcaaaaata tgattgattg 240

atggatggat cggaggaagg aaaagatatg tgatcaaaac gagtatagta aaatgttcat 300

ggtggaatct agatatatag atatcacttg taatttttaa aaatgttatt gcatttttga 360

aatttgttat gttaaaatat tgggtgaaaa ttctgatgcc ctggccatgt cccaaatctc 420

cttgatggcc caagccactc tctcagtttt ttaaaaaatt gttttatcaa ggtctctgga 480

ttcttcatgg gaatgacttc cagtttatat tttttggctt ggttccaaaa agctatcagc 540

taaggaatgc atatacttac ttcccctatg ggtaaagtaa atgagaattt tagaagccaa 600

ctcacatttt tagcctgtac agaatctgca attcaccaag ctacttctga ctcatgtcta 660

taaagttctt ccctgttctt ttctcacttc acatgtactc tttgcaagaa ttcatccact 720

tgtgtagttt cagtctgttg atgactaccc aatctataaa ttccagcaga gaatgatctt 780

ttgagtttta gacatgtaga tacactgctg ctc 813

<210> 112

<211> 939

<212> DNA

<213> Artificial sequence

<400> 112

ggggttaaat taagatgact tatgtctctg atcttttttt ccttctaagt aataactacc 60

ttgcagagtt attacttgaa ttggccataa ggtaaagagg acaccaaata cagtaaataa 120

taacataata ataaagaaac cttatgaatt ctattcagtg atatccatat tgaggtattt 180

aaggtgaatg atactgatgt cagcagttta cctggatatg catcaaaaat atgattgatt 240

gatggatgga tcggaggaag gaaaagatat gtgatcaaaa cgagtatagt aaaatgttca 300

tggtggaatc tagatatata gatatcactt gtaattttta aaaatgttat tgcatttttg 360

aaatttgtta tgttaaaata ttgggtgaaa attctgatgc cctggccatg tcccaaatag 420

tctcacttcc ccagcaccac cactttatga aaacaaggac ttactaagat catcagtgac 480

tttgtaatag ctaattagtg tattttaatt cgtccatctt cttgactata ttttaacatt 540

gatcctgttg gtcaactctg ctaatcaaaa ctttatcctc cttggttccc agaacaatat 600

tatcttgaat atctcatttc tctaatcata taataattgt gaggtgcttg gcacaatgcc 660

tagtgcgtag taagaactca gtaaaatatc atctgccatc gacaccataa aaattaattt 720

acttactcaa caaatacttt tgtatgaagt ttgtgctagg taggcccagt aattggtact 780

tggtatagag caatgaaaag ccctaccctc ataaagctta tattcttgga agcagaagtt 840

ggaagacaga cattgacaaa taaaaattaa atacatgatg tgtcagatgg tcatacacac 900

agtgtgggaa gacaaagagg aaaacaagtg gagagagag 939

<210> 113

<211> 928

<212> DNA

<213> Artificial sequence

<400> 113

tggggttcta taaaagaaga cttaatgtct ctgatctttt tttccttcta agtaataact 60

accttgcaga gttattactt gaattggcca taaggtaaag aggacaccaa atacagtaaa 120

taataacata ataataaaga aaccttatga attctattca gtgatatcca tattgaggta 180

tttaaggtga atgatactga tgtcagcagt ttacctggat atgcatcaaa aatatgattg 240

attgatggat ggatcggagg aaggaaaaga tatgtgatca aaacgagtat agtaaaatgt 300

tcatggtgga atctagatat atagatatca cttgtaattt ttaaaaatgt tattgcattt 360

ttgaaatttg ttatgttaaa atattgggtg aaaattctga tgccctggcc atgtcccaaa 420

ttcacttccc cagcaccacc actttatgaa aacaaggact tactaagatc atcagtgact 480

ttgtaatagc taattagtgt attttaattc gtccatcttc ttgactatat tttaacattg 540

atcctgttgg tcaactctgc taatcaaaac tttatcctcc ttggttccca gaacaatatt 600

atcttgaata tctcatttct ctaatcatat aataattgtg aggtgcttgg cacaatgcct 660

agtgcgtagt aagaactcag taaaatatca tctgccatcg acaccataaa aattaattta 720

cttactcaac aaatactttt gtatgaagtt tgtgctaggt aggcccagta attggtactt 780

ggtatagagc aatgaaaagc cctaccctca taaagcttat attcttggaa gcagaagttg 840

gaagacagac attgacaaat aaaaattaaa tacatgatgt gtcagatggt catacacaca 900

gtgtgggaag aacaaaggag gaaaacaa 928

<210> 114

<211> 522

<212> DNA

<213> Artificial sequence

<400> 114

aggggttcca ttaggatgac tttaattgtc tctgaatctt tttttccttc taagtaaata 60

actaccttgc agagttatta cttgaattgg ccataaggta aagaggacac caaatacagt 120

aaataataac ataataataa agaaacctta tgaattctat tcagtgatat ccatattgag 180

gtatttaagg tgaatgatac tgatgtcagc agtttacctg gatatgcatc aaaaatatga 240

ttgattgatg gatggatcgg aggaaggaaa agatatgtga tcaaaacgag tatagtaaaa 300

tgttcatggt ggaatctaga tatatagata tcacttgtaa tttttaaaaa tgttattgca 360

tttttgaaat ttgttatgtt aaaatattgg gtgaaaattc tgatgccctg gccatgtccc 420

aaatgccaag agtttgagac cagcctgggc aacatagtga gataccattt ctacaaaaca 480

taaaaaaaaa aaaaaaaagc caattttttt gtccccggct ac 522

<210> 115

<211> 422

<212> DNA

<213> Artificial sequence

<400> 115

gcggtgactc gttcatgcac tggttgaaat atgctcccca ccccagtaac atcagcatca 60

gacctctgca accagaaaga ttccataggt aatgaaatcc aaatgctggc atttggaagt 120

caaaccagtg tattttgaaa tggaaaggca aggggaagtg catggagcaa gtacagcatg 180

tgctacaact tcagttcata ccctccttaa aatactgtgc aactaaactg tcaattgccc 240

attgatagag atagtatctt tttcagcttt acaccttccg aattcaacac tctcactcca 300

cacacccaat gggaaaattt gtggagggca atatgactcg tcacttcatt tcccattata 360

tatgaatgga aattaacagc gcttatagac agtatctcct caaactaaga cttgagaaaa 420

aa 422

<210> 116

<211> 690

<212> DNA

<213> Artificial sequence

<400> 116

ccgtgaatcg tcatgcactg gttgaaaata tgctccccac cccagtaaca tcagcatcag 60

acctctgcaa ccagaaagat tccataggta atgaaatcca aatgctggca tttggaagtc 120

aaaccagtgt attttgaaat ggaaaggcaa ggggaagtgc atggagcaag tacagcatgt 180

gctacaactt cagttcatac cctccttaaa atactgtgca actaaactgt caattgccca 240

ttgatagaga tagtatcttt ttcagcttta caccttccga attcaacacc tccttgatgg 300

cccaagccac tctctcagtt ttttaaaaaa ttgttttatc aaggtctctg gattcttcat 360

gggaatgact tccagtttat attttttggc ttggttccaa aaagctatca gctaaggaat 420

gcatatactt acttccccta tgggtaaagt aaatgagaat tttagaagcc aactcacatt 480

tttagcctgt acagaatctg caattcacca agctacttct gactcatgtc tataaagttc 540

ttccctgttc ttttctcact tcacatgtac tctttgcaag aattcatcca cttgtgtagt 600

ttcagtctgt tgatgactac ccatctataa ttccagcaga gaatgatctt ttgagtttta 660

gacatgtaga tcacagcaga gctgaaatgg 690

<210> 117

<211> 994

<212> DNA

<213> Artificial sequence

<400> 117

gcgttacttc atcatgcact ggttgaaata tgctccccac cccagtaaca tcagcatcag 60

acctctgcaa ccagaaagat tccataggta atgaaatcca aatgctggca tttggaagtc 120

aaaccagtgt attttgaaat ggaaaggcaa ggggaagtgc atggagcaag tacagcatgt 180

gctacaactt cagttcatac cctccttaaa atactgtgca actaaactgt caattgccca 240

ttgatagaga tagtatcttt ttcagcttta caccttccga attcaacaca gtctcacttc 300

cccagcacca ccactttatg aaaacaagga cttactaaga tcatcagtga ctttgtaata 360

gctaattagt gtattttaat tcgtccatct tcttgactat attttaacat tgatcctgtt 420

ggtcaactct gctaatcaaa actttatcct ccttggttcc cagaacaata ttatcttgaa 480

tatctcattt ctctaatcat ataataattg tgaggtgctt ggcacaatgc ctagtgcgta 540

gtaagaactc agtaaaatat catctgccat cgacaccata aaaattaatt tacttactca 600

acaaatactt ttgtatgaag tttgtgctag gtaggcccag taattggtac ttggtataga 660

gcaatgaaaa gccctaccct cataaagctt atattcttgg aagcagaagt tggaagacag 720

acattgacaa ataaaaatta aatacatgat gtgtcagatg gtcatacaca cagtgtggaa 780

gaacaaagag gaaaacaagt ggagagagag agggaggtgg aagaggagtg ctgccatgaa 840

aatgtggtaa tcaaaaaagg tcttactgaa aaaggtggca tttaagcaaa ttctaaaaga 900

cctgaggatg tgggcctatg tatattgggg ggggaaaagt aatccaggag agtcctaaat 960

aattaaaatg cccaagcagg aattttcttg gcat 994

<210> 118

<211> 889

<212> DNA

<213> Artificial sequence

<400> 118

cgggtgaaat tcttcatgca ctggttgaaa atatgctccc caccccagta acatcagcat 60

cagacctctg caaccagaaa gattccatag gtaatgaaat ccaaatgctg gcatttggaa 120

gtcaaaccag tgtattttga aatggaaagg caaggggaag tgcatggagc aagtacagca 180

tgtgctacaa cttcagttca taccctcctt aaaatactgt gcaactaaac tgtcaattgc 240

ccattgatag agatagtatc tttttcagct ttacaccttc cgaattcaac actcacttcc 300

ccagcaccac cactttatga aaacaaggac ttactaagat catcagtgac tttgtaatag 360

ctaattagtg tattttaatt cgtccatctt cttgactata ttttaacatt gatcctgttg 420

gtcaactctg ctaatcaaaa ctttatcctc cttggttccc agaacaatat tatcttgaat 480

atctcatttc tctaatcata taataattgt gaggtgcttg gcacaatgcc tagtgcgtag 540

taagaactca gtaaaatatc atctgccatc gacaccataa aaattaattt acttactcaa 600

caaatacttt tgtatgaagt ttgtgctagg taggcccagt aattggtact tggtatagag 660

caatgaaaag ccctaccctc ataaagctta tattcttgga agcagaagtt ggaagacaga 720

cattgacaaa taaaaattaa atacatgatg tgtcagatgg tcatacacac agtgtggaag 780

aacaaagagg aaaacaagtg gagagagaga gggaggtgga aagaggagtg ctgccatgaa 840

aatgttggta atcaaaaaag gtcttactga agaggtggct atttaagca 889

<210> 119

<211> 730

<212> DNA

<213> Artificial sequence

<400> 119

gcgtggatct tcatgcactg gttgaaaata tgctccccac cccagtaaca tcagcatcag 60

acctctgcaa ccagaaagat tccataggta atgaaatcca aatgctggca tttggaagtc 120

aaaccagtgt attttgaaat ggaaaggcaa ggggaagtgc atggagcaag tacagcatgt 180

gctacaactt cagttcatac cctccttaaa atactgtgca actaaactgt caattgccca 240

ttgatagaga tagtatcttt ttcagcttta caccttccga attcaacacg ccaagagttt 300

gagaccagcc tgggcaacat agtgagatac catttctaca aaacataaaa aaaaaaaaaa 360

aaaagccaat tttttttccc cccctcttgg gaaacttggg gaaaaagatt tttttgaccc 420

agggatttct tttcaggggg ggggggggct taaaaaagcc cagggtcccc cccccggggg 480

gacgtaggga aaccccctcc ttaaaattta attaaatttt aagggtaaaa gggcccccaa 540

accccccgtt cacccccccc attttttaaa accccatttt aaatcctcct taggaaaatt 600

acccttgttg ctctttttag ggggggaagg gtttgggtat gtttaaaaat taatccgttt 660

aacaactctt tttggacctt aggaattaaa aaatgttcct tttggctctt ggggaaaaaa 720

aaaaataaat 730

<210> 120

<211> 622

<212> DNA

<213> Artificial sequence

<400> 120

gcggggcaag tttatgaatg tagtagtttt atgccaagaa catttccctc gtgcacctgt 60

gttttaataa gatagaatat aaaatagcaa aaggggcccg acttttgtga tgatatttac 120

tcataaggta gtaagtcaga tgtgatgaca ttttctttga cttagagctg ctatacttgg 180

gtcagatttc agttcagtaa atttgcagtg aagttgtctt tctaacatgg tgtcatcctg 240

gaactgccct gctcccacag ttacatacag gctagggagt gggtaggagt gggggtgaaa 300

tcctcttaat gtttatggtg tcagtagatt caaactaaaa ttagccttac agccatactc 360

ctaataaggg gcccctggca tatttaattg atttaacaaa tttatcaaaa atagataaac 420

tgaaatctgc cttgaaatta attactgtat ctctattttt ataagaaaat attttggacc 480

cgttcctctc actccacaca cccaatggga aaatttgtgg agggcaatat gactcgtcac 540

ttcatttccc attatatatg aatggaaatt aacagcgctt atagacagta tctcctcaaa 600

ctaagccctt tggtgggggt gg 622

<210> 121

<211> 893

<212> DNA

<213> Artificial sequence

<400> 121

ggcgggggga aaaattatga tgtagtagtt ttatgccaag aacatttccc tcgtgcacct 60

gtgttttaat aagatagaat ataaaatagc aaaaggggcc cgacttttgt gatgatattt 120

actcataagg tagtaagtca gatgtgatga cattttcttt gacttagagc tgctatactt 180

gggtcagatt tcagttcagt aaatttgcag tgaagttgtc tttctaacat ggtgtcatcc 240

tggaactgcc ctgctcccac agttacatac aggctaggga gtgggtagga gtgggggtga 300

aatcctctta atgtttatgg tgtcagtaga ttcaaactaa aattagcctt acagccatac 360

tcctaataag gggcccctgg catatttaat tgatttaaca aatttatcaa aaatagataa 420

actgaaatct gccttgaaat taattactgt atctctattt ttataagaaa atattttgga 480

cccgttccct ccttgatggc ccaagccact ctctcagttt tttaaaaaat tgttttatca 540

aggtctctgg attcttcatg ggaatgactt ccagtttata ttttttggct tggttccaaa 600

aagctatcag ctaaggaatg catatactta cttcccctat gggtaaagta aatgagaatt 660

ttagaagcca actcacattt ttagcctgta cagaatctgc aattcaccaa gctacttctg 720

actcatgtct ataaagttct tccctgttct tttctcactt cacatgtact ctttgcaaga 780

attcatccac ttgtgtagtt tcagtctgtt gatgactacc catctataat tccagcagag 840

aatgatcttt tgagttttag acatgtagac cctgccagtg aaagaaaaaa agg 893

<210> 122

<211> 1323

<212> DNA

<213> Artificial sequence

<400> 122

caggtttaga catgtgcctt ccggtgtggg aaagaaaaaa tttaatgaat gtagtagttt 60

tatgccaaga acatttccct cgtgcacctg tgttttaata agatagaata taaaatagca 120

aaaggggccc gacttttgtg atgatattta ctcataaggt agtaagtcag atgtgatgac 180

attttctttg acttagagct gctatacttg ggtcagattt cagttcagta aatttgcagt 240

gaagttgtct ttctaacatg gtgtcatcct ggaactgccc tgctcccaca gttacataca 300

ggctagggag tgggtaggag tgggggtgaa atcctcttaa tgtttatggt gtcagtagat 360

tcaaactaaa attagcctta cagccatact cctaataagg ggcccctggc atatttaatt 420

gatttaacaa atttatcaaa aatagataaa ctgaaatctg ccttgaaatt aattactgta 480

tctctatttt tataagaaaa tattttggac ccgttccagt ctcacttccc cagcaccacc 540

actttatgaa aacaaggact tactaagatc atcagtgact ttgtaatagc taattagtgt 600

attttaattc gtccatcttc ttgactatat tttaacattg atcctgttgg tcaactctgc 660

taatcaaaac tttatcctcc ttggttccca gaacaatatt atcttgaata tctcatttct 720

ctaatcatat aataattgtg aggtgcttgg cacaatgcct agtgcgtagt aagaactcag 780

taaaatatca tctgccatcg acaccataaa aattaattta cttactcaac aaatactttt 840

gtatgaagtt tgtgctaggt aggcccagta attggtactt ggtatagagc aatgaaaagc 900

cctaccctca taaagcttat attcttggaa gcagaagttg gaagacagac attgacaaat 960

aaaaattaaa tacatgatgt gtcagatggt catacacaca gtgtggaaga acaaagagga 1020

aaacaagtgg agagagagag ggaggtggaa gaggagtgct gccatgaaaa tgtggtaatc 1080

aaaaaaggtc ttactgaaaa ggtggcattt aagcaaattc taaaagacct gaggatgtgg 1140

gccatatgta taattggggg ggaaaaagta gtccaggaga gtcctaataa gttaaaatgc 1200

cccaaagcag gaatattctt ggcatgttga aggaacctta aaagggagat cagttaggca 1260

gaaaaggatc aagcgagcag gaaggtagtt gacaataaat ttagaggggt aactggcatc 1320

tga 1323

<210> 123

<211> 964

<212> DNA

<213> Artificial sequence

<400> 123

cgcgggtgga aatttatgat gtagtagttt tatgccaaga acatttccct cgtgcacctg 60

tgttttaata agatagaata taaaatagca aaaggggccc gacttttgtg atgatattta 120

ctcataaggt agtaagtcag atgtgatgac attttctttg acttagagct gctatacttg 180

ggtcagattt cagttcagta aatttgcagt gaagttgtct ttctaacatg gtgtcatcct 240

ggaactgccc tgctcccaca gttacataca ggctagggag tgggtaggag tgggggtgaa 300

atcctcttaa tgtttatggt gtcagtagat tcaaactaaa attagcctta cagccatact 360

cctaataagg ggcccctggc atatttaatt gatttaacaa atttatcaaa aatagataaa 420

ctgaaatctg ccttgaaatt aattactgta tctctatttt tataagaaaa tattttggac 480

ccgttcctca cttccccagc accaccactt tatgaaaaca aggacttact aagatcatca 540

gtgactttgt aatagctaat tagtgtattt taattcgtcc atcttcttga ctatatttta 600

acattgatcc tgttggtcaa ctctgctaat caaaacttta tcctccttgg ttcccagaac 660

aatattatct tgaatatctc atttctctaa tcatataata attgtgaggt gcttggcaca 720

atgcctagtg cgtagtaaga actcagtaaa atatcatctg ccatcgacac cataaaaatt 780

aatttactta ctcaacaaat acttttgtat gaagtttgtg ctaggtaggc ccagtaattg 840

gtacttggta tagagcaatg aaaagcccta ccctcataaa gcttatattc ttggaagcag 900

aagttggaag acagacattg acaaataaaa atttaaatac atgatgtgtc agatggtcat 960

acac 964

<210> 124

<211> 900

<212> DNA

<213> Artificial sequence

<400> 124

cgcgggtgga aattatgatg tagtagtttt atgccaagaa catttccctc gtgcacctgt 60

gttttaataa gatagaatat aaaatagcaa aaggggcccg acttttgtga tgatatttac 120

tcataaggta gtaagtcaga tgtgatgaca ttttctttga cttagagctg ctatacttgg 180

gtcagatttc agttcagtaa atttgcagtg aagttgtctt tctaacatgg tgtcatcctg 240

gaactgccct gctcccacag ttacatacag gctagggagt gggtaggagt gggggtgaaa 300

tcctcttaat gtttatggtg tcagtagatt caaactaaaa ttagccttac agccatactc 360

ctaataaggg gcccctggca tatttaattg atttaacaaa tttatcaaaa atagataaac 420

tgaaatctgc cttgaaatta attactgtat ctctattttt ataagaaaat attttggacc 480

cgttccgcca agagtttgag accagcctgg gcaacatagt gagataccat ttctacaaaa 540

cataaaaaaa aaaaaaaaaa acccagtttg tgactccccc ccatttggaa attggaggaa 600

aaagattttt ttgaaccaag aatttctttt aagggggggg ggggcctata atatggccgg 660

gtaatcccgc cggggggaaa ggagggaaac ccccccctta aaattaaatt aaatttaaaa 720

gctaaaaata acccaaaacc cccgttaccc cccccaattt ttaaaaccca ttttaaattt 780

ccccttgtcg aaagggcctt gtatcccttt ttaggaataa aagattgggt ggttaaaaaa 840

taatccatta ccaatccctt gggacctagc aaattaaaat ggctcttcgc cccctcagaa 900

<210> 125

<211> 417

<212> DNA

<213> Artificial sequence

<400> 125

agcattctgc tgtgagaaca gcaggaggcc ctcaccagac accaaatgcc agagccttta 60

tcttggactt cccagcctcc agaactgtga gtgaatacat tggtattatt tgtaaattac 120

ccagtctcag gcattttgtt ataacagcac aaacagacta agacaatcat acagtgagaa 180

attaatcaac aactaataag caaagaggta gattaatctt gaaactatga tatagagtgt 240

tccatttggc tgctggaagt tttatttctt ggtctgggtg atggtctctc actccacaca 300

cccaatggga aaatttgtgg agggcaatat gactcgtcac ttcatttccc attatatatg 360

aatggaaatt aacagcgctt atagacagta tctcctcaaa ctaaagccat gagaaaa 417

<210> 126

<211> 685

<212> DNA

<213> Artificial sequence

<400> 126

ccatctgcat gtgagaacag caggaaggcc ctcaccagac accaaatgcc agagccttta 60

tcttggactt cccagcctcc agaactgtga gtgaatacat tggtattatt tgtaaattac 120

ccagtctcag gcattttgtt ataacagcac aaacagacta agacaatcat acagtgagaa 180

attaatcaac aactaataag caaagaggta gattaatctt gaaactatga tatagagtgt 240

tccatttggc tgctggaagt tttatttctt ggtctgggtg atggtcctcc ttgatggccc 300

aagccactct ctcagttttt taaaaaattg ttttatcaag gtctctggat tcttcatggg 360

aatgacttcc agtttatatt ttttggcttg gttccaaaaa gctatcagct aaggaatgca 420

tatacttact tcccctatgg gtaaagtaaa tgagaatttt agaagccaac tcacattttt 480

agcctgtaca gaatctgcaa ttcaccaagc tacttctgac tcatgtctat aaagttcttc 540

cctgttcttt tctcacttca catgtactct ttgcaagaat tcatccactt gtgtagtttc 600

agtctgttga tgactaccca tctataattc cagcagagaa tgatcttttg agttttagac 660

atgtagaaca agctgcctga aaaaa 685

<210> 127

<211> 979

<212> DNA

<213> Artificial sequence

<400> 127

cacatttctg cctgtgagat acagcaggag gccctcacca gacaccaaat gccagagcct 60

ttatcttgga cttcccagcc tccagaactg tgagtgaata cattggtatt atttgtaaat 120

tacccagtct caggcatttt gttataacag cacaaacaga ctaagacaat catacagtga 180

gaaattaatc aacaactaat aagcaaagag gtagattaat cttgaaacta tgatatagag 240

tgttccattt ggctgctgga agttttattt cttggtctgg gtgatggtca gtctcacttc 300

cccagcacca ccactttatg aaaacaagga cttactaaga tcatcagtga ctttgtaata 360

gctaattagt gtattttaat tcgtccatct tcttgactat attttaacat tgatcctgtt 420

ggtcaactct gctaatcaaa actttatcct ccttggttcc cagaacaata ttatcttgaa 480

tatctcattt ctctaatcat ataataattg tgaggtgctt ggcacaatgc ctagtgcgta 540

gtaagaactc agtaaaatat catctgccat cgacaccata aaaattaatt tacttactca 600

acaaatactt ttgtatgaag tttgtgctag gtaggcccag taattggtac ttggtataga 660

gcaatgaaaa gccctaccct cataaagctt atattcttgg aagcagaagt tggaagacag 720

acattgacaa ataaaaatta aatacatgat gtgtcagatg gtcatacaca cagtgtggaa 780

gaacaaagag gaaaacaagt ggagagagag agggaggtgg aagaggagtg ctgccatgaa 840

aatgtggtaa tcaaaaaagg tcttactgaa aaggtggcat ttaagcaatt ctaaagacct 900

gaggatgtgg gccatatgta taattggggg ggaaaagtag tccaggagag tcctaataag 960

ttaaaatgcc ccaaagcag 979

<210> 128

<211> 989

<212> DNA

<213> Artificial sequence

<400> 128

accttctgcg tgtgagatac agcaggaagg ccctcaccag acaccaaatg ccagagcctt 60

tatcttggac ttcccagcct ccagaactgt gagtgaatac attggtatta tttgtaaatt 120

acccagtctc aggcattttg ttataacagc acaaacagac taagacaatc atacagtgag 180

aaattaatca acaactaata agcaaagagg tagattaatc ttgaaactat gatatagagt 240

gttccatttg gctgctggaa gttttatttc ttggtctggg tgatggtctc acttccccag 300

caccaccact ttatgaaaac aaggacttac taagatcatc agtgactttg taatagctaa 360

ttagtgtatt ttaattcgtc catcttcttg actatatttt aacattgatc ctgttggtca 420

actctgctaa tcaaaacttt atcctccttg gttcccagaa caatattatc ttgaatatct 480

catttctcta atcatataat aattgtgagg tgcttggcac aatgcctagt gcgtagtaag 540

aactcagtaa aatatcatct gccatcgaca ccataaaaat taatttactt actcaacaaa 600

tacttttgta tgaagtttgt gctaggtagg cccagtaatt ggtacttggt atagagcaat 660

gaaaagccct accctcataa agcttatatt cttggaagca gaagttggaa gacagacatt 720

gacaaataaa aattaaatac atgatgtgtc agatggtcat acacacagtg tggaagaaca 780

aagaggaaaa caagtggaga gagagaggga ggtggaagag gagtgctgcc atgaaaatgt 840

ggtaatcaaa aaaggtctta ctgaaaaggt ggcatttaag caaattctaa agacctgagg 900

atgtgggcca tatgtataat tgggggggaa aagtagtcca ggagagtcct aataagttaa 960

aatgccccaa agcaggaata ttcttggca 989

<210> 129

<211> 720

<212> DNA

<213> Artificial sequence

<400> 129

gccgtttgcc gggtgagata cagcaggatg gccctcacca gacaccgaat gccagagcct 60

ttatcttgga cttcccagcc tccagaactg tgagtgaata cattggtatt atttgtaaat 120

tacccagtct caggcatttt gttataacag cacaaacaga ctaagacaat catacagtga 180

gaaattaatc aacaactaat aagcaaagag gtagattaat cttgaaacta tgatatagag 240

tgttccattt ggctgctgga agttttattt cttggtctgg gtgatggtcg ccaagagttt 300

gagaccagcc tgggcaacat aatgagatac catttctaca aaacataaaa aaaaaaaaaa 360

aaacgccgtt tttttttccc ccccttttgg gaaaccttga ggaaagattt ttttggaccc 420

aaaaagttct gttacgggtc cgggggattt ttttaatttc ccttttcctc ccagtggggg 480

gagaatggga aaacccctct cataaaaaaa aataaattta aaaccaaaaa aggcccccca 540

agccccccga taaccctctt aaatttgaaa acccctttat taattcccct ttaggcaaaa 600

gttgtggggg ggccctattt agggagggaa gatatttgga agttaaaaaa tcccttccct 660

ttgacacccc ctttagggac ccaacaagga gataaaaatg gtttttttgg gtttcgggaa 720

<210> 130

<211> 661

<212> DNA

<213> Artificial sequence

<400> 130

gggagttaat attgttatgc tgcagggtta ttgtaggtgt caatgaaatg atgtgtctgg 60

cactataaaa gcacagagcc cggtgcctgg ctattagtaa ctgtttaata aatgttaatt 120

cctttctctg cccaggacat cagtaggcag atgtagcaat ttaaaacttc tagtgttact 180

ttaaattcct gaatgaaggt agaggactga aaagatatca tggtattcaa aagtatgatc 240

cattgcttct taagaataga gttcagaaaa gcttgacaga ttcctgtact ctgaggcagc 300

accatagccg gtaatctgta ggatggctat tggttttgtg ctcacaaatg cttgcttggg 360

caggccccag gaaatctggt agactgtaag cccagtaaga tttcaaatct tactttacgg 420

cagtgttttt caccttgact gtacattgaa atcacctgga tgctttgaaa aataacagcg 480

tcagtgtcca acctccagaa atactgatta agttggtctg gaatggagtc tcactccaca 540

cacccaatgg gaaaatttgt ggagggcaat atgactcgtc acttcatttc ccattatata 600

tgaatggaaa ttaacagcgc ttatagacag tatctcctca aactaagcct ttgtgaaaga 660

a 661

Claims

1. A gRNA for treating Duchenne muscular dystrophy, the gRNA consisting of a gRNA whose target sequence is shown in SEQ ID NO. 22 and a gRNA whose target sequence is shown in SEQ ID NO. 59.

2. An expression vector for treating duchenne muscular dystrophy, said expression vector expressing a gRNA of claim 1.

3. The expression vector of claim 2, wherein the base vector of the expression vector is an AAV vector.

4. A CRISPR-Cas9 system for treating duchenne muscular dystrophy, comprising a gRNA of claim 1 and a Cas9 protein.

5. The CRISPR-Cas9 system according to claim 4, wherein the Cas9 protein is a SaCas9 protein.

6. An expression vector for treating duchenne muscular dystrophy, said expression vector expressing a Cas9 protein and a gRNA of claim 1.

7. The expression vector of claim 6, wherein the C as9 protein is SaCas9 protein, and the basic vector of the expression vector is an AAV vector.

8. Use of a gRNA of claim 1 or an expression vector of claim 2 or 3 in the manufacture of a medicament for preventing or treating duchenne muscular dystrophy.

9. Use of the CRISPR-Cas9 system of claim 4 or 5, or the expression vector of claim 6 or 7, in the manufacture of a medicament for the prevention or treatment of duchenne muscular dystrophy.