Patent Description:
Duchenne muscular dystrophy (hereinafter referred to as DMD) is a disease causing atrophy of muscle fibers due to loss of function of the dystrophin gene. The skeletal muscle isoform (Dp427m) of the dystrophin gene is constituted by <NUM> exons. In cases where shifting of the reading frame occurs in this gene due to partial deletion of the exons or the like, normal production of the dystrophin protein becomes impossible, leading to development of DMD (Non-patent Document <NUM>).

Aiming at radical cure of DMD, various studies have been carried out for gene therapies in which a functional exogenous dystrophin gene is added to replace a dysfunctional endogenous dystrophin gene. Since the full-length cDNA of dystrophin has a size of as long as <NUM> kb, attempts are being made to reduce its size by elimination of unnecessary domain portions, and to introduce the thus prepared minidystrophin or microdystrophin into muscular tissue using various vectors for gene transfer (AAV, lentivirus, Sleeping Beauty transposon vectors, and the like). However, since introduction of a huge gene is difficult, no effective therapeutic method has been established so far.

In a study in progress, an antisense oligonucleotide is used to prevent reading of part of a particular exon during splicing of mRNA in order to restore dystrophin having the normal function (exon skipping). However, since the antisense oligonucleotide is only temporarily effective, a method for repairing the gene itself has been demanded for the radical cure.

As methods for repairing the gene itself, genome editing techniques such as TALEN and CRISPR-Cas systems have recently been developed. In these techniques, a particular sequence position is recognized in the genome sequence, and then DNA double-strand break is induced to cause local induction of a DNA repair mechanism through non-homologous recombination (non-homologous end joining, NHEJ) or homologous recombination (homology directed repair, HDR), thereby enabling addition of a base(s) to or deletion of a base(s) from the cleaved site.

For CRISPR-Cas genome editing techniques, the type II and type V CRISPR systems of bacteria and archaebacteria are widely used. They can bind to a target DNA dependently on a spacer sequence contained in a guide RNA (gRNA or sgRNA), to induce a double-strand DNA break by the action of a Cas nuclease (Cas9 in cases of type II, and Cpfl in cases of type V). In the type II CRISPR system, the guide RNA is a complex containing crRNA and tracrRNA, or an sgRNA (single guide RNA) containing crRNA and tracrRNA linked to each other.

It has been reported that exon skipping for dystrophin using a genome editing technique was carried out in myoblasts [Non-patent Documents <NUM> and <NUM>] or at the mdx mouse level [Non-patent Documents <NUM> to <NUM>].

In these studies, both ends of the exon to be skipped are cleaved using two guide RNAs, to induce a large deletion including the whole exon. However, such a method increases the risk of non-specific cleavage since two gRNAs are required. Moreover, cleavage by only one of the gRNAs cannot induce exon skipping, and the two gRNA sequences need to act in the same genome. Moreover, at least several hundred bases need to be deleted, and, in cases where the region contains an unknown regulatory region or miRNA-coding region, there is a risk of occurrence of unexpected side effects.

On the other hand, the present inventors have previously reported that, by using a genome editing technique such as TALEN or CRISPR-Cas9 in iPS cells derived from DMD patients, a dystrophin gene mutation can be repaired by (<NUM>) exon skipping, (<NUM>) frameshift induction, and (<NUM>) knock-in of a deleted exon [Non-patent Document <NUM>]. Among these, the method of (<NUM>) employs a method in which the splice acceptor of the exon is deleted. Since exon skipping can be sufficiently induced in cases where deletion of several bases to several ten bases can be induced with one gRNA, the method is superior to the methods reported by [Non-patent Documents <NUM> to <NUM>] in terms of the three facts: a higher efficiency, a smaller risk of side-effect mutations, and requirement of only small DNA base deletion.

On the other hand, although a splice acceptor is an attractive target site for induction of exon skipping, it contains a polypyrimidine sequence (consecutive T/C's), and similar sequences are contained in a large number of exon sequences. Therefore, the method has a problem in that a gRNA having a high specificity cannot be easily designed. The double-nicking method, in which the specificity is increased by combination of two units of nickase-modified CRISPR-Cas containing a mutation introduced into the DNA cleavage domain of CRISPR-Cas such that a single-strand break rather than a DNA double-strand break is induced [<NPL>. ], is known. Since type V AsCpfl is known to have a higher specificity than Type II SpCas9 in human cells [<NPL>. <NUM><NPL>. ], it is thought that the problem of the specificity can be avoided by targeting a site near the splicing acceptor using the double-nicking method or Cpfl.

It is also known that the cleavage activity and the cleavage length vary depending on the spacer sequence in the guide RNA and the type of the CRISPR-Cas, and guide sequences and design methods that enable efficient induction of exon skipping have been empirically unknown.

An object of the present invention as defined in the claims is to provide an efficient method of exon skipping.

The present inventors intensively studied to solve the above problems. The present inventors discovered that, by using, as a target gene for exon skipping, a marker gene containing a sequence which is inserted in a coding region and which contains a first intron, an exon to be analyzed, and a second intron, and by designing the marker gene such that the marker gene functions when the exon to be analyzed is skipped, the exon skipping can be efficiently analyzed based on the phenotype of the marker gene. As a result, the present inventors discovered that, when targeted exon skipping is carried out for a gene of interest in a genome using CRISPR-Cas and guide RNA, by arranging the guide RNA such that the site of cleavage by the CRISPR-Cas is positioned within <NUM> bases from the splice acceptor site or the splice donor site of the target exon, the efficiency of the exon skipping can be increased, thereby completing the present invention.

According to the method of the present invention as defined in the claims, the efficiency of exon skipping can be increased in exon skipping utilizing a genome editing technique, so that the method is effective for use in the treatment of diseases and the like. Further, by using, as a target gene for the exon skipping, a marker gene containing a sequence which is inserted in a coding region and which contains a first intron, an exon to be analyzed, and a second intron, and by designing the marker gene such that the marker gene functions when the exon is skipped, the exon skipping can be efficiently analyzed based on the phenotype of the marker gene.

The method of the present invention as defined in the claims is a method of skipping a target exon of a gene of interest in a genome, comprising using CRISPR-Cas and guide RNA, wherein the guide RNA contains a spacer sequence such that the site of cleavage by the CRISPR-Cas is positioned within <NUM> bases from the splice acceptor site immediately before the target exon or the splice donor site immediately after the target exon.

As CRISPR systems, class <NUM>, which acts as a complex formed by a plurality of factors, and class <NUM>, which acts even as a single factor, are known. Examples of class <NUM> include type I, type III, and type IV, and examples of class <NUM> include type II, type V, and type VI (<NPL>| <NPL>). At present, for use in genome editing in mammalian cells, class <NUM> CRISPR-Cas, which acts as a single factor, is mainly used. Representative examples of the class <NUM> CRISPR-Cas include type II Cas9 and type V Cpfl.

As a CRISPR-Cas9 system, class <NUM> type II Cas9 derived from Streptococcus pyogenes, which is widely used as a genome editing tool, may be used. Class <NUM> type II Cas9 systems derived from other bacteria have also been reported, and, for example, Cas9 derived from Staphylococcus aureus (Sa), Cas9 derived from Neisseria meningitidis (Nm), or Cas9 derived from Streptococcus thermophilus (St) may also be used.

Further, as a class <NUM> type V CRISPR-Cas system, Cpfl has been identified, and it is reported that, by using Cpfl derived from Acidaminococcus sp. (As) or Cpfl derived from Lachnospiraceae, genome editing in human cells is possible dependently on the gRNA sequence. Thus, these kinds of CRISPR-Cpfl may also be used.

CRISPR-Cas9 has two nuclease domains, the RuvC domain and the HNH domain, each of which is involved in cleavage of one strand of the double-stranded DNA. CRISPR-Cpfl has the RuvC domain and the Nuc domain. In cases where Asp at the 10th position in the RuvC domain of Cas9 derived from Streptococcus pyogenes is substituted with Ala (D10A), no cleavage occurs in the DNA strand to which the gRNA does not bind. In cases where His at the 840th position in the HNH domain is substituted with Ala (H840A), no cleavage occurs in the DNA strand to which the gRNA binds [<NPL>. This property was used for development of the double nicking (or paired nickases) method, in which nickases each of which cleaves only one strand of a double-stranded DNA are used, in a state where they are positioned close to each other, to cleave the respective separate DNA strands, to induce a DNA double-strand break in a target region [<NPL>. By this, genome editing such as targeting by insertion of an arbitrary sequence by knock-in became possible while the risk of inducing a sequence mutation at a site other than the target site was reduced [<CIT>].

Thus, in the method of the present invention as defined in the claims, D10A Cas9 nickase may be used as CRISPR-Cas9, and two kinds of guide RNAs for cleavage of the sense strand and the antisense strand, respectively, may be used (double nicking method). A nickase-type Cas9 or a nickase-type Cpfl applicable to the double nicking method can be obtained also by introducing a mutation to an active amino acid residue of the RuvC domain of a system other than the Cas9 derived from Streptococcus pyogenes.

A DNA encoding the above CRISPR-Cas can be obtained by performing cloning based on a sequence encoding CRISPR-Cas deposited in GenBank or the like. Further, a commercially available plasmid containing CRISPR-Cas may be obtained from Addgene or the like and used; a DNA encoding CRISPR-Cas may be obtained by PCR using the plasmid as a template, or the DNA may be artificially prepared using an artificial gene synthesis technique known to those skilled in the art.

A DNA encoding Cas nickase may be obtained by introducing a mutation to an active amino acid residue of a nuclease domain of CRISPR-Cas by a known molecular biological technique, or may be obtained by cloning from a plasmid or the like containing a CRISPR-Cas gene to which a mutation was introduced in advance. Further, in order to increase the expression efficiency of the CRISPR-Cas in the host, codon alteration may be carried out.

The CRISPR-Cas may be introduced into the cell as mRNA, protein, or DNA. The guide RNA may be introduced into the cell as RNA or DNA. In cases of introduction using a vector, examples of the vector include vectors capable of replicating in eukaryotic cells, vectors capable of maintaining an episome, and vectors that can be incorporated into the host cell genome. Examples of virus vectors therefor include adenovirus vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors, and adeno-associated virus vectors. Examples of transposon vectors therefor include piggyBac vectors, piggyBat vectors, Sleeping Beauty vectors, TolII vectors, and LINE vectors. For use in treatment, introduction using a vector showing constant expression is not preferred because of an increased risk of side effects. Since use in treatment requires induction of DNA cleavage only immediately after the administration, introduction as Cas9 mRNA/gRNA or Cas9 protein/gRNA, introduction as an episomal vector, or the like is preferred.

The vector may contain a selection marker. The "selection marker" means a genetic element that provides a selectable phenotype to the cell into which the selection marker is introduced. The selection marker is generally a gene whose gene product gives resistance to an agent which inhibits growth of cells or which kills cells. Specific examples of the selection marker include the Puro resistance gene, Neo resistance gene, Hyg resistance gene, Bls gene, hisD gene, Gpt gene, and Ble gene. Examples of drugs useful for selecting the presence of selection markers include puromycin for the Puro resistance gene, G418 for the Neo resistance gene, hygromycin for the Hyg resistance gene, blasticidin for the Bls gene, histidinol for hisD, xanthine for Gpt, and bleomycin for Ble.

A guide RNA (gRNA or sgRNA) is a complex containing tracrRNA and crRNA, or tracrRNA and crRNA artificially linked to each other, in the CRISPR-Cas9 method. ] In the present invention as defined in the claims, the guide RNA means a product prepared by linking a spacer sequence having a sequence corresponding to the gene of interest to a scaffold sequence.

As the scaffold sequence of the guide RNA of SpCas9, a known sequence, for example, the sequence of
<IMG>
can be used. Alternatively, an altered scaffold sequence (<NPL>)
<IMG>
may be used.

The guide RNA does not require tracrRNA in CRISPR-Cpf1.

As the scaffold sequence of the guide RNA of AsCpfl, a known sequence, for example, the sequence of <NUM>'-GTAATTTCTACTCTTGTAGAT-<NUM>' (SEQ ID NO:<NUM>) or <NUM>'-GGGTAATTTCTACTCTTGTAGAT-<NUM>' (SEQ ID NO:<NUM>) can be used. The DNA may be, for example, artificially prepared using an artificial gene synthesis technique known to those skilled in the art.

In the method of the present invention, the spacer sequence of the guide RNA is arranged such that the site of cleavage by the CRISPR-Cas is positioned within <NUM> bases, preferably within <NUM> bases, more preferably within <NUM> bases, from the splice acceptor site immediately before the target exon or the splice donor site immediately after the target exon. It may also be designed for the exonic splicing enhancer (ESE) sequence portion.

With such arrangement, cleavage occurs at a position near the splice acceptor site or the donor site of the target exon, and the splice acceptor site or the donor site is disrupted in the repair process, resulting occurrence of skipping of the target exon when the splicing reaction occurs in the process of maturation of pre-mRNA into mRNA.

The splice acceptor site is defined as the two bases immediately before the target exon, and its examples include the AG sequence.

The splice donor site is defined as the two bases immediately after the target exon, and its examples include the GT sequence.

The exonic splicing enhancer (ESE) sequence is defined as a binding site of SR protein (SRSF1 to <NUM> genes) present in the target exon. The binding site of SR protein can be obtained by searching databases, and examples of such databases include RESCUE-ESE [<NPL>] and ESEfinder [<NPL>].

The spacer sequence of Type II Cas9 can be designed as RNA having a continuous base sequence of <NUM> to <NUM> bases whose <NUM>'-end corresponding to the base immediately before the PAM sequence (for example, NGG in the case of S. pyogenes Cas9, or NNGRRT in the case of Staphylococcus aureus Cas9) in the sequence of the sense strand or the antisense strand of the gene of interest (for example, NNNNNNNNNNNNNNNNNNNNNGG (SEQ ID NO:<NUM>)). (The N's represent the spacer sequence.

However, since the cleavage occurs even without <NUM>% matching of the sequence, a mismatch(es) of one or two bases is/are acceptable (especially in the <NUM>'-side). For a transcription start site from the human H1 PolIII promoter, the <NUM>'-end of the spacer sequence is preferably not C or T. In cases where the corresponding base in the genome is C or T, it is preferably converted to G.

The spacer sequence of the guide RNA of type V Cfp1 can be designed as RNA having a continuous base sequence of <NUM> to <NUM> bases whose <NUM>'-end corresponds to the base immediately after the PAM sequence (for example, TTTV in the case of Acidaminococcus sp. Cpfl) (for example, TTTTVNNNNNNNNNNNNNNNNNNNN (SEQ ID NO:<NUM>)). In the case of Cpf1, tracrRNA is not required.

Regarding the site of DNA cleavage by S. pyogenes Cas9, the cleavage occurs between the third base and the fourth base as counted in the <NUM>'→<NUM>' direction from the <NUM>'-end base of the spacer sequence, which is regarded as <NUM>. Accordingly, "the cleavage site is positioned within <NUM> bases from the acceptor site or donor site of the target exon" means that the number of bases present between the bases at the acceptor site or the donor site (for example, GT or AG (in the case of the antisense strand, AC or CT)) and the fourth base as counted in the <NUM>'→<NUM>' direction from the base corresponding to the <NUM>'-end base of the spacer sequence is not more than <NUM> bases.

Regarding the site of DNA cleavage by AsCpfl, the cleavage occurs at the 19th sense-strand base and the 23rd antisense-strand base as counted in the <NUM>'→<NUM>' direction from the <NUM>'-end base of the spacer sequence, which is regarded as <NUM>.

A description is given with reference to <FIG>.

Using exon <NUM> of the human dystrophin (hDMD) gene as a target, the acceptor site immediately before the exon <NUM> is disrupted to carry out skipping of the exon <NUM>.

In this process, in Sp-sgRNA-DMD1, a spacer sequence corresponding to the <NUM> bases immediately before the PAM sequence (AGG) (tggtatcttacagGAAC/TCC) (SEQ ID NO:<NUM>) is designed. In this case, there are four bases between the acceptor sequence (ag) and the cleavage site (C/T).

Similarly, in Sp-sgRNA-DMD2, a spacer sequence corresponding to the <NUM> bases immediately before the PAM sequence (TGG) (atcttacagGAACTCCA/GGA) (SEQ ID NO: <NUM>) is designed. In this case, there are eight bases between the acceptor sequence (ag) and the cleavage site (A/G).

Similarly, in Sp-sgRNA-DMD3, a spacer sequence corresponding to the <NUM> bases immediately before the PAM sequence (TGG) (cagGAACTCCAGGATGG/CAT) (SEQ ID NO: <NUM>) is designed. In this case, there are <NUM> bases between the acceptor sequence (ag) and the cleavage site (G/C).

Similarly, in Sp-sgRNA-DMD4, a spacer sequence corresponding to the <NUM> bases immediately before the PAM sequence (CGG) (TCCAGGATGGCATTGGG/CAG) (SEQ ID NO: <NUM>) is designed. In this case, there are <NUM> bases between the acceptor sequence (ag) and the cleavage site (G/C).

Sp-sgRNA-DMDS is arranged for the antisense strand, and a spacer sequence corresponding to the <NUM> bases immediately before the PAM sequence (AGG) (GTTCctgtaagatacca/aaa) (SEQ ID NO: <NUM>) is designed.

In this case, there are <NUM> bases between the acceptor sequence (ct) and the cleavage site (a/a).

In each sequence ID number, T is read as U when an RNA sequence is meant.

By adding a scaffold sequence to the above spacer sequence, a guide RNA can be obtained. Plasmids which contains a scaffold sequence therein, and with which a desired guide RNA can be expressed by inserting a DNA sequence corresponding to an arbitrary spacer sequence, are commercially available (for example, Addgene plasmid <NUM>), and they can be simply used for introduction of a guide RNA into cells.

Two or more kinds of guide RNAs are used. In such a case, two or more kinds of guide RNAs satisfying the condition "the site of cleavage by the CRISPR-Cas is positioned within <NUM> bases from the splice acceptor site immediately before the target exon or the splice donor site immediately after the target exon" may be used for one site to be disrupted (target exon). By using two or more different kinds of guide RNAs for the same site to be disrupted, and causing DNA double-strand breaks simultaneously at two or more sites, exon skipping can be caused with a very high efficiency. The two or more kinds of guide RNAs may be arranged either for one of the sense strand and the antisense strand, or for both of these.

In cases where D10A Cas9 nickase is used, and two kinds of guide RNAs for cleaving the sense strand and the antisense strand, respectively, are used, the two kinds of guide RNAs are designed such that they satisfy the requirement that the cleavage site of at least one of them is positioned within <NUM> bases from the acceptor site or the donor site before or after the target exon. In this case, the distance between the guide RNA-binding site (spacer sequence) in the sense strand and the guide RNA-binding site (spacer sequence) in the antisense strand is preferably -<NUM> to <NUM> bases, more preferably <NUM> to <NUM> bases. The acceptor site or the donor site is preferably located between the cleavage site in the sense strand and the cleavage site in the antisense strand. The spacer sequence of the guide RNA for cleavage of the sense strand and the spacer sequence of the guide RNA for cleavage of the antisense strand may overlap with each other, but they preferably do not overlap with each other.

The gene of interest is human dystrophin gene Dystrophin gene is a causative gene of Duchenne muscular dystrophy. Since it is known that the phenotype of the mutant gene can be masked by skipping of exon <NUM>, exon <NUM> of the human dystrophin gene is suitably used as a target of exon skipping.

Specific examples of the spacer sequence contained in the guide RNA that can be used in the method of the present invention as defined in the claims include the base sequence of bases from <NUM> to <NUM> in the base sequence of any of SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. Their complementary sequences may also be used. When two kinds of guide RNAs are used in combination, the combination of the spacer sequences include the combinations of sgRNA-DMD1 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO: <NUM>), sgRNA-DMD2 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO: <NUM>), sgRNA-DMD4 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>), sgRNA-DMD8 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>), or sgRNA-DMD9 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>) with sgRNA-DMD23 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO: <NUM><NUM>).

Further, among these, preferred examples of the combination of the spacer sequences for use in the double nicking method include sgRNA-DMD4 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>).

Transfection of cells with DNA, RNA, or vectors expressing these can be carried out by using known arbitrary means, and commercially available transfection reagents may be used. For example, Lipofectamine <NUM> (Thermo Fisher), StemFect (STEMGEN), FuGENE <NUM>/HD (Promega), jetPRIME Kit (Polyplus-transfection), DreamFect (OZ Biosciences), GenePorter <NUM> (OZ Biosciences), or Calcium Phosphate Transfection Kit (OZ Biosciences) can be used. Electroporation may be also used. For example, NEPA21 (Nepa Gene), 4D-Nucleofector (Lonza), Neon (Thermo Fisher), Gene Pulser Xcell (BioRad), or ECM839 (BTX Harvard Apparatus) can be used. Regarding the transfection of cells, a complex may be formed with CRISPR-Cas protein and gRNA in advance, and the cells may then be transfected with the complex. Further, microinjection or electroporation may be carried out for introduction of the DNA or RNA into fertilized eggs.

The cells are preferably a mammalian cells, more preferably human cells. The cells may be primary cultured cells isolated from an established cell line or from a mammalian tissue, or may be mesenchymal cells or pluripotent stem cells such as induced pluripotent stem (iPS) cells. For example, in cases where the dystrophin gene is to be targeted, skeletal muscle cells, mesenchymal cells, or iPS cells derived from a DMD patient may be established, and then guide RNA and CRISPR-Cas, or a guide RNA pair and Cas nickase, for induction of exon skipping of the present invention as defined in the claims may be simultaneously introduced into the cells, to induce exon skipping of the dystrophin gene, thereby enabling recovery of the dystrophin protein. By transplanting such repaired cells or an induced product therefrom to a patient, atrophic muscle cells can be complemented.

Further, in another mode, guide RNA and CRISPR-Cas, or a guide RNA pair and Cas nickase, for induction of exon skipping of the present invention as define in the claims are simultaneously introduced into a muscular tissue of a DMD patient, to induce exon skipping of the dystrophin gene, thereby enabling recovery of the dystrophin protein in the body of the patient.

The present invention as defined in the claims is described below concretely by way of Examples.

Using a Perl script, base sequences of <NUM> to <NUM>-mer (k-mer) with all combinations were generated. Using the Bowtie program (Langmead et al. , <NUM>), the k-mer sequences generated were mapped on human genome hg19 without accepting a mismatch. Subsequently, k-mer sequences mapped only once on human genome hg19 were extracted, and a unique k-mer database was constructed (Li HL et al. , Stem Cell Reports, <NUM>). Using the ngs. r program (Shen L et al. , BMC Genomics, <NUM>), which runs with the R language, the distribution of unique k-mers in <NUM> bp before and after all human exons were investigated and plotted.

DNA synthesis (GenScript) was carried out to prepare the pUC57-SphcCas9 vector, which has a Cas9 cDNA derived from Streptococcus pyogenes optimized for the human codon frequencies inserted therein, the Cas9 cDNA having an SV40 large T antigen-derived nuclear localization signal peptide (PKKKRKV) (SEQ ID NO:<NUM>) at the C-terminus. This was cleaved with SalI-Xbal restriction enzymes, and then ligated to the SalI-Xbal site of pENTR2B (A10463, Thermo Fisher) to construct the pENTR-SphcCas9 vector. Subsequently, the SphcCas9 cDNA portion of the pENTR-SphcCas9 vector was inserted into the pHL-EF1α-GW-iC-A vector, the pHL-EF1α-GW-iP-A vector, and the pHL-EF1α-GW-A vector by the Gateway LR clonase reaction, to construct the pHL-EF1α-SphcCas9-iC-A vector (SpCas9-IRES-mCheery-polyA), the pHL-EF 1α-SphcCas9-iP-A vector (SpCas9-IRES-PuroR-polyA) (Addgene, <NUM>), and the pHL-EF1α-SphcCas9-A vector (SpCas9-polyA). The EF1α promoter is more suitable than virus-derived promoters (the CMV promoter and the like) since a higher expression level can be obtained in pluripotent stem cells.

Further, for preparation of the D10A mutant of SpCas9 (nickase), the DNA sequence SphcCas9-D10A (NcoI-Sbfl), in which the GaC codon (Asp, D) was converted to the GcC codon (Ala, A), was synthesized by gBlock (IDT).

The SphcCas9-D10A(NcoI-Sbfl) sequence was cleaved with the NcoI-Sbfl restriction enzymes, and then inserted into the NcoI-SbfI site of the pENTR-SphcCas9 vector using the In-Fusion reaction, to construct the pENTR-SphcCas9-D10A vector. Subsequently, the SphcCas9-D10A cDNA portion of the pENTR-SphcCas9-D10A was inserted into the pHL-EF1α-GW-iC-A vector and the pHL-EF1α-GW-iP-A vector by the Gateway LR clonase reaction, to construct the pHL-EF1α-SphcCas9-D10A-iC-A vector (SpCas9-IRES-mCheery-polyA) and the pHL-EF1α-SphcCas9-D10A-iP-A vector (SpCas9-IRES-PuroR-polyA).

For cloning of gRNA of SpCas9 into an expression vector, <NUM> pmol each of the following arbitrary Sp-sgRNA-XXX-fwd primer (one of SEQ ID NOs: <NUM> to <NUM>) and the Sp-sgRNA-Universal-rev primer were mixed together, and thermal cycling reaction was performed using KOD Plus Neo DNA polymerase (Toyobo) (heat denaturation at <NUM>: <NUM> followed by {<NUM>: <NUM> sec, <NUM>: <NUM> sec, <NUM>: <NUM> sec} × <NUM> cycles, further followed by incubation at <NUM>). The resulting PCR product was subjected to electrophoresis in <NUM>% agarose gel, and a DNA band with a size of about <NUM> bp was excised and purified. The purified PCR product was inserted, using the In-Fusion reaction (Takara-Clontech), into the pHL-H1-ccdB-mEF1a-RiH vector (Addgene <NUM>) cleaved with BamHI-EcoRI, to construct a pHL-H1-[SpCas9-gRNA]-mEF1a-RiH vector which expresses an arbitrary gRNA.

DNA synthesis (GenScript) was carried out to prepare the pUC-Kan-SahcCas9 vector, which has a Cas9 cDNA derived from Staphylococcus aureus optimized for the human codon frequencies inserted between the Gateway attL1 site and attL2 site, the Cas9 cDNA having an SV40 large T NLS at the N-terminus, and a nucleoplasmin NLS and a <NUM>×HA tag at the C-terminus. Subsequently, the SaCas9 cDNA portion of the pUC-Kan-SahcCas9 vector was inserted into the pHL-EF1α-GW-A and pHL-EF1α-GW-iP-A vectors by the Gateway LR clonase reaction, to construct the HL-EF1α-SaCas9-A and HL-EF1α-SaCas9-iC-A vectors, respectively.

For cloning of gRNAs of SaCas9 into an expression vector, <NUM> pmol each of the following arbitrary sgRNA-DMD-SA-X-fwd primer (one of SEQ ID NOs:<NUM> to <NUM>) and the SA1-gRNA-Universal-Rev primer were mixed together, and thermal cycling reaction was performed using KOD Plus Neo DNA polymerase (Toyobo) (heat denaturation at <NUM>: <NUM> followed by {<NUM>: <NUM> sec, <NUM>: <NUM> sec, <NUM>: <NUM> sec} × <NUM> cycles, further followed by incubation at <NUM>). The resulting PCR product was subjected to electrophoresis in <NUM>% agarose gel, and a DNA band with a size of about <NUM> bp was excised and purified. The purified PCR product was inserted, using the In-Fusion reaction (Takara-Clontech), into the pHL-H1-ccdB-mEF1a-RiH vector (Addgene <NUM>) cleaved with BamHI-EcoRI, to construct a pHL-H1-[SaCas9-gRNA]-mEF1a-RiH vector which expresses the arbitrary gRNA.

DNA synthesis (GenScript) was carried out to prepare the pUC57-hcAsCpf1 vector, which has a Cpfl cDNA derived from Acidaminococcus sp. BV3L6 optimized for the human codon frequencies is inserted between the Gateway attL1 site and attL2 site, the Cpfl cDNA having a nucleoplasmin NLS (KRPAATKKAGQAKKKK) (SEQ ID NO:<NUM>) and a <NUM>×HA tag (YPYDVPDYA YPYDVPDYAYPYDVPDYA) (SEQ ID NO:<NUM>) sequence at the C-terminus. Subsequently, the hcAsCpf1 cDNA portion of the pENTR-hcAsCpf1 vector was inserted into the pHL-EF1α-GW-A and pHL-EF1α-GW-iP-A vectors by the Gateway LR clonase reaction, to construct the HL-EF1α-hcAsCpf1-A and HL-EF1α-hcAsCpf1-iC-A vectors, respectively.

For cloning of gRNAs of AsCpf1 into an expression vector, <NUM> pmol each of the following arbitrary AsCpf1-gRNA-XXX-rev primer (one of SEQ ID NOs:<NUM> to <NUM>) and the AsCpf <NUM>-gRNA-Universal-GGG-fwd primer (or the AsCpf1-gRNA-Universal-G-fwd primer) were mixed together, and thermal cycling reaction was performed using KOD Plus Neo DNA polymerase (Toyobo) (heat denaturation at <NUM>: <NUM> followed by {<NUM>: <NUM> sec, <NUM>: <NUM> sec, <NUM>: <NUM> sec} × <NUM> cycles, further followed by incubation at <NUM>). The resulting PCR product was subjected to electrophoresis in <NUM>% agarose gel, and a DNA band with a size of about <NUM> bp was excised and purified. The purified PCR product was inserted, using the In-Fusion reaction (Takara-Clontech), into the pHL-H1-ccdB-mEF1a-RiH vector cleaved with BamHI-EcoRI, to construct a pHL-H1-[AsCpf1-gRNA]-mEF1a-RiH vector which expresses the AsCpfl gRNA.

The SSA-DMD-all-ss oligo DNA and the SSA-DMD-all-as oligo DNA, which have the target sequences of Sp-gRNA-DMD1 to <NUM>, were annealed with each other, and then ligated into the BsaI site present in firefly Luc2 cDNA of the pGL4-SSA vector (Addgene <NUM>, Ochiai et al. , Genes Cells, <NUM>), to construct the pGL4-SSA-DMD-all vector. In the pGL4-SSA-DMD-all vector, the firefly Luc2 cDNA is divided, and does not show Luc activity. However, when cleavage of the target DNA portion in the pGL4-SSA vector is induced by guide RNA, the DNA cleavage is repaired by the SSA (single strand annealing) pathway, resulting recovery of the firefly Luc2 cDNA.

A mixture of <NUM> ng of the pGL4-SSA-DMD-All vector, <NUM> ng of the phRL-TK vector, which expresses Renilla Luc, <NUM> ng of the pHL-EF1a vector, which expresses CRISPR-Cas, and <NUM> ng of the pHL-H1-sgRNA-mEF1a-RiH vector, which expresses sgRNA, was prepared, and then diluted with <NUM>µl of Opti-MEM. With <NUM>µl of Opti-MEM, <NUM>µl of Lipofectamine <NUM> was diluted, and the resulting dilution was incubated at room temperature for <NUM> to <NUM> minutes, followed by mixing the dilution with the above DNA solution, and then incubating the resulting mixture at room temperature for additional <NUM> minutes. The cell number of 293T cells suspended by trypsin-EDTA treatment was counted, and then the cells were diluted to <NUM>,<NUM> cells/<NUM>µl with a medium, followed by plating the cells on a <NUM>-well plate containing the above DNA-Lipofectamine complex at <NUM>µl/well. After culturing the cells for <NUM> hours with <NUM>% CO<NUM> at <NUM>, the <NUM>-well plate was allowed to cool to room temperature, and then Dual-Glo Reagent was added thereto, followed by incubation at room temperature for <NUM> minutes to lyse the cells to cause the luciferase reaction. To a white <NUM>-well plate, <NUM>µl of the supernatant was transferred, and the luminescence intensities of Firefly and Renilla were measured using Centro LB960 (Berthold Technologies). Since firefly Luc emits light only when DNA cleavage is induced by CRISPR, the luminescence value of firefly was normalized against the luminescence value of Renilla to measure the DNA cleavage efficiency.

293T cells were cultured inDMEM medium supplemented with <NUM> to <NUM>% FBS.

DMD-iPS cells (clone ID: CiRA00111) were cultured, on SNL feeder cells whose growth was inhibited by mitomycin C treatment, using Knockout SR medium {prepared by adding <NUM> of Knockout SR (Thermo Fisher, <NUM>), <NUM> of L-glutamine (Thermo Fisher, <NUM>), <NUM> of non-essential amino acid mixture (Thermo Fisher, <NUM>), <NUM> of <NUM>-mercaptoethanol (Thermo Fisher, <NUM>), <NUM> of penicillin-streptomycin (Thermo Fisher, <NUM>), and <NUM> ng/ml human basic FGF (Wako, <NUM>) to <NUM> of DMEM/F12 medium (Thermo Fisher, <NUM>)}. Alternatively, using StemFit AK03N (Ajinomoto) medium, the cells may be cultured on iMatrix-<NUM> (Nippi, <NUM>) without the use of feeder cells.

For iPS cells established from a DMD patient who lacks exon <NUM>, <NUM> Y-<NUM> (Sigma) was added to the medium not less than one hour before transfection. Immediately before electroporation, the iPS cells were detached with CTK solution, and then dispersed using <NUM>% trypsin-EDTA, followed by counting the cells for providing <NUM> × <NUM><NUM> cells for each condition. To these cells, electroporation of <NUM>µg of the pHL-EF1α-SphcCas9-iP-A vector (Addgene, <NUM>) and <NUM>µg of the pHL-H1-[Sp-gRNA]-mEF1α-RiH vector was carried out using a NEPA21 electroporator (Nepa Gene) with a poration pulse voltage of <NUM> V, a pulse width of <NUM> milliseconds, and a number of pulses of <NUM>. In the cases where double-nicking was carried out, electroporation was carried out with <NUM>µg of the pHL-EF1α-SphcCas9-D10A-iP-A vector and a total of <NUM>µg, that is, <NUM>µg each, of two kinds of pHL-H1-[Sp-gRNA]-mEF1α-RiH vectors. The iPS cells subjected to the electroporation were cultured for not less than several days, and genome DNA was extracted therefrom, followed by performing primary PCR amplification using the DMD-MiSeq-Rd1-fwd-X and DMD-MiSeq-Rd2-rev-X primers, and then performing secondary PCR amplification using the Multiplex P5 fwd primer and the Multiplex P7 rev primer. The resulting PCR product was excised from the gel, and then purified, followed by quantification using a Qubit <NUM> Fluorometer (Thermo Fisher) and a KAPA Library Quantification Kit for Illumina (KAPA Biosystems). The samples were mixed to the same amount, and the DNA concentration was adjusted to <NUM>, followed by performing alkali denaturation of the DNA by treatment with <NUM> N NaOH for <NUM> minutes. Each denatured DNA sample was diluted to <NUM> pM, and then <NUM> pM PhiX spike-in DNA was added thereto, followed by performing MiSeq sequencing reaction using a MiSeq Reagent Kit v2 for <NUM> × <NUM> bp (Illumina). From the FASTQ sequence file generated as a result of the sequencing, low-quality reads were removed by using the fastq_quality_filter program in the FASTX-Toolkit. After removing the PhiX sequence used as the spike-in, the fastx_barcode_splitter program was used to divide the samples according to the barcode sequences. The sequences of the samples were mapped using the BWA program, and the insertion/deletion patterns of the sequences were extracted from the MD tag information in the CIGAR code.

Specific sequences containing the barcode for the X.

Luc2 cDNA was amplified by PCR from pGL4-CMV-luc2 (Promega), and then cloned into the pENTR-D-TOPO vector (Thermo Fisher Scientific Inc. ), to construct the pENTR-D-TOPO-Luc2 vector. The pENTR-D-TOPO-Luc2 was cleaved with NarI and AgeI, and then the following intron sequence synthesized by gBlock (IDT) and the DMD exon <NUM> sequence were inserted thereto, to construct the pENTR-D-TOPO-Luc2-DMD-intron-Ex45[+] vector. Subsequently, the gBlock sequence was cleaved at the two SalI sites present in both sides of hEx45, and then the vector side was re-ligated to construct the pENTR-D-TOPO-Luc2-DMD-intron-Ex45[-] vector.

NarI-AgeI-DMD-Ex45-gBlock (Sequence of <FIG>)
<IMG>
<IMG>.

The piggyBac <NUM>'TR (Terminal repeat) and <NUM>'TR sequences derived from Trichoplusia ni were synthesized (IDT) as three separate gBlocks sequences (gBlock11 to <NUM>-PV-<NUM>'TR-<NUM>'TR), and the three fragments were linked to each other by PCR, followed by insertion into the AatII-PvuII site in the pUC <NUM> vector by the In-Fusion reaction, to construct the pPV-synthesized vector.

The PB-EF1a-GW-iP vector (Masui H et al. , PLOS ONE, <NUM> Aug <NUM>; <NUM>(<NUM>): e104957. ) was cleaved with NheI-PacI, and then ligated to the NheI-PacI site of pPV-synthesized, to construct the pPV-EF1a-GW-iP vector. Subsequently, a rabbit-derived hemoglobin poly A signal was amplified from the pCXLE-EGFP vector (<NPL>. ) using the pHL-PacI-rHBB-pA-IF-fw primer (<NUM>'-GTATACCTCGAGTTAAATTCACTCCTCAGGTGC-<NUM>' (SEQ ID NO:<NUM>)) and the pPV-PacI-rHBB-pA-IF-rev primer (<NUM>'-CGAGCTTGTTGGTTAATTAAGTCGAGGGATCTCCATAA-<NUM>' (SEQ ID NO:<NUM>)), and then inserted into the PacI site of the pPV-EF1a-GW-iP vector using the In-Fusion reaction, to construct the pPV-EF1α-GW-iP-A vector. By the Gateway LR reaction using the pPV-EF1α-GW-iP-A vector and pENTR-D-TOPO-Luc2-DMD-intron-Ex45[+], the pPV-EF1a-Luc2-hDMD-Ex45[+]-iP-A vector was constructed. Further, by the Gateway LR reaction using the pPV-EF1α-GW-iP-A vector and pENTR-D-TOPO-Luc2-DMD-intron-Ex45[-], the pPV-EF1a-Luc2-hDMD-Ex45-[-]-iP-A vector was constructed.

Using the piggyBac vector pPV-EF1a-Luc2-hDMD-Ex45[-] vector as a template, PCR was carried out separately using, as primers, the combination of Luc2-NcoI-IF-Fwd and Luc2-V323I-fwd, and the combination of Luc2-V323I-rev and Luc2-SalI-IF-Rev. The two amplified fragments were mixed together, and the primers at both ends (Luc2-NcoI-IF-Fwd and Luc2-SalI-IF-Rev) were used to prepare a fragment having a mutation, followed by inserting the fragment into the NcoI-SalI cleavage site of the pPV-EF1a-Luc2-hDMD-Ex45[-] vector by the In-Fusion reaction, to construct the pPV-EF1a-Luc2(V323I)-hDMD-Ex45[-] vector.

PCR amplification was carried out using pPV-EF1a-Luc2-hDMD-Ex45[+]-iP-A as a template, and using the DMD-Ex45-SalI-IF-F and DMD-Ex45-SalI-IF-R primers. The resulting product was inserted into the SalI cleavage site of the pPV-EF1a-Luc2(V323I)-hDMD-Ex45[-] vector by the In-Fusion reaction, to construct the pPV-EF1a-Luc2(V323I)-hDMD-Ex45[+] (<NUM> kb) vector.

Using, as a template, human genomic DNA prepared from 1383D2 cells, and using the following primers (HDMD-SR-XkbFrag-fwd & rev), exon <NUM> and the introns in the vicinity thereof were amplified. The resulting product was inserted into the SalI cleavage site of the pPV-EF1a-Luc2(V323I)-hDMD-Ex45[-] vector by the In-Fusion reaction, to construct the pPV-EF1a-Luc2(V323I)-hDMD-Ex45[+] (<NUM> kb), pPV-EF1a-Luc2(V323I)-hDMD-Ex45[+] (<NUM> kb), and pPV-EF1a-Luc2(V323I)-hDMD-Ex45[+] (<NUM> kb) vectors.

The sequences of the exon skipping reporter cDNA portion constructed are shown below.

A mixture of <NUM> ng of the pPV-EF1a-Luc2(V323I)hDMD-Ex45[+] vector, <NUM> ng of the phRL-TK vector, which expresses Renilla Luc, <NUM> ng of the pHL-EF1a vector (Addgene), which expresses CRISPR-Cas, and <NUM> ng of the pHL-H1-sgRNA-mEF1a-RiH vector, which expresses a guide RNA, was prepared, and then diluted with <NUM>µl of Opti-MEM. With <NUM>µl of Opti-MEM, <NUM>µl of Lipofectamine <NUM> was diluted, and the resulting dilution was incubated at room temperature for <NUM> to <NUM> minutes, followed by mixing the dilution with the above DNA solution, and then incubating the resulting mixture at room temperature for additional <NUM> minutes. The cell number of 293T cells suspended by trypsin-EDTA treatment was counted, and then the cells were diluted to <NUM>,<NUM> cells/<NUM>µl with a medium, followed by plating the cells on a <NUM>-well plate containing the above DNA-Lipofectamine complex at <NUM>µl/well. After culturing the cells for <NUM> hours with <NUM>% CO<NUM> at <NUM>, the <NUM>-well plate was allowed to cool to room temperature, and then Dual-Glo Reagent was added thereto, followed by incubation at room temperature for <NUM> minutes to lyse the cells to cause the luciferase reaction. To a white <NUM>-well plate, <NUM>µl of the supernatant was transferred, and the luminescence intensities of Firefly and Renilla were measured using Centro LB960 (Berthold Technologies). Since firefly Luc emits light only when exon skipping is induced, the luminescence value of firefly was normalized against the luminescence value of Renilla to measure the exon skipping efficiency.

In order to develop a therapeutic method for Duchenne muscular dystrophy, induction of exon skipping in the dystrophin gene was studied. <FIG> shows its overview.

The splice acceptor is constituted by a branching sequence, polypyrimidine (C/U) sequence, and an "AG" acceptor sequence. Since, among these, the "AG" acceptor sequence is most highly conserved during the splicing reaction, deletion of these two bases may enable induction of exon skipping.

In designing of gRNAs for CRISPR systems, from the viewpoint of the off-target risk, that is, recognition and cleavage of sequences other than the target sequence in the genome, the unique k-mer method [<NPL>] was used to investigate the sequence specificity in the region around the splice acceptor. As a result, it became clear that the region near the splice acceptor has an especially low specificity compared to the exon regions and other intron regions (<FIG>).

In order to simply and highly sensitively detect the exon skipping efficiency in the dystrophin gene, a reporter vector was constructed using the firefly luciferase (Luc) gene. A vector in which Luc cDNA was divided into two parts, and a synthetic intron sequence was inserted thereto (Luc + Int), and a vector in which a sequence around human dystrophin exon <NUM> was further inserted thereto (Luc + hEx45), were prepared (<FIG>). Each vector was introduced into 293T cells, and mRNA was recovered, followed by performing reverse transcription and then PCR amplification. As a result, it was found that the first half of the Luc cDNA contains a pseudo-splicing donor sequence, causing extra splicing (<FIG>, Panel (a)). In order to disrupt this pseudo-splicing donor sequence, splicing donor sequences and acceptor sequences were extracted from all exon sequences contained in the human dystrophin gene from the Ensemble Biomart database (http://www. org), and common bases were analyzed with Weblogo software (http://weblogo. threeplusone. As a result, they were found to be matching well with known splicing donor and acceptor sequences (<FIG>, Panel (b)). Thus, it was expected that, by converting the "G" at the center of the pseudo-splicing donor sequence "AGGTA" to a "sequence other than G", the pseudo-splicing donor sequence can be prevented from functioning as a splicing donor. However, since this base is the first base of the codon "GTA", which encodes a Val amino acid, alteration of this base inevitably changes the amino acid sequence. Alteration to "A" results in generation of the codon "ATA", which encodes an Ile amino acid (<FIG>, Panel (c)), and alteration to "C" or "T" results in generation of a codon "CTA" or "TTA", which encodes a Leu Amino acid. In order to confirm that this amino acid conversion does not affect the Luc activity, the present inventors downloaded the spatial structure of luciferase protein (PDB code: 1BA3) from the PDB database. By using Chimera software, it could be confirmed that the G967A(V323I) mutant amino acid site is distant from the active residue [<NPL>. ], and hence that there is no direct interaction (<FIG>).

Subsequently, in order to investigate whether the expected splicing pattern actually occurs or not, the vectors shown in <FIG>, Panel (a) were introduced into 293T cells, and mRNA was extracted therefrom, followed by performing reverse transcription and then PCR amplification. As a result of investigation of the cDNA size by gel electrophoresis, the vectors into which the G967A(V323I) mutation was not introduced (lanes <NUM> and <NUM>) showed a band corresponding to the non-spliced transcript with high intensity. On the other hand, it could be confirmed that splicing with the expected size occurred in almost the entire transcripts from the vectors into which the G967A(V323I) mutation was introduced (lanes <NUM> and <NUM>) (<FIG>, Panel (b)). Further, according to the result of investigation of the luciferase activity of each vector, the insertion of the intron sequence or the introduction of the G967A(V323I) mutation hardly caused changes. On the other hand, in the cases where the human exon <NUM> sequence was inserted, the vector without the G967A(V3231) mutation showed some luciferase activity through the pseudo-splicing donor, and therefore exhibited a high background level. In contrast, by the introduction of the G967A(V323I) mutation, the Luc cDNA sequence containing human exon <NUM> became the majority, and therefore only very low luciferase activity was found (<FIG>, Panel (c)).

Subsequently, analysis of the splicing pattern depending on the lengths of the intron sequences before and after exon <NUM> was carried out. Vectors were constructed by inserting, other than the originally constructed <NUM>-kb sequence, a sequence with a length of <NUM> kb, <NUM> kb, or <NUM> kb (<FIG>). According to the result of analysis of the splicing patterns of these vectors (<FIG>), the vectors mostly showed almost expected splicing patterns (the band of <NUM> bp), but, as the intron size increased, the band of the residual intron (<NUM> bp) tended to disappear. On the other hand, as a result of investigation of the luciferase activity, it was found that, as the intron size increases, the introduction activity into the cells decreases, resulting in a rather high background activity. With any of the vectors, an increase in the luciferase activity could be found when exon skipping was induced using CRISPR-sgRNA-DMD1 (<FIG>). Thus, the vectors were found to be useful as reporter vectors that enable simple and sensitive measurement.

In order to induce exon skipping of dystrophin while minimizing the off-target risk, a plurality of gRNAs were designed at the splice acceptor site of exon <NUM> (<FIG>), and an SSA (Single Strand Annealing) assay was carried out for the cleavage pattern by an ordinary wild-type SpCas9 and a gRNA, and for cases where a D10A nickase-type SpCas9 and two gRNAs were used in combination. The DNA cleavage activities for the target site were measured. As a result, as shown in <FIG>, any of five kinds of gRNAs exhibited a high DNA cleavage activity. Further, it was found that, in the double nicking method, the cleavage activity is low when two gRNAs are overlapping, and that induction of efficient DNA cleavage requires the presence of a certain distance.

Subsequently, in order to investigate DNA cleavage patterns obtained under various conditions, the target site was amplified by PCR, and sequence analysis with a next-generation sequencer MiSeq was carried out. As a result, when two gRNAs were designed such that they were arranged at an appropriate distance in the double nicking method, DNA cleavage patterns with occurrence of deletion between the nicking induction sites of the gRNAs were frequently observed. Thus, it was discovered that, in cases where a splice acceptor sequence, especially the "AG" acceptor sequence, is included in this region, efficient induction of exon skipping is possible (<FIG>).

For studying gRNA sequences, types of CRISPR (SpCas9, AsCpf1, and the like), and genome editing methods (double-nicking) that enable efficient induction of exon skipping, a study was carried out using the exon skipping reporters developed as described above.

As shown in <FIG>, five kinds of gRNA sequences of SpCas9 were tested, and, as shown in <FIG>, comparative analysis was carried out for SpCas9, SaCas9, AsCpf1, and the SpCas9 double-nicking method. As shown in <FIG>, in order to measure the exon skipping efficiency for all sequences that can be designed in human exon <NUM> (sgRNA sequences including the NGG PAM sequence), <NUM> kinds of gRNAs were designed (<FIG>).

The <NUM> kinds of gRNAs were introduced into human 293T cells, and the target DNA cleavage activity was measured by a T7E1 assay (<FIG>). As a result, although sgRNA-DMD6 showed a low DNA cleavage activity, other gRNAs basically showed cleavage activities of not less than <NUM>%. In view of this, the <NUM> kinds of gRNAs were subjected to measurement of the exon skipping efficiency in 293T cells using the Luc2 (G967A) +hEx45 (<NUM> kb) reporter. As a result, it was found that the gRNA design site and the distance from the splice acceptor or the splicing donor are important for the exon skipping efficiency (<FIG>).

Claim 1:
A method of skipping a target exon <NUM> of a human dystrophin gene in a genome, comprising using CRISPR-Cas and guide RNA, wherein the guide RNA contains a spacer sequence such that the site of cleavage by the CRISPR-Cas is positioned within <NUM> bases from the splice acceptor site immediately before the target exon or the splice donor site immediately after the target exon, wherein two or more kinds of the guide RNA are used, and
wherein the spacer sequences of the two or more kinds of guide RNA include the combinations of sgRNA-DMD1 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO: <NUM>), sgRNA-DMD2 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO: <NUM>), sgRNA-DMD4 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>), sgRNA-DMD8 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>), or sgRNA-DMD9 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>) with sgRNA-DMD23 (the base sequence of bases from <NUM> to <NUM> in SEQ ID NO:<NUM>) or sgRNA-DMD20 (the base sequence of bases from <NUM> to <NUM> in SEQ ID N°<NUM>) wherein the method is not a method of treatment of the human/animal body by therapy and wherein the method is not a process for modifying the germ line genetic identity of human beings.