Method for screening and determining regulatory sites of exon skipping splicing in duchenne muscular dystrophy

Abstract

The present invention provides a method for screening regulatory sites of exon skipping splicing in Duchenne muscular dystrophy. Specifically, the present invention provides a method for screening potential gene editing sites that induce exon skipping on the basis of mini-genes. The present invention screens potential gene editing sites that can induce all or part of exon skipping on the basis of mini-genes. gRNAs can be designed for these sites, and after these sites are used together with an editing tool enzyme, an mRNA that is slightly shortened but has a correct reading frame can be generated. Such an mRNA can produce a functional Dystrophin protein when expressed, and restore the normal function of muscle cells, thereby treating DMD disease.

Description

Method for screening and determining a splicing regulatory site for exon skipping in Duchenne muscular dystrophy TECHNICAL FIELD

[0001] The present application relates to the field of gene editing, in particular, to a method for screening and determining a splicing regulatory site for exon skipping in Duchenne muscular dystrophy. BACKGROUND

[0002] Many human diseases are caused by mutations in the sequence of genes encoding proteins, leading to reduced levels of protein or proteins that lack some or all activity, of which RNA splicing mutations account for about one-third of all monogenic diseases. RNA splicing is a crucial step in the process of gene expression, which processes the precursor messenger RNA (pre-mRNA) into mature messenger RNA (mRNA) by removing intronic sequences and connecting exonic sequences. There are splicing-related sequences on pre-mRNA that help the recognition of the spliceosome, and these sequences mainly exist at the junction of exons and introns, including the splice donor at the 5' end of the intron (3' end of the exon), the splice acceptor at the 3' end of the intron (5' end of the exon), and the branch point site (BP) located upstream of the splice acceptor. In addition, there are many elements involved in splicing regulation, called splicing-regulatory elements (SREs). There are multiple exons and introns on a pre-mRNA, and the selection of splicing sites is regulated by cis-regulatory elements and trans-acting factors. According to the location and function of the cis-regulatory elements, they can be classified as exon splicing enhancers (ESEs), intron splicing enhancers (ISEs), exon splicing silencers (ESSs) or intron splicing silencers (ISSs). These cis-acting elements recruit splicing factors to facilitate or inhibit the recognition of nearby splicing sites. Trans-acting elements mainly include two families, the serine and arginine-rich (SR) protein family and the heterogeneous ribonucleoproteins (hnRNPs), which can bind to pre-mRNA to regulate the assembly of the spliceosome and the recognition of splicing sites.

[0003] Spinal Muscular Atrophy (SMA), Huntington’s Disease (HD), etc. In these cases, mutations in the splicing region (including exon and intron sequences) can cause incorrect mRNA splicing, thus triggering diseases. Modulating the splicing of RNA, such as inducing exon restoration or skipping, can be used to treat many such diseases. However, the splicing of mRNA is precise and complex, and in mammalian genes, in addition to the relatively conserved 5’SS and 3’SS, other splicing-related elements are greatly degenerated, and it is difficult to determine which sites will affect splicing. Currently, gene therapy can target the splicing regulatory region, and through editing of the nucleotides in the regulatory region, it can to some extent regulate the splicing of mRNA. For example, through gRNA-dependent CRISPR / Cas system or gRNA-independent ZFN, TALEN and derivative systems to edit DNA so that the RNA transcribed from the DNA has an editing mark to regulate splicing. In the same way, editing of RNA or using small molecules, small nucleotides and other drugs to combine with the sequence related to splicing can also play a regulatory role. However, these treatment methods cannot locate the key sites that truly affect the splicing of mRNA when designed, resulting in an increased risk of bystander mutations, off-targets, etc.

[0004] DMD is a rare disease that has been clinically found for a long time but lacks effective treatment means for a long time. It is a fatal degenerative neuromuscular disease, and about one in every 4,000 newborn males worldwide can be detected as a child. Among them, China is one of the countries with the highest number of DMD patients, with 400-500 children born each year, and the cumulative number of patients is as high as 70-80 thousand. Its pathogenesis is that the gene on the X chromosome responsible for regulating and encoding dystrophin protein appears abnormal mutations, leading to the absence of dystrophin protein, causing muscle weakness and atrophy. DMD patients generally develop at the age of 3-5 years old, and the earliest symptoms are progressive leg muscle weakness, and the ability to walk is lost at about 12 years old. After that, the symptoms will gradually worsen, and eventually die of heart and lung failure. Patients usually have a lifespan of 20 to 30 years. DMD mutation types are more, including small deletions, insertions, point mutations, and repeat sequences, or deletion or sequence repetition of one or more exons in large fragments. The hot spot area of mutation is between exons 45-53, followed by exons 2-20. BMD (Becker Muscular Dystrophy) is a relatively mild muscle atrophy disease, with an incidence of about 1 / 10 of DMD. BMD is also caused by mutations in the DMD gene, and the abnormal Dystrophin protein produced after mutation still has some function, so the clinical symptoms appear later and are relatively mild. The main manifestations are bilateral lower extremity weakness, often accompanied by gastrocnemius muscle pseudohypertrophy and Gower sign. BMD progresses slowly, and some even have no obvious muscle atrophy in their fifties, and generally have less impact on patient lifespan. DMD has no effective treatment means, and the traditional corticosteroid therapy has limited effect and large side effects. Currently, exon skipping is the fastest developing treatment strategy. DMD can restore the reading frame of the Dystrophin gene or skip the mutated exons through exon skipping, achieve the effect of restoring Dystrophin protein, and make the patient change from DMD phenotype to a mild phenotype, achieving the effect of treatment. According to statistics, about 80% of DMD patients can be treated by exon skipping therapy.

[0005] Currently, there are four exon skipping antisense oligonucleotide (ASO) drugs approved by the FDA for accelerated approval in the United States. ASO is a single-stranded nucleic acid polymer of 18-30 nucleotides, which can regulate gene expression through various mechanisms. The four ASO drugs on the market include Amondys 45 (Casimersen) skipping exon 45 of DMD, Exondys 51 (Eteplirsen) skipping exon 51, Vyondys 53 (Golodirsen) skipping exon 53, and Viltepso 53 (Viltolarsen), which mainly binds to the specific exon region of pre-mRNA during the maturation of DMD pre-mRNA, preventing the effective binding of the splicing or regulatory complex, resulting in the final mRNA not containing this exon, thus restoring the reading frame of the damaged DMD mRNA. Although these four ASO therapies can restore the coding frame of the Dystrophin protein by skipping the corresponding exon, the protein restoration level is extremely low, and the clinical efficacy is not clear. The treatment requires continuous administration throughout the body, and the treatment is expensive. For example, Eteplirsen costs $7.5-15 million per person per year, which greatly limits the treatable patient population. In addition, compared to skeletal muscle cells, the efficiency of Dystrophin protein repair in cardiac muscle cells is lower, and improving skeletal muscle performance will further exacerbate cardiac muscle damage, posing a threat to the overall health of patients.

[0006] Elevidys (SRP-9001) is the first DMD gene replacement therapy approved, mainly for the treatment of DMD patients aged 4-5 years. Its principle is to use adeno-associated virus (rAAVrh74) to deliver a gene that can express truncated dystrophin (micro-dystrophin) to muscle cells. Since AAV gene therapy can only effectively package fragments less than 4.7 kbp, the micro-dystrophin carried by Elevidys is about 3.6 kbp in length, which is only about 1 / 3 of the normal human Dystrophin protein, and cannot completely replace the function of the full-length protein. In addition, exogenous DNA will be diluted with cell division, and the promoter may be methylated and silenced, so continuous injection is required to achieve sustained efficacy. However, the human body produces very high anti- AAV antibodies after AAV injection, theoretically making it impossible to administer a second dose. The results of Elevidys clinical trials show that it only has efficacy in DMD patients aged 4-5 years, and no therapeutic effect in patients aged 6-7 years. This also shows the great limitations of replacement therapy. In contrast, directly editing the DMD gene of the cell and continuously expressing the Dystrophin protein through the repaired gene can truly achieve the goal of one-time treatment and lifelong effectiveness.

[0007] In order to achieve a permanent cure effect, gene editing technology is applied to the treatment of DMD. As early as 2015, scientists used double-cut CRISPR / Cas9 gene editing method to successfully restore Dystrophin expression in DMD patient myoblasts. The gene editing method is to make the target gene produce two nicks with the help of Cas9 protein under the mediation of two gRNAs, and the two fragments cut by DNA non-homologous end joining (NHEJ) repair mechanism are connected together, the protein reading frame is restored, and finally a slightly shortened functional Dystrophin protein can be expressed. Subsequently, scientists also used single-cut CRISPR / Cas9 gene editing technology to treat mouse DMD models. The site designed by single-cut CRISPR / Cas9 is mainly selected in the predicted splice-related elements, by destroying the exon splicing enhancer site, the purpose of exon skipping is achieved, or by insertion and deletion (Insertion-deletion, Indel) to cause frame shift to restore the reading frame. However, whether it is double-cut or single-cut gene editing, it is to induce cell NHEJ or HDR by DNA double-strand break (Double Strand Break, DSB) to achieve the effect of gene editing, and the consequence of DSB is to cause various unexpected gene editing (unediting, deletion, Indel, large fragment deletion, inversion, transposition, etc.). Therefore, when using double-cut or single-cut gene editing technology, the influence of off-target and translocation cannot be ignored. In addition, the generation of DSB in stem cells will activate the p53 signaling pathway and ultimately induce cell apoptosis, and this result other than targeted region editing is one of the important reasons that hinder the development of gene editing drugs.

[0008] Base Editor (BE) for DMD treatment is still in the early stage of development. The earliest BE treatment for DMD originated from the study of CRISPR-guided cytidine deaminase for genetic regulation of mRNA splicing. Through Targeted AID-mediated Mutagenesis (TAM) mediated by cytosine deaminase (AID), the 51st exon-deleted DMD iPSC was edited, and only the base of the 5' splice site of the 50th exon was edited, which achieved efficient exon skipping and restored protein expression. After treatment, the mice not only recovered significantly in myocardial and skeletal muscle function, but also significantly prolonged their lifespan. In addition, there are also adenine editors (ABE) and prime editors (PE) for editing splice sites, but since the sites affecting splicing will also have other sites affected by bystander mutations, it is unknown whether these bystander mutations will affect the editing effect of the main splice site. As the most advanced gene therapy technology at present, base editor has irreplaceable advantages, bringing another choice for DMD patients, and is currently the best solution to permanently cure DMD in theory. However, due to the lack of research on the specific splice site on the DMD gene, it is difficult to determine the key base that determines splicing. Therefore, it often leads to the inability of base editors to find effective editing sites, or the lack of suitable base editors for editing sites, resulting in low editing efficiency, inability to induce efficient exon skipping, and increased off-target risk.

[0009] On the other hand, in the field of gene therapy, adeno-associated virus (AAV) is a highly potential gene delivery vector, and its packaging capacity is always a core bottleneck restricting its application. AAV belongs to a non-enveloped single-stranded DNA defective virus, and its genome is less than 5 kb in length, and the actual effective loading capacity is only 4.7 kb (including the viral terminal inverted repeat sequence ITR), which limits the packaging capacity of exogenous genes. The gene editing system mediated by AAV Cas9 protein (such as base editor) faces multiple technical challenges. For example, the clinically widely used Cas9 from Streptococcus pyogenes (SpCas9) has a coding gene (cDNA) length of 4.1 kb. If it is constructed into a single vector with a promoter, sgRNA expression cassette, nuclear localization signal (NLS), and ITR element, the total length will exceed 5.5 kb, which is significantly higher than the upper limit of AAV loading. Although researchers have tried to split the editing elements (such as packaging Cas9 and sgRNA modules separately) by using a double AAV vector system, the co-transduction efficiency in vivo is low, and high-dose AAV is needed to maintain editing activity. It is worth noting that high-dose AAV may cause dose-dependent toxicity risk (such as immunogenicity and liver toxicity), which seriously restricts the clinical transformation of this technology. Although several miniaturized Cas9 homologues (such as Cje1Cas9 from Campylobacter jejuni, containing 984 amino acids) have been discovered in recent years, their coding gene length (2.95 kb) is theoretically suitable for single AAV vector delivery, but the high complexity of the N3VRYAC type PAM sequence recognized by them leads to insufficient genome coverage, making it difficult to meet the demand for broad-spectrum targeting. Another type of new miniaturized editing tool (such as IscB-ωRNA system) has the advantage of small protein size (about 400 amino acids), but it relies on a strict 6-base target sequence adjacent motif (TAM), further compressing the target recognition window, limiting the application potential of single-vector delivery systems.

[0010] Current studies have shown that Cas9 from Staphylococcus aureus (SaCas9, 1053 amino acids) is a more ideal single-vector delivery candidate element, but its targeting range and editing activity of wild type and KKH mutant are still significantly weaker than SpCas9. More importantly, when SaCas9 is integrated into a base editing system, the additional introduction of a deaminase module (such as APOBEC or TadA) will further increase the load of the vector, making single AAV delivery face capacity bottlenecks again. Under this background, developing element miniaturization strategies has become a breakthrough - including the use of compact promoters (such as H1, U6 variants), truncated Cas enzyme domains, optimized deaminase sequences, and shortened sgRNA expression cassettes, etc. The above technical paths are becoming the research focus of current gene editing vector engineering.

[0011] In summary, there is an urgent need in the art to screen key sites that can affect the splicing regulation of DMD mRNA, and only need to mutate these sites to induce exon skipping, produce slightly shortened but in-frame mRNA, which can produce functional Dystrophin protein when expressed, restore normal muscle cell function. In addition, by optimizing the delivery vector, improving the editing efficiency, further improve the therapeutic effect. SUMMARY

[0012] Therefore, in view of the above problems, it is urgent to screen key sites that can affect the splicing regulation of DMD mRNA, and only need to mutate these sites to induce exon skipping, produce slightly shortened but in-frame mRNA, which can produce functional Dystrophin protein when expressed, restore normal muscle cell function. On the other hand, by optimizing the polIII promoter, the flexibility of single vector editing tool construction is increased while improving the editing efficiency.

[0013] The first aspect of the present application provides a method for screening potential gene editing sites affecting exon splicing based on a mini gene, comprising:

[0014] editing the 1-20 (preferably 1-10, more preferably 1-5) base editing sites of the intron before the splice acceptor (SA) of the exon and / or the 1-20 (preferably 1-10, more preferably 1-5) base editing sites of the exon after the splice acceptor (SA) of the exon in the mini gene;

[0015] editing the 1-20 (preferably 1-10, more preferably 1-5) base editing sites of the exon before the splice donor (SD) of the exon and / or the 1-20 (preferably 1-10, more preferably 1-5) base editing sites of the intron after the splice donor (SD) of the exon in the mini gene, and obtaining potential gene editing sites affecting exon splicing based on the proportion of exon skipping;

[0016] The exon includes the 45th exon (E45), the 51st exon (E51), the 53rd exon (E53), the 48th exon (E48), the 54th exon (E54), and / or the 44th exon (E44).

[0017] In another preferred embodiment, the exon is an exon of the DMD gene.

[0018] In another preferred embodiment, the mini gene is a truncated DMD gene.

[0019] In another preferred embodiment, the mini gene contains exons 43-50 and introns of 30-1000 bp (preferably, 40-500 bp, more preferably, 50-300 bp) flanking each exon.

[0020] In another preferred embodiment, the mini gene contains exons 49-56 and introns of 30-1000 bp (preferably, 40-500 bp, more preferably, 50-300 bp) flanking each exon.

[0021] In another preferred embodiment, the sequence of the mini gene is SEQ ID NO. 123-124.

[0022] In another preferred embodiment, the gene editing comprises editing of DNA and editing of RNA.

[0023] In another preferred embodiment, the gene editing comprises indel and mutation.

[0024] In another preferred embodiment, the mutation comprises A>G, A>C, A>T, C>T, G>A, G>C and / or T>C mutation.

[0025] In another preferred embodiment, the mutation is performed on one or more positions of the exon 45 (E45) selected from the group consisting of SA(-1), SA(-1, +9), SA(-1, +1, +9, +10), SA(-1, +1), SA(-2), SD(+1), SD(-1, +1), SD(+2), SA(-1, +1, +9, +10, +13, +14).

[0026] In another preferred embodiment, the mutation is performed on one or more positions of the exon 51 selected from the group consisting of SD(+2), SA(-1, +1), SA(-1, +11), SD(-1, +1), SD(+1, +5, +7).

[0027] In another preferred embodiment, the mutation is performed on one or more positions of the exon 53 selected from the group consisting of SA(-1), SD(+4), SD(+5), SD(-1, +1, +5), SA(-1, +3, +7), SA(-2, +4, +5, +6), SA(-2), SD(+1), SD(-1, +1), SA(-1, +3, +7, +14), SA(-1, +3, +7, +14, +20).

[0028] In another preferred embodiment, the mutation is performed on one or more positions of the exon 48 selected from the group consisting of SD(+1), SD(-6, -1, +1, +4, +5, +6).

[0029] In another preferred embodiment, the following positions of the 54thexon are mutated: SD(+1).

[0030] In another preferred embodiment, one or more positions of the 44thexon selected from the group consisting of: SA(-1, +1), SA(-4, -1, +1, +3), SA(-4, -1, +1, +3, +8, +12), SD(-1, +1), SD(-1, +1, +5), SD(-11, -10, -1, +1, +5) are mutated.

[0031] In another preferred embodiment, when the proportion of exon skipping is ≥ 30%, preferably ≥ 40%, more preferably ≥ 50%, such as 60%, 70%, 80%, 90%, 95%, 100%, it indicates that the edited site is a potential gene editing site affecting exon splicing.

[0032] The second aspect of the present application provides a gRNA affecting exon splicing, which targets the potential gene editing site obtained by the method of the first aspect of the present application.

[0033] In another preferred embodiment, the gene editing site comprises one or more selected from the group consisting of:

[0034] (a) one or more positions of the 45thexon (E45) selected from the group consisting of: SA(-1), SA(-1, +9), SA(-1, +1, +9, +10), SA(-1, +1), SA(-2), SD(+1), SD(-1, +1), SD(+2), SA(-1, +1, +9, +10, +13, +14);

[0035] (b) one or more positions of the 51stexon selected from the group consisting of: SD(+2), SA(-1, +1), SA(-1, +11), SD(-1, +1), SD(+1, +5, +7);

[0036] (c) one or more positions of the 53rdexon selected from the group consisting of: SA(-1), SD(+4), SD(+5), SD(-1, +1, +5), SA(-1, +3, +7), SA(-2, +4, +5, +6), SA(-2), SD(+1), SD(-1, +1), SA(-1, +3, +7, +14), SA(-1, +3, +7, +14, +20);

[0037] (d) one or more positions of the 48thexon selected from the group consisting of: SD(+1), SD(-6, -1, +1, +4, +5, +6);

[0038] (e) the following positions of exon 54: SD(+1);

[0039] (f) one or more positions of exon 44 selected from the group consisting of: SA(-1, +1), SA(-4, -1, +1, +3), SA(-4, -1, +1, +3, +8, +12), SD(-1, +1), SD(-1, +1, +5), SD(-11, -10, -1, +1, +5).

[0040] In another preferred embodiment, the gRNA guides the base editor to target the potential gene editing site obtained from the method of the first aspect of the application to affect exon splicing.

[0041] In another preferred embodiment, the targeting sequence of the gRNA has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the sequence set forth in any one of SEQ ID NO. 111-117, or is the reverse complement of a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the sequence set forth in any one of SEQ ID NO. 111-117.

[0042] In another preferred embodiment, the targeting sequence of the gRNA is set forth in any one of SEQ ID NO. 111-117, or is the reverse complement of the sequence set forth in any one of SEQ ID NO. 111-117.

[0043] In another preferred embodiment, the gRNA further comprises one or more targeting sequences selected from the group consisting of SEQ ID NO. 127-136.

[0044] In another preferred embodiment, the gRNA can be directly synthesized by chemical means, or prepared by other means, such as prepared by in vitro IVT means.

[0045] In another preferred embodiment, the gRNA comprises a targeting sequence targeting a specific sequence of a genome.

[0046] In another preferred embodiment, the gRNA comprises unmodified and modified gRNAs.

[0047] In another preferred embodiment, the modified gRNA comprises chemical modification of a base.

[0048] In another preferred embodiment, the chemical modification comprises methylation modification, methoxy modification, fluorination modification, or thio modification.

[0049] In another preferred embodiment, the base editor comprises a protein having a nuclease- active domain.

[0050] In another preferred embodiment, the nuclease-active domain is a RuvC domain and / or a HNH domain and / or a FoKI domain.

[0051] In another preferred embodiment, the protein having a nuclease-active domain comprises a FoKI protein, a Cas protein.

[0052] In another preferred embodiment, the protein having a nuclease-active domain comprises a Type II Cas protein, a Type V Cas protein, or a Type VI Cas protein.

[0053] In another preferred embodiment, the protein having a nuclease-active domain comprises a Cas9, a Cas12, a Cas13, a Cas14.

[0054] In another preferred embodiment, the protein having a nuclease-active domain comprises a Cas9, a CasX, a CasY, a Cas12, a Casl2a, a Casl2b, a Casl2c, a Casl2g, a Casl2h, a Casl2i, a Cas13, a Casl3a1, a Casl3a2, a Casl3b, a Casl3c, a Casl3d, a Casl4.

[0055] In another preferred embodiment, the protein having a nuclease-active domain comprises a transposase protein.

[0056] In another preferred embodiment, the protein having a nuclease-active domain comprises a IscB, a IsrB, a IshB, a TnpB, a Fanzor.

[0057] In another preferred embodiment, the base editor comprises an adenine base editor, a guanine base editor, a cytosine base editor, a thymine base editor.

[0058] In another preferred embodiment, the base editor comprises an adenine deaminase, a guanine deaminase, a cytosine deaminase.

[0059] In another preferred embodiment, the base editor comprises an APOBEC, an AID, an ADAT2, an ADAT3, an ADAR2, a TadA, a UNG, a UGI.

[0060] In another preferred embodiment, the cytosine base editor comprises a cytosine base editor TAM.

[0061] In another preferred embodiment, the cytosine base editor TAM includes cytosine deaminase hAIDx, a SaCas9 mutant (e.g., KKH-nSaCas9), and uracil glycosylation inhibitor UGI.

[0062] In another preferred embodiment, the amino acid sequence of the cytosine base editor TAM is shown in SEQ ID NO.122.

[0063] A third aspect of the present invention provides a nucleic acid that encodes the gRNA described in the second aspect of the present invention, or a precursor encoding the gRNA described in the second aspect of the present invention.

[0064] A fourth aspect of the present invention provides a vector comprising the gRNA described in the second aspect of the present invention or the nucleic acid described in the third aspect of the present invention.

[0065] In another preferred embodiment, the vector further includes a nucleic acid encoding a base editor.

[0066] In another preferred embodiment, the carrier comprises one or more carriers, including:

[0067] a) A first regulatory element, operatively linked to the gRNA,

[0068] b) A second regulatory element operatively connected to the base editor;

[0069] Components (a) and (b) are located on the same or different carriers.

[0070] In another preferred embodiment, the first and / or second control elements include a promoter.

[0071] In another preferred embodiment, the vector contains one or more promoters selected from the group consisting of SEQ ID NO.149-169.

[0072] In another preferred embodiment, the vector is a vector for targeting and editing potential gene editing sites obtained by the method described in the first aspect of the present invention.

[0073] In another preferred embodiment, the vector comprises a plasmid or a viral vector.

[0074] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, SV40, poxvirus, or combinations thereof.

[0075] The fifth aspect of the present invention provides a gene editing system comprising the gRNA described in the second aspect of the present invention.

[0076] In another preferred embodiment, the gene editing system further comprises a base editor.

[0077] In another preferred embodiment, the gene editing system further comprises one or more promoters selected from the group consisting of SEQ ID NO. 149-169.

[0078] In another preferred embodiment, the base editor comprises a protein having a nuclease active domain.

[0079] In another preferred embodiment, the protein having a nuclease active domain is a RuvC domain and / or a HNH domain and / or a FoKI domain.

[0080] In another preferred embodiment, the protein having a nuclease active domain comprises a FoKI protein, a Cas protein.

[0081] In another preferred embodiment, the protein having a nuclease active domain comprises a Type II Cas protein, a Type V Cas protein or a Type VI Cas protein.

[0082] In another preferred embodiment, the protein having a nuclease active domain comprises a Cas9, a Cas12, a Cas13, a Cas14.

[0083] In another preferred embodiment, the protein having a nuclease active domain comprises a Cas9, a CasX, a CasY, a Cas12, a Casl2a, a Casl2b, a Casl2c, a Casl2g, a Casl2h, a Casl2i, a Cas13, a Casl3a1, a Casl3a2, a Casl3b, a Casl3c, a Casl3d, a Casl4.

[0084] In another preferred embodiment, the protein having a nuclease active domain comprises a transposase protein.

[0085] In another preferred embodiment, the protein having a nuclease active domain comprises a IscB, a IsrB, a IshB, a TnpB, a Fanzor.

[0086] In another preferred embodiment, the base editor comprises an adenine base editor, a guanine base editor, a cytosine base editor, a thymine base editor.

[0087] In another preferred embodiment, the base editor comprises an adenine deaminase, a guanine deaminase, a cytosine deaminase.

[0088] In another preferred embodiment, the base editor comprises an APOBEC, an AID, an ADAT2, an ADAT3, an ADAR2, a TadA, a UNG, a UGI.

[0089] In another preferred embodiment, the cytosine base editor comprises a cytosine base editor TAM.

[0090] In another preferred embodiment, the cytosine base editor TAM comprises a cytosine deaminase hAIDx, a SaCas9 mutant (e.g., KKH-nSaCas9), and a uracil glycosylase inhibitor UGI.

[0091] In another preferred embodiment, the gene editing comprises in vivo gene editing, in vitro gene editing.

[0092] The sixth aspect of the present application provides a cell, which is obtained by editing the cell with the gene editing system of the fifth aspect of the present application.

[0093] In another preferred embodiment, the cell is an in vitro cultured cell.

[0094] In another preferred embodiment, the cell comprises a primary cell and a passaged cell.

[0095] In another preferred embodiment, the cell comprises a mammalian cell.

[0096] In another preferred embodiment, the cell comprises a human cell.

[0097] In another preferred embodiment, the cell comprises a muscle cell and a nerve cell.

[0098] In another preferred embodiment, the cell comprises an AC16 cell, an iPSC induced muscle cell.

[0099] The seventh aspect of the present application provides a composition comprising a protein component or an mRNA component for encoding a protein component; and a gRNA component or an mRNA component for encoding a gRNA component; the gRNA or the mRNA component encoding the gRNA comprises the gRNA of the second aspect of the present application, and the protein component or the mRNA component for encoding a protein component comprises a base editor.

[0100] In another preferred embodiment, the gRNA component is selected from the group consisting of: the gRNA of the second aspect of the present application, or a nucleic acid encoding the aforementioned gRNA; and the base editor is selected from the group consisting of: a base editor, or a nucleic acid encoding a base editor.

[0101] In another preferred embodiment, the base editor comprises a protein having a nuclease active domain.

[0102] In another preferred embodiment, the nuclease active domain is a RuvC domain and / or a HNH domain and / or a FoKI domain.

[0103] In another preferred embodiment, the protein having a nuclease active domain comprises a FoKI protein, a Cas protein.

[0104] In another preferred embodiment, the protein having a nuclease active domain comprises a Type II Cas protein, a Type V Cas protein, or a Type VI Cas protein.

[0105] In another preferred embodiment, the protein having a nuclease active domain comprises a Cas9, a Cas12, a Cas13, a Cas14.

[0106] In another preferred embodiment, the protein having a nuclease active domain comprises a Cas9, a CasX, a CasY, a Cas12, a Casl2a, a Casl2b, a Casl2c, a Casl2g, a Casl2h, a Casl2i, a Cas13, a Casl3a1, a Casl3a2, a Casl3b, a Casl3c, a Casl3d, a Casl4.

[0107] In another preferred embodiment, the protein having a nuclease active domain comprises a transposase protein.

[0108] In another preferred embodiment, the protein having a nuclease active domain comprises a IscB, a IsrB, a IshB, a TnpB, a Fanzor.

[0109] In another preferred embodiment, the base editor comprises an adenine base editor, a guanine base editor, a cytosine base editor, a thymine base editor.

[0110] In another preferred embodiment, the base editor comprises an adenine deaminase, a guanine deaminase, a cytosine deaminase.

[0111] In another preferred embodiment, the base editor comprises an APOBEC, an AID, an ADAT2, an ADAT3, an ADAR2, a TadA, a UNG, a UGI.

[0112] In another preferred embodiment, the cytosine base editor comprises a cytosine base editor TAM.

[0113] In another preferred embodiment, the cytosine base editor TAM comprises a cytosine deaminase hAIDx, a SaCas9 mutant (such as: KKH-nSaCas9), and a uracil glycosylase inhibitor UGI.

[0114] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0115] In another preferred embodiment, the pharmaceutical composition is in a liquid form.

[0116] In another preferred embodiment, the dosage form of the pharmaceutical composition comprises an injection or a needle injection.

[0117] In another preferred embodiment, the dosage form of the pharmaceutical composition is an intravenous injection dosage form.

[0118] The eighth aspect of the present application provides a composition comprising:

[0119] The system of the fifth aspect of the present application or the cell of the sixth aspect of the present application; and a pharmaceutically acceptable carrier.

[0120] In another preferred embodiment, the system or the cell in the composition accounts for 1-99 wt%, preferably 10-90 wt%, more preferably 30-70 wt% of the total weight of the composition.

[0121] The ninth aspect of the present application provides a delivery composition comprising an active ingredient and a delivery medium, wherein the active ingredient comprises the gRNA of the second aspect of the present application, the vector of the fourth aspect of the present application, or the system of the fifth aspect of the present application.

[0122] In another preferred embodiment, the delivery composition further comprises a base editor.

[0123] In another preferred embodiment, the delivery medium comprises a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, or a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0124] The tenth aspect of the present application provides a host cell comprising the gRNA of the second aspect of the present application, the nucleic acid of the third aspect of the present application, the vector of the fourth aspect of the present application, the system of the fifth aspect of the present application, the composition of the seventh aspect of the present application, or the delivery composition of the ninth aspect of the present application.

[0125] In another preferred embodiment, the host cell comprises a mammalian cell.

[0126] In another preferred embodiment, the host cell comprises a human cell.

[0127] In another preferred embodiment, the cell comprises a muscle cell and a nerve cell.

[0128] In another preferred embodiment, the cell comprises an AC16 cell, an iPSC-induced muscle cell

[0129] The eleventh aspect of the present application provides a CRISPR complex comprising the gRNA of the second aspect of the present application, a base editor, and the potential gene editing site obtained by the method of the first aspect of the present application.

[0130] The twelfth aspect of the present application provides a kit comprising:

[0131] a first container, and an active ingredient or a medicament containing the active ingredient, the active ingredient or the medicament being located in the first container, the active ingredient comprising the gRNA of the second aspect of the present application, the nucleic acid of the third aspect of the present application, the vector of the fourth aspect of the present application, the system of the fifth aspect of the present application, the cell of the sixth aspect of the present application, the composition of any one of the seventh aspect or the eighth aspect of the present application, or the delivery composition of the ninth aspect of the present application, or the host cell of the tenth aspect of the present application, or the CRISPR complex of the eleventh aspect of the present application.

[0132] In another preferred embodiment, the active ingredient or the medicament is a single preparation.

[0133] In another preferred embodiment, the dosage form of the active ingredient or the medicament is an injection dosage form or a needle injection.

[0134] In another preferred embodiment, the dosage form of the active ingredient or the medicament is an intravenous injection dosage form.

[0135] In another preferred embodiment, the kit further comprises an instruction, which describes how to administer the active ingredient or the medicament to a subject, so as to (i) affect exon splicing; and / or (ii) prevent and / or treat DMD diseases.

[0136] In another preferred embodiment, the subject is a cell.

[0137] In another preferred embodiment, the subject is a human cell.

[0138] In another preferred embodiment, the subject comprises a human muscle cell.

[0139] In another preferred embodiment, the subject is a human or a non-human mammal.

[0140] In another preferred embodiment, the administration is to contact the active ingredient or the medicament with a cell, such as a human muscle cell, or to inject the active ingredient or the medicament into a human body by injection.

[0141] In a thirteenth aspect, the present application provides a method of gene editing a cell, comprising contacting the cell with the gRNA of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the system of the fifth aspect, the composition of any one of the seventh aspect or the eighth aspect, or the delivery composition of the ninth aspect, or the host cell of the tenth aspect, or the CRISPR complex of the eleventh aspect, or the kit of the twelfth aspect.

[0142] In another preferred embodiment, the contacting is performed in vitro.

[0143] In another preferred embodiment, the cell is a cell cultured in vitro.

[0144] In another preferred embodiment, the cell comprises a primary cell and a passaged cell.

[0145] In another preferred embodiment, the cell comprises a mammalian cell.

[0146] In another preferred embodiment, the cell comprises a human cell.

[0147] In another preferred embodiment, the cell comprises a muscle cell and a neural cell.

[0148] In another preferred embodiment, the cell comprises an AC16 cell, an iPSC induced muscle cell.

[0149] In a fourteenth aspect, the present application provides a kit for gene editing, comprising the gRNA of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the system of the fifth aspect, the composition of any one of the seventh aspect or the eighth aspect, or the delivery composition of the ninth aspect, or the host cell of the tenth aspect, or the CRISPR complex of the eleventh aspect, or the kit of the twelfth aspect.

[0150] In another preferred embodiment, the kit further comprises a label or an instruction.

[0151] In another preferred embodiment, the label or the instruction indicates administration of the gRNA of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the system of the fifth aspect, the composition of any one of the seventh aspect or the eighth aspect, or the delivery composition of the ninth aspect, or the host cell of the tenth aspect, or the CRISPR complex of the eleventh aspect, or the kit of the twelfth aspect to a subject to be edited, thereby performing gene editing.

[0152] In another preferred embodiment, the editing subject comprises a cell.

[0153] In another preferred embodiment, the cell comprises a primary cell and a passaged cell.

[0154] In another preferred embodiment, the cell comprises a mammalian cell.

[0155] In another preferred embodiment, the cell comprises a human cell.

[0156] In another preferred embodiment, the cell comprises a muscle cell and a neural cell.

[0157] In another preferred embodiment, the cell comprises an AC16 cell, an iPSC induced muscle cell.

[0158] The fifteenth aspect of the present application provides use of the gRNA of the second aspect of the present application, the nucleic acid of the third aspect of the present application, the vector of the fourth aspect of the present application, the system of the fifth aspect of the present application, the composition of any one of the seventh aspect or the eighth aspect of the present application, or the delivery composition of the ninth aspect of the present application, or the host cell of the tenth aspect of the present application, or the CRISPR complex of the eleventh aspect of the present application, or the kit of the twelfth aspect of the present application, or the kit of the fourteenth aspect of the present application, in (i) affecting exon splicing, and / or (ii) preventing and / or treating DMD disease.

[0159] The sixteenth aspect of the present application provides a method of treating DMD disease, comprising administering to a subject an effective amount of the system of the fifth aspect of the present application, the cell of the sixth aspect of the present application, the composition of any one of the seventh aspect or the eighth aspect of the present application, or the delivery composition of the ninth aspect of the present application, or the host cell of the tenth aspect of the present application, or the CRISPR complex of the eleventh aspect of the present application, or the kit of the twelfth aspect of the present application, or the kit of the fourteenth aspect of the present application.

[0160] In another preferred embodiment, the administration comprises injection administration.

[0161] In another preferred embodiment, the subject is a human or a non-human mammal.

[0162] In another preferred embodiment, the non-human mammal comprises a rodent and a primate, preferably a mouse, a rat, a rabbit, a monkey.

[0163] The seventeenth aspect of the present application provides a gene editing system, wherein the system targets a potential gene editing site obtained by the method of the first aspect of the present application.

[0164] In another preferred embodiment, the gene editing system comprises a gRNA- independent gene editing system.

[0165] In another preferred embodiment, the gene editing system comprises a zinc finger protein (ZFP) and / or a transcription activator-like effector (TALE).

[0166] In another preferred embodiment, the gene editing system comprises a protein with a nuclease active domain.

[0167] In another preferred embodiment, the protein with a nuclease active domain comprises a RuvC domain and / or a HNH domain and / or a FoKI domain.

[0168] In another preferred embodiment, the protein with a nuclease active domain comprises a Cas protein and / or a FoKI protein.

[0169] In another preferred embodiment, the gene editing system comprises a ZFN, a TAL EN, and a derivative editing system thereof.

[0170] In another preferred embodiment, the gene editing system comprises a gRNA- independent base editor.

[0171] In another preferred embodiment, the base editor comprises an adenine base editor, a guanine base editor, a cytosine base editor, a thymine base editor.

[0172] In another preferred embodiment, the base editor comprises a zinc finger protein (ZFP) and / or a transcription activator-like effector (TALE).

[0173] In another preferred embodiment, the base editor comprises a protein with a nuclease active domain.

[0174] In another preferred embodiment, the base editor comprises a ZFN, a TAL EN, and a derivative editing system thereof.

[0175] In another preferred embodiment, the base editor comprises an adenine deaminase, a guanine deaminase, a cytosine deaminase.

[0176] In another preferred embodiment, the base editor comprises an APOBEC, an AID, an ADAT2, an ADAT3, an ADAR2, a TadA, a UNG, a UGI.

[0177] In another preferred embodiment, the cytosine base editor comprises a cytosine base editor TAM.

[0178] In another preferred embodiment, the cytosine base editor TAM comprises cytosine deaminase hAIDx, a SaCas9 mutant (e.g., KKH-nSaCas9), and a uracil glycosylase inhibitor UGI.

[0179] In another preferred embodiment, the cytosine base editor TAM comprises a cytosine deaminase hAIDx, a SaCas9 mutant (e.g., KKH-nSaCas9), and a uracil glycosylase inhibitor UGI.

[0180] In another preferred embodiment, the gene editing system comprises the gRNA of the second aspect of the application.

[0181] In another preferred embodiment, the gene editing system comprises the gRNA of the second aspect of the application.

[0182] In another preferred embodiment, the gRNA targets a potential gene editing site of exon 6, exon 7, exon 16, exon 46, and exon 52 of the human DMD gene.

[0183] In another preferred embodiment, the gRNA targets a potential gene editing site of exon 6, exon 7, exon 16, exon 46, and exon 52 of the human DMD gene.

[0184] In another preferred embodiment, the gRNA can be directly synthesized by chemical means or prepared by other means, such as prepared by in vitro IVT means.

[0185] In another preferred embodiment, the gRNA comprises unmodified and modified gRNAs.

[0186] In another preferred embodiment, the modified gRNA comprises chemical modification of the bases.

[0187] In another preferred embodiment, the chemical modification comprises methylation modification, methoxy modification, fluorination modification, or thio modification.

[0188] In another preferred embodiment, the gRNA guides the base editor to target a potential gene editing site to affect exon splicing.

[0189] In another preferred embodiment, the gRNA guides the base editor to target a potential gene editing site to affect exon splicing.

[0190] In another preferred embodiment, the promoter sequence further comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NO. 149-169.

[0191] In a twenty-first aspect of the present application, there is provided an expression cassette comprising the promoter of the nineteenth aspect of the present application and a gene of interest, wherein the gene of interest is operably linked to the promoter.

[0192] In another preferred embodiment, the gene of interest is located 3' to the promoter.

[0193] In another preferred embodiment, the gene of interest is at least one of gRNA, tRNA, ncRNA, rRNA, miRNA, siRNA, IncRNA, piRNA, snoRNA, snRNA, scRNA, and mRNA.

[0194] In a twenty-first aspect of the present application, there is provided an expression cassette comprising the promoter of the nineteenth aspect of the present application and a gene of interest, wherein the gene of interest is operably linked to the promoter.

[0195] In a twenty-second aspect of the present application, there is provided a vector comprising the nucleic acid of the twenty-first aspect of the present application.

[0196] In a twenty-third aspect of the present application, there is provided a cell comprising the vector of the twenty-second aspect of the present application.

[0197] In a twenty-fourth aspect of the present application, there is provided a gene editing system comprising the gRNA of the nineteenth aspect of the present application, the promoter of the twentieth aspect of the present application, and / or the expression cassette of the twenty-first aspect of the present application.

[0198] In a twenty-fifth aspect of the present application, there is provided a method of gene editing a cell, the method comprising editing a cell using the gene editing system of the twenty-fourth aspect of the present application.

[0199] It should be understood that, within the scope of the present application, all combinations between the above-mentioned technical features of the present application and the technical features specifically described hereinafter (e.g., in the Examples) can be interchanged, thereby constituting new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0200] FIG. 1 shows a description of a splice element.

[0201] Figure 2 shows the schematic diagram of DMD mini-gene structure. (A) Schematic diagram of DMD mini-gene plasmid pGAT-DMDminiE43E50. (B) Schematic diagram of DMD mini-gene plasmid pGAT-DMDminiE49E56.

[0202] Figure 3 shows the electropherogram of each exon skipping of point mutation mini-gene and its quantitative analysis.

[0203] Figure 4 shows the sequencing results of splicing of each exon of point mutation mini-gene.

[0204] Figure 5 shows the schematic diagram of base editor TAM structure.

[0205] Figure 6 shows the gRNAs designed for inducing E51 and E53 skipping.

[0206] Figure 7 shows the electropherogram of mini-gene verification gRNA-induced E51 and E53 skipping and its quantitative analysis.

[0207] Figure 8 shows the sequencing results of mini-gene verification gRNA-induced E51 and E53 skipping.

[0208] Figure 9 shows the schematic diagram of (A) fluorescent reporter plasmid pGAT-3xgRNA-mCherry and (B) fluorescent reporter plasmid pGAT-BE-EGFP structure.

[0209] Figure 10 shows the iPSC-induced skeletal muscle successfully transfected by double plasmid enrichment by flow cytometry sorting.

[0210] Figure 11 shows the gRNAs that can induce E51 and E53 skipping in iPSC-induced skeletal muscle verification.

[0211] Figure 12 shows the sequencing results of gRNA-induced E51 and E53 skipping in iPSC-induced skeletal muscle verification.

[0212] Figure 13 shows the gRNAs that can induce E53 skipping in iPSC-induced skeletal muscle verification using AAV delivery system.

[0213] Figure 14 shows the sequencing results of gRNA-induced E53 skipping in iPSC-induced skeletal muscle verification using AAV delivery system.

[0214] Figure 15 shows the schematic diagram of model mouse gene structure.

[0215] Figure 16 shows the results of gRNA that can edit E53 target genomic DNA in model mouse verification using AAV delivery system.

[0216] Figure 17 shows the results that E53 target genomic DNA editing can transcribe E53 skipped mRNA.

[0217] Figure 18 shows the results that E53 skipped mRNA can restore dystrophin protein expression.

[0218] Figure 19 shows the results that different gRNAs were verified in HEK293T cells using TAM base editor to edit the corresponding DMD exon splicing key sites.

[0219] Figure 20 shows the schematic diagram of the arrangement of each element when constructing a polIII mutant.

[0220] Figure 21 shows the schematic diagram of the target for human DMD gene exon 50.

[0221] Figure 22 shows the effect of different polIII promoter mutants on TAM base editor editing. DETAILED DESCRIPTION

[0222] The inventors have made extensive and in-depth research, and for the first time accidentally found that based on the mini gene, potential gene editing sites affecting the skipping of all or part of the exon can be screened, gRNAs are designed for these sites, and gene editing of these sites can produce slightly shortened but in-frame mRNA, which can produce functional Dystrophin protein when expressed, restore normal muscle cell function, and thus treat DMD disease. In addition, the inventors found a shorter and more active promoter than the wild type by recombining and arranging the commonly used polIII promoter, and verified that it can improve the treatment effect of the gene editing system on DMD disease. On this basis, the inventors completed the present application.

[0223] TERMS

[0224] The term "in vitro transcription (IVT)" refers to a technology that simulates the in vivo transcription process to transcribe RNA using linear DNA as a template in an in vitro cell-free system containing RNA polymerase and NTP.

[0225] The term "cell transfection" refers to a technology for introducing exogenous molecules such as DNA, RNA, etc. into eukaryotic cells using transfection reagents.

[0226] The term "base editor (BE)," refers to an agent that binds to a polynucleotide and has base modification activity. In some embodiments, a base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a protein (e.g., Cas9, Cas12, Cas13, Cas14, FoKI) containing a polynucleotide-binding domain that recognizes a polynucleotide (e.g., a guide RNA and a ZFP, TALE recognizes nucleotide units) and binds thereto. In some embodiments, the domain with base editing activity is capable of deaminating a base within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating a cytosine (C) or an adenosine (A) within a DNA. In some embodiments, the base editor is a TAM editor.

[0227] The term "base editing activity" refers to an activity for chemically altering a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is a cytidine deaminase activity, e.g., converting a target C G to T A. In another embodiment, the base editing activity is an adenosine or adenine deaminase activity, e.g., converting a target A T to G C. In another embodiment, the base editing activity is a cytidine deaminase activity, e.g., converting a target C G to T A, and an adenosine or adenine deaminase activity, e.g., converting A T to G C.

[0228] The term "gRNA," also known as a guide RNA, crRNA, or guide sequence, generally a guide RNA can comprise or consist essentially of or consist of a direct repeat and a guide sequence. In some cases, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of a CRISPR / Cas complex to the target sequence. When optimally aligned, the degree of complementarity between a guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0229] SA / SD(x, y)

[0230] In the mini-gene constructed in the present application, the splicing acceptor site (SA) refers to a sequence connected with the 5' end of an exon and an intron, wherein the first position of the intron is defined as -1, and the first position of the exon is defined as +1. The splicing donor site (SD) refers to a sequence connected with the 3' end of an exon, wherein the first position of the intron is defined as +1, and the last position of the exon is defined as -1. As shown in Figure 1.

[0231] For example, SA(-1) refers to the first base of the intron before the splicing acceptor (SA) of the exon in the mini-gene.

[0232] For example, SA(-1, +9) refers to the first base of the intron before the splicing acceptor (SA) of the exon in the mini-gene, and the 9th base of the exon after the splicing acceptor (SA) of the exon.

[0233] For example, SA(-1, +1, +9, +10) refers to the first base of the intron before the splicing acceptor (SA) of the exon in the mini-gene, and the 1st, 9th and 10th bases of the exon after the splicing acceptor (SA) of the exon.

[0234] For example, SD(-1, +1) refers to the 1st base of the exon before the splicing donor (SD) of the exon in the mini-gene, and the 1st base of the intron after the splicing donor (SD) of the exon.

[0235] For example, SD(+2) refers to the 2nd base of the intron after the splicing donor (SD) of the exon in the mini-gene.

[0236] For example, SD(-6, -1, +1, +4, +5, +6) refers to the 6th and 1st bases of the exon before the splicing donor (SD) of the exon in the mini-gene, and the 1st, 4th, 5th and 6th bases of the intron after the splicing donor (SD) of the exon.

[0237] Gene editing system

[0238] The present application provides a gene editing system, which comprises the gRNA of the second aspect of the present application, and the gRNA can target the potential gene editing site obtained by the method of the first aspect of the present application.

[0239] In a preferred embodiment, the gene editing system further comprises a base editor.

[0240] The gene editing system of the present application can significantly (i) induce exon skipping; and / or (ii) prevent and / or treat DMD disease.

[0241] As the selection of different gene therapy, those skilled in the art can understand and select other possible conventional gene editing systems or small molecule drugs targeting the potential gene editing site or adjacent sequence obtained by screening of the present application, and achieve the purpose of RNA splicing regulation of the sequence of the present application.

[0242] In the sequence of the gRNA of the present application, SEQ ID NO. 111-117 is designed according to the sequence of the site to be edited, which causes mutation of the site to be edited by targeting the site to be edited, thereby producing slightly shortened but frame-corrected mRNA. This mRNA can produce functional Dystrophin protein when expressed, restore the normal function of muscle cells, and thus treat DMD disease.

[0243] In one embodiment, the gRNA is designed for E51-SA-1+11, E51-SA-s1, for E51-SA-1+1, E51-SA-s2, for E53-SA-1, SD-1+1+5, E53-1, E53-2, E53-3 and E53-4, the binding positions of which are shown in Figure 6, and the sequences are shown in SEQ ID NO. 111-116; another gRNA (SEQ ID NO. 117) is designed for E53-SA-1+3+7 (E53-S18).

[0244] The inventors have confirmed by experiments that the gRNA designed for the above-mentioned gene editing site has significant (i) induction of exon skipping; and / or (ii) prevention and / or treatment of DMD disease.

[0245] Pharmaceutical composition and administration method

[0246] In another aspect, the present application also provides a pharmaceutical composition comprising (a) a safe and effective amount of the gene editing system of the present application or the cell of the present application; and (b) a pharmaceutically acceptable carrier or excipient. The dose of the gene editing system of the present application is usually 10 micrograms-100 milligrams / dose, preferably 100-1000 micrograms / dose.

[0247] In a preferred embodiment, the concentration of the cell in the pharmaceutical composition is 1×10 3 -1×10 8 cells / Kg body weight, more preferably 1×10 5 -1×10 7about 0.01 mg / kg to 50 mg / kg, preferably 0.05 mg / kg to 10 mg / kg body weight of the gene editing system of the present application. In addition, the gene editing system of the present application can be used alone or in conjunction with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

[0248] In the present application, the pharmaceutical composition of the present application can be administered in the form of a cell therapy agent, for example, the gene editing system of the present application can be used to engineer cells, and then the engineered cells are administered to a subject in need thereof by intravenous injection. In certain embodiments, the gene editing system of the present application can be used to engineer cells in vivo or isolated cells. In certain embodiments, the cells can be muscle cells and neural cells such as AC16 cells, iPSC induced muscle cells, and the engineering can include gene editing (preferably mutation).

[0249] The pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. This term refers to such carriers that are nontoxic to the cell or tissue to which they are administered and do not interfere with the effectiveness of the biological activity of the therapeutic composition. These carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to, saline, buffered fluid, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0250] The pharmaceutically acceptable carrier in the therapeutic composition can contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances can be present in the carrier such as wetting or emulsifying agents, pH buffering substances, and the like.

[0251] Generally, the therapeutic composition can be prepared as injectables, either as liquid solutions or suspensions; or solid forms suitable for reconstitution into liquid solutions or suspensions prior to injection. Once the composition of the present application has been formulated, it can be administered by conventional routes, including, but not limited to, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated can be an animal; particularly a human.

[0252] When the pharmaceutical composition of the present application is used for actual treatment, various pharmaceutical compositions of different dosage forms can be used depending on the use. Preferably, it is an intravenous injection. These pharmaceutical compositions can be prepared by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the preparation process can be performed in a conventional manner depending on the dosage form.

[0253] The pharmaceutical composition of the present application can also be administered in the form of a sustained-release agent. For example, the gene editing system of the present application can be incorporated into a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methyl cellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, etc. can be exemplified, and preferably biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be exemplified.

[0254] In one embodiment, the pharmaceutical composition can include a buffer such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The pharmaceutical composition of the present application is preferably formulated for intravenous administration.

[0255] When the pharmaceutical composition of the present application is used for actual treatment, the dose of the gene editing system of the present application as an active ingredient can be reasonably determined depending on the body weight, age, sex, and degree of symptoms of each patient to be treated.

[0256] The main advantages of the present application include:

[0257] (1) The present application first discovered that based on the mini-gene, potential gene editing sites that can affect the skipping of all or part of the exon can be screened. With respect to these sites, appropriate gene editing tools can be used to edit the sites at the DNA level or the pre-mRNA level, thereby rapidly and accurately achieving exon skipping of the mutant DMD gene, restoring the reading frame of the mRNA, producing functional Dystrophin protein, restoring the normal function of muscle cells, and thereby treating DMD disease.

[0258] (2) This invention first obtains key site sequence information affecting exon jumping through mini-genes. Then, based on the site, different gRNAs are designed and appropriate editing tools are selected to perform site editing verification in different cells, including but not limited to muscle cells derived from human iPSCs, and gene editing drugs that can induce exon jumping are screened.

[0259] (3) This invention successfully screened out novel recombinant polIII promoters by arranging and replacing the elements of different types of polIII promoters. Compared with the parental promoters, these promoters have significantly enhanced activity and shorter length, which can not only effectively improve the working efficiency of gene editing systems, but also be more flexibly integrated into single-vector systems of gene editing tools.

[0260] To facilitate understanding of the invention, a more comprehensive description of the invention will be provided below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise specified, the cell lines listed in the context of this invention are cultured according to existing techniques. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0261] Unless otherwise specified, all materials and reagents used in the examples are commercially available products.

[0262] Example 1. Screening for sequences affecting exon skipping using DMD mini-genes

[0263] The present application simulates the splicing of DMD mRNA in cells through a mini gene, and then screens the key bases that can skip all or part of the exons through point mutation of the sequence related to the splicing region. In order to better simulate the splicing of dystrophin mRNA in muscle cells, the present application selects AC16 cell strain. AC16 is a human myocardial cell strain fused with SV40 (purchased from the Chinese Academy of Sciences Cell Library), which retains the nuclear DNA and mitochondrial DNA of primary myocardial cells, expresses myocardial cell-specific peptide hormones BNP, L-type calcium channel alpha 1C subunit and gap junction protein, and stops proliferation and can form multinucleated syncytia when grown in a medium without mitogen. In the mini gene constructed in the present application, the splicing acceptor site (SA) refers to a sequence connected to the 5' end of the exon and the intron, wherein the first position of the intron is defined as -1, and the first position of the exon is +1. The splicing donor site (SD) refers to a sequence connected to the 3' end of the exon, wherein the first position of the intron is defined as +1, and the last position of the exon is -1. As shown in Figure 1.

[0264] 1.1 Construction of mini gene plasmid

[0265] In order to study the DMD treatment hotspot exon region, the present application constructs two mini genes pGAT-DMDminiE43E50 and pGAT-DMDminiE49E56 by intercepting part of the exons of DMD and the introns of 300 bp before and after the exons. The sequence maps are shown in Figures 2A-2B.

[0266] 1.2 Construction of splicing mutation mini gene screening plasmid

[0267] The present application constructs different point mutation mini genes for exons 44, 45, 48, 51, 53 and 54 of DMD to study the related sites that affect splicing of each exon. According to the instructions of the rapid point mutation kit (Tiangen, KM101), different point mutation plasmids are constructed. Specifically, point mutation primers are designed, and then the mini gene plasmids pGAT-DMDminiE43E50 and pGAT-DMDminiE49E56 are used as templates for PCR loop amplification to obtain each point mutation mini gene. The loop amplification PCR reaction system is shown in Table 1, the point mutation primer sequences are shown in SEQ ID NO. 1-100, and the double-end mutation mini gene is mutated at the corresponding sites using two pairs of primers.

[0268] Table 1. Loop amplification PCR reaction system

[0269] 1.3 Screening of sites affecting splicing of DMD exons in AC16 cells through point mutation mini gene

[0270] Mutant plasmids of different exon splicing sites were transfected into human myocardial cell line AC16 cells to simulate the transcription and splicing process, and the key sites affecting the splicing of each exon were studied.

[0271] 1.3.1 Passage and culture of AC16

[0272] Take the 100mm culture dish for example. Discard the culture medium, slowly add 3mL D-PBS along the wall of the culture dish and shake gently. Discard the D-PBS and add 1mL trypsin containing EDTA, and digest for 1 minute at room temperature. Terminate the reaction with DMEM-F12 complete medium containing 10% FBS, 1% Penicillin-Streptomycin (Gibco, C11330500BT), collect the cells, centrifuge at 1200rpm for 5 minutes, discard the supernatant and resuspend with 4mL complete medium, and count under a microscope. 1x10 6 AC16 cells were plated in a 100mm culture dish containing 10mL complete medium, and cultured in a 37°C cell incubator for 3 to 4 days, and the next passage was performed when the cell confluence was 80%-90%. The AC16 cells used in the following examples were between P8-P15.

[0273] 1.3.2 Mini-gene plasmid transfection of AC16 cells

[0274] The day before transfection, normally passage the AC16 cells, and plate 5x10 5 cells / mL of medium in each well of a 6-well plate, and place in the incubator for culture. About 24h later, when the cell density reaches about 70%, use JetPRIME transfection reagent (Polyplus, 101000046) for plasmid transfection. Dilute 2μg of the target plasmid with 200μL of jetPRIME buffer, and mix well by vortexing, then add 6μL of jetPRIME reagent and mix well. Incubate at room temperature for 10 minutes, then add the plasmid-reagent complex to the AC16 cells. After 24 hours of transfection, discard the culture medium and add 3mL of DMEM-F12 complete medium. After 48 hours, collect the cells for analysis.

[0275] 1.3.3 Analysis of splicing of each exon

[0276] Total RNA was extracted from cells according to the E.Z.N.A. HP Total RNA Kit (Omega bio-tek, R6812) kit instructions, and the next step was performed after passing the test. Reverse transcription PCR was performed according to the PrimeScript RT reagent Kit with gDNA Eraser (TAKARA, RR047A) kit instructions, and the reaction system is shown in Table 2. According to the TakaRa Ex PremierTM DNA Polymerase Dye plus (TAKARA, RR371A) kit instructions, different primers were used to PCR amplify the related transcription products, and the primer sequences are shown in SEQ ID NO. 101-110, and the reaction system is shown in Table 3. If there is a visible electrophoresis band, the jumping ratio is obtained by ImageJ software gray scale analysis. Since small fragment jumping cannot be effectively distinguished by electrophoresis, all bands on the electrophoresis are further confirmed by sanger sequencing to determine the type of small jumping band. The electrophoresis results and quantitative analysis are shown in Figure 3, and the electrophoresis band sequencing results are shown in Figure 4.

[0277] Table 2. Total RNA reverse transcription

[0278] Table 3. PCR reaction system

[0279] Result analysis:

[0280] Since DMD exons E45, E51, E53, and E54 are 3n+2 nucleotides, jumping E45, E51, E53, and E54 or 3n+2 nucleotides can effectively restore the reading frame of the DMD gene and achieve the effect of restoring Dystrophin protein expression. Similarly, exon E44 is 3n+1 nucleotides, and jumping E44 or 3n+1 nucleotides can have the same therapeutic effect. The present application determines the key bases that can affect exon skipping by point mutation of the sequence related to the splicing region of each exon, and the mutation site and type are shown in Table 4.

[0281] In this embodiment, the jumping proportion of the electrophoretogram of multiple band samples in the same lane is obtained by gray scale analysis of ImageJ software, each band sample is recovered by gel recovery, Sanger sequencing is performed, the results are analyzed by EditR (https: / / moriaritylab.shinyapps.io / editr_v10 / ), and the band jumping proportion is the average peak fitting proportion of the first 5 bases. The site with jumping is a potential available site. In order to avoid the situation that small fragment jumping and non-jumping fragments cannot be distinguished after editing by electrophoresis, Sanger sequencing is also performed after all fragments are recovered by gel recovery, and further confirmation is performed.

[0282] Figure 3A shows that E45-SD+1 and E45-SA-1 do not show visible jumps on the electropherogram, Sanger sequencing shows that the mutation at the E45-SD+1 position can induce a 32 bp 3' end jump of E45, and the mutation at the E45-SA-1 position can induce a 1 bp 5' end jump of E45, the band jump type and ratio are shown in Figure 3B. Figure 3C shows that E45-SA-1+9 does not show visible jumps on the electropherogram. E45-SA-1+1+9+10 and E45-SA-1+1+9+10, SD-1+1 show visible jump bands, the jump type and ratio are shown in Figure 3D. Sanger sequencing analysis of each band shows that the mutation at the E45-SA-1+9 position can induce a 1 bp 5' end jump of E45, and the mutation at the E45-SA-1+1+9+10 and E45-SA-1+1+9+10, SD-1+1 position can induce the entire E45 jump (Figure 3C bands 4-5), the band jump type and ratio are shown in Figure 3E. Figure 3F (bands 1-4) shows that E45-SA-1+1, E45-SA-2 and E45-SD+2 do not show visible jumps on the electropherogram. Sanger sequencing analysis of each band after gel recovery shows that the mutation at the E45-SA-1+1 and E45-SA-2 position can induce a 9 bp 5' end jump of E45, and the mutation at the E45-SD+2 position can induce a 32 bp 3' end jump of E45, the band jump type and ratio are shown in Figure 3G (bands 1-4). Figure 3F (bands 5-7) shows that E51-SD+2 does not show visible jumps on the electropherogram. Sanger sequencing analysis of each band after gel recovery shows that the mutation at the E51-SD+2 position can induce a 138 bp 3' end jump of E45, the band jump type and ratio are shown in Figure 3G (bands 5-7). Figure 3H (bands 1-3) shows that E45-SA-1+1+9+10+13+14 shows visible jump bands on the electropherogram, the jump type and ratio are shown in Figure 31. Sanger sequencing analysis of each band shows that the mutation at the E45-SA-1+1+9+10+13+14 position can induce the entire E45 jump (Figure 3H band 2), the band jump type and ratio are shown in Figure 3J (bands 1-3). Figure 3K shows that E51-SD-1+1 shows visible jump bands on the electropherogram, and E51-SA-1+1 does not show jump bands. Sanger sequencing analysis of each band shows that the mutation at the E51-SD-1+1 position can induce a 138 bp 3' end jump of E51, and the mutation at the E51-SA-1+1 position can induce an 11 bp 5' end jump of E51, the band jump type and ratio are shown in Figure 3L. Figure 3M shows that E51-SA-1+11, SD-1+1 shows visible jump bands on the electropherogram, the jump type and ratio are shown in Figure 3N.E51-SA-1+11 No jumping bands were observed, each band was analyzed by Sanger sequencing, mutations at E51-SA-1+11, SD-1+1 positions can induce the whole E51 jumping (Figure 3M, band 4) and partial E51 5’ end jumping 47bp, mutations at E51-SA-1+11 positions can induce E51 5’ end jumping 47bp, band jumping types and ratios are shown in Figure 30. Figure 3Q (bands 2-3, 9-10) shows that E51-SD+1+5+7 and E51-SD+2 jumping bands were observed on the electropherogram, each band was analyzed by Sanger sequencing, mutations at E51-SD+1+5+7 and E51-SD+2 positions can induce E51 3’ end jumping 138bp, band jumping types and ratios are shown in Figure 3S (bands 2-3, 9-10). Figure 3Q (bands 4-8, 11) shows that E53-SA-1, SD+5 and E53-SA-1, SD-1+1+5 jumping bands were observed on the electropherogram, jumping ratios are shown in Figure 3R. Each band was analyzed by Sanger sequencing, mutations at E53-SA-1, SD+5 and E53-SA-1, SD-1+1+5 positions can induce the whole E51 jumping (Figure 3Q, bands 7-8). Mutations at E53-SA-1 and E53-SA-1, SD+4 positions can induce E53 5’ end jumping 7bp, band jumping types and ratios are shown in Figure 3S (bands 4-8, 11). Figure 3T shows that E53-SA-1+3+7 and E53-SA-2+4+5+6 jumping bands were not observed on the electropherogram. Each band was analyzed by Sanger sequencing, mutations at E53-SA-1+3+7 and E53-SA-2+4+5+6 positions can induce E53 5’ end jumping 14bp, band jumping types and ratios are shown in Figure 3U. Figure 3W shows that mutations at E53-SA-2 positions can induce E53 5’ end jumping 7bp, mutations at E53-SD+1 positions can induce E53 3’ end jumping 90bp, band jumping types and ratios are shown in Figure 3X. Figure 3Y shows that E53-SA-1+3+7, SD-1+1 jumping bands were observed on the electropherogram, band jumping types and ratios are shown in Figure 3Z. Each band was analyzed by Sanger sequencing, mutations at E53-SA-1+3+7, SD-1+1 positions can induce the whole E53 jumping (Figure 3Z, band 2), band jumping types and ratios are shown in Figure 3AA. Figure 3AB shows that E53-SA-1+3+7+14, E53-SA-1+3+7+14+20 and E53-SD-1+1+5 jumping bands were not observed on the electropherogram.Each band was analyzed by Sanger sequencing, mutations at E53-SA-1+3+7+14 position can induce 20 bp skipping at 5' end of E53, mutations at E53-SA-1+3+7+14+20 position can induce 30 bp skipping at 5' end of E53, mutations at E53-SD-1+1+5 position can induce 90 bp skipping at 3' end of E53, band skipping type and ratio are shown in Figure 3AC. Figure 3AD shows that E48-SD+1 and E48-SD-6-1+1+4+5+6 can induce 78 bp skipping at 3' end of E47 and whole E48, band skipping type and ratio are shown in Figure 3AE. Figure 3AF shows that E54-SD+1 can induce whole E54 skipping, band skipping type and ratio are shown in Figure 3AG. Figure 3AI shows that E44-SA-4-1+1+3+8+12, E44-SD-1+1, E44-SD-1+1+5, E44-SD-11-10-1+1+5 can induce whole E44 skipping, band skipping type and ratio are shown in Figure 3AJ. Each band was analyzed by Sanger sequencing, mutations at E44-SA-1+1 and E44-SA-4-1+1+3 position can induce 12 bp skipping at 5' end of E44, mutations at E44-SA-4-1+1+3+8+12, E44-SD-1+1, E44-SD-1+1+5 and E44-SD-11-10-1+1+5 position can induce whole E44 skipping (Figure 3AI bands 4, 7, 9-10), band skipping type and ratio are shown in Figure 3AK. Since the mini-genes cannot completely mimic the full-length DMD gene, due to the complexity of splicing system and splicing process, not all mutations of mini-genes can lead to effective skipping at mini-gene level, but it does not mean that editing corresponding sites of DMD gene in muscle cells will not lead to skipping.

[0283] Table 4. Results of point mutation mini-gene exon skipping (for sense strand)

[0284] Conclusion: Through the screening system of mini-genes of DMD, key sites that can induce skipping and partial skipping of exons E45, E51, E53, E48, E54 and E44 of DMD were successfully screened.

[0285] Example 2 Detection of exon skipping of each exon by BE editing mini-genes

[0286] This embodiment designs targeted gRNA according to the key sites screened in Example 1 affecting the splicing of E51 and E53, and then co-transfected into myocardial cells AC16 cells with base editor (BE) and mini-genes to detect the mRNA splicing of the edited mini-genes, in order to verify the reliability of the screening results of the mini-genes and the effectiveness of the designed gRNA. The BE used here contains cytosine deaminase AIDx, KKH-SaCas9 and uracil glycosylase inhibitor UGI to form TAM editor, the structure of which is shown in Figure 5, and the sequence is shown in SEQ ID NO. 122. The gRNA is designed near the DMD gene mutation site based on the PAM recognized by KKH-SaCas9. Specifically, E51-SA-s1 is designed according to E51-SA-1+11, E51-SA-s2 is designed according to E51-SA-1+1, and E53-1, E53-2, E53-3 and E53-4 are designed according to E53-SA-1, SD-1+1+5, the binding positions of which are shown in Figure 6, and the gRNA sequences are shown in SEQ ID NO. 111-116.

[0287] 2.1 Passage and culture of AC16 The passage and culture of AC16 were as described in Example 1.

[0288] 2.2 Transfection of mini-gene plasmid in AC16 cells

[0289] The day before transfection, the AC16 cells were normally passaged once, and 5x10 5 cells / mL of medium were plated in each well of a 6-well plate and incubated in an incubator; about 24 h later, when the cell density reached about 70%, plasmid transfection was performed using JetPRIME transfection reagent (Polyplus, 101000046). The gRNA plasmid and TAM plasmid were prepared according to the instructions of the endotoxin-free plasmid extraction kit (Tiangen, DP118). 0.02 μg of mini-gene, 1.0 μg of gRNA plasmid and 1.0 μg of TAM plasmid were diluted with 200 μL of jetPRIME buffer, and vortexed thoroughly. Then 6.1 μL of jetPRIME reagent was added and mixed thoroughly. After incubation at room temperature for 10 minutes, the plasmid-reagent complex was added to the AC16 cells. After 24 hours of transfection, the culture medium was discarded and 3 mL of DMEM-F12 complete medium (Gibco, C11330500BT) was added. After 72 hours of transfection, the cells were collected for analysis.

[0290] 2.3 Analysis of splicing of each exon

[0291] The reaction system is shown in Table 2 and Table 3, the primer sequence is shown in SEQ ID NO. 103-106, the electrophoresis result is shown in Figure 7, and the electrophoresis band sequencing result is shown in Figure 8, as described in Example 1.

[0292] Result analysis: Figure 7A shows that the products edited by gRNA E51-SA-s1 and E51-SA-s2 cannot be observed in the electrophoretogram. After gel recovery, Sanger sequencing is performed, E51-SA-s1 can induce E51 5' end to jump 47bp, and E51-SA-s2 can induce E51 5' end to jump 11bp, and the band jump type and ratio are shown in Figure 7B. Figure 7C shows that the products edited by gRNA E53-1, E53-1 / 3, E53-1 / 4 and E53-2 / 4 can be observed in the electrophoretogram, and the jump ratio is shown in Figure 7D. Each band is analyzed by sanger sequencing, and the whole E53 can be induced to jump after being edited by gRNA E53-1, E53-1 / 3, E53-1 / 4 and E53-2 / 4 (Figure 7C bands 3, 8, 10, 13), and the band jump type and ratio are shown in Figure 7E.

[0293] Conclusion: Through the mini-gene screening system of DMD, the gRNA and base editor combination used for E51 and E53 exon skipping are successfully screened.

[0294] Example 3: Gene editing verification of gRNA effect on DMD gene splicing on human induced pluripotent stem cells iPSC

[0295] In this embodiment, the combination of TAM editor and gRNA is used to edit the splicing related sites screened by iPSC induced skeletal muscle, and the effect of gRNA on skipping the corresponding DMD exon is verified. The mCherry and EGFP tags are added to the synthesized pGAT-3xgRNA and pGAT-BE backbone plasmids respectively, and then the double fluorescent cells are screened by FACS to ensure that the gRNA and BE are co-transfected into the same cell.

[0296] 3.1 Flow cytometry sorting double fluorescent screening plasmid construction

[0297] The plasmid for muscle cell transfection in the present application has a fluorescent tag, wherein the gRNA vector pGAT-3xgRNA-mCherry contains three gRNA insertion sites and has an mCherry fluorescent tag, and the structural diagram is shown in Figure 9A. The same gRNA can be inserted into this plasmid by using BbsI, BsmbI and PaqCI three enzyme cutting sites to construct the gRNA expression plasmid, and the gRNA sequence for each exon is shown in SEQ ID NO. 111 and SEQ ID NO. 117.

[0298] SEQ ID NO. 117 is designed based on SEQ ID NO. 27 (E53-SA-1+3+7 point mutations are introduced).

[0299] The vector pGAT-BE-EGFP expressing BE protein carries an EGFP fluorescent tag, and its structure map is shown in FIG. 9B.

[0300] 3.2 IPS cell culture and passage

[0301] Take the 6-well plate passage as an example. The Matrigel coated 6-well plate (coating concentration is 0.013 mg / cm 2 ), is placed in the biosafety cabinet about 1 hour in advance to recover to room temperature; when the confluence of the clone group is 85%, the iPSC hole culture medium is aspirated, 2 mL / hole of DPBS is added, and it is gently shaken and aspirated. Add 2 mL / hole of Nuwacell EDTA passage working solution to completely cover the bottom of the hole, and incubate in a 37°C incubator for 8 min. After digestion, gently take the cell culture plate back to the biosafety cabinet to avoid shaking and shaking the cells, and tilt and aspirate. Timely add 2 mL / hole of pre-warmed Blebbistatin (Nuwacell, RP01008) and ncTarget complete culture medium (Nuwacell, RP01020), and horizontally cross-shake the 6-well plate to make the cells detach from the matrix, and pass at a ratio of 1:12. 0.166 mL of cells are inoculated into a new Matrigel coated 6-well plate, and 2 mL of pre-warmed Blebbistatin+ncTarget complete culture medium is added to the hole.

[0302] 3.3 Skeletal muscle cell induction

[0303] The method of inducing iPSC cells (Zhongsheng traceable origin) into skeletal muscle cells is carried out according to the Skeletal Muscle Differentiation Kit instructions of Genea Biocells company.

[0304] 3.4 SKM cell plasmid transfection

[0305] Transfection was performed in the first 24 hours of myoblast induced differentiation into myotubes. 2 pg of plasmid of interest and 4 pL P3000 were diluted with 125 pL of serum-free medium Opti-MEM (Gibco, 31985.070), 6 pL Lipo3000 were diluted with 125 pL of serum-free medium Opti-MEM and mixed well. The plasmid and Lipo3000 diluted with Opti-MEM were mixed immediately and incubated at room temperature for 15 minutes. The plasmid-liposome complex was added to the cells. After 24 hours of transfection, the culture medium was discarded and 2 mL of SKM-03 induction medium containing 1% Penicillin-Streptomycin (MYOCEA, SKM-03) was added; after 7 days of transfection, the cells were harvested for analysis. EGFP and mCherry double-fluorescent positive cells were screened by flow sorting, and the sorted cells were subjected to relevant detection.

[0306] 3.5 gRNA induced target gene exon skipping efficiency analysis

[0307] The cells successfully transfected with plasmid were enriched by flow sorting through a double-fluorescent system, in which P3 was strong double-positive and P5 was weak double-positive. The results of flow sorting are shown in FIG. 10. The total RNA of the cells was extracted according to the E.Z.N.A. HP Total RNA Kit (Omega bio-tek, R6812) kit instructions, and after passing the test, the next step of reverse transcription was performed. The reverse transcription system is shown in Table 2, the PCR system and reaction conditions are shown in Table 3, the primer sequences used for each exon detection are shown in SEQ ID NO. 118-121, the electrophoresis results are shown in FIG. 11 and Table 5, and the electrophoresis band sequencing results are shown in FIG. 12.

[0308] Result analysis: FIG. 11A (bands 1-3) shows that the product edited by gRNA E51-SA-s1 cannot be observed in the electrophoretogram. After gel recovery, sanger sequencing was performed, E51-SA-s1 can induce E51 5' end to skip 47bp or 11bp two results, and the band skipping type and ratio are shown in FIG. 11C (bands 1-3). FIG. 11A (bands 4-8) shows that the product edited by gRNA E53-S18 can be observed in the electrophoretogram, and the skipping ratio is 52%, as shown in FIG. 11B. After sanger sequencing analysis, the whole E53 can be induced to skip after editing by gRNA E53-S18 (FIG. 11A bands 6, 8), and the band skipping type and ratio are shown in FIG. 11C (bands 4-8).

[0309] Table 5. DMD exon skipping results of iPSC induced muscle cells induced by E51 and E53 gRNA combined with BE

[0310] Conclusion: Both E51-SA-s1 and E53-S18 gRNAs can induce exon skipping of E51 and E53 respectively in iPSC induced skeletal muscle cells by co-transfecting with BE plasmid, and achieve the purpose of restoring the reading frame of pathogenic DMD gene at the mRNA level. It is proved that the sites affecting exon skipping screened by mini-gene can achieve exon skipping on the genome of cells after mutation by base editor.

[0311] Example 4. Verification of the effect of gRNA on DMD gene splicing by gene editing of defective human induced pluripotent stem cells iPSC using adeno-associated virus

[0312] In order to realize the effective conversion to human Duchenne muscular dystrophy (DMD) treatment, this embodiment uses adeno-associated virus (AAV) as a delivery vector, carries TAM base editor and gRNA, and verifies the effect of gRNA on DMD gene splicing in skeletal muscle cells differentiated from DMD exon 52 deleted induced pluripotent stem cells (iPSC). Since in the mRNA splicing process, the 5' splice site (5' SS) or the 3' splice site (3' SS) usually plays a dominant role, and under the condition of weak splicing site, exon splicing enhancer (ESE) or intron splicing enhancer (ISE) site will also cause the change of splicing product, based on the ESE finder analysis result, the gRNA E53-S26 is designed for the skipping of DMD gene exon 53, and the nucleotide sequence is shown in SEQ ID NO. 175 of the sequence table.

[0313] 4.1 AAV virus packaging

[0314] AAV9-TAM, AAV9-E53-S18, AAV9-E53-2, AAV9-E53-4, AAV9-E53-S26, RC plasmid and helper plasmid for AAV virus packaging were constructed and entrusted to Suzhou Jinyu Zhi for synthesis. The same process flow was used for three-plasmid virus preparation, which is summarized as follows. Three-plasmid transfection was used to suspend VPC2.0 cells (ThermoFisher, Cat. No. A49784), and the cells were harvested 72 hours after transfection. After lysis, clarification filtration, affinity chromatography and ultrafiltration concentration, AAV virus samples were obtained. AAV virus sample titer detection was performed by qPCR.

[0315] 4.2 IPS cell culture and passage and skeletal muscle cell induction as described in Example 3.

[0316] 4.3 AAV infection of iPSC-induced skeletal muscle

[0317] When the myoblast cells in the 24-well plate were differentiated into skeletal muscle cells for 5 days, they were digested with 500 μL of 0.05% trypsin containing EDTA at 37°C for 2 minutes. The cells were resuspended using 500 μL of 1640 medium (Gibco, 11875-093) containing 10% FBS, 1% Penicillin-Streptomycin, and centrifuged at 1200 rpm for 5 min. The culture supernatant was aspirated, and the cells were resuspended in 500 μL of D-PBS. A live cell count was performed. The AAV concentration and infection volume were adjusted with 250 μL of serum-free DMEM high-glucose basal medium (Cytiva, SH30243.01) so that the ratio of AAV9-TAM and AAV9-E53-S18 was 1:3, the ratio of AAV9-TAM and AAV9-E53-S26 was 1:3, and the ratio of AAV9-TAM, AAV9-E53-2, and AAV9-E53-4 was 1:1.5:1.5, and the final infection of skeletal muscle virus MOI was 1E6 or 3E6. The adjusted concentration of AAV was added to the cells and transferred to a 24-well culture plate. After 4 hours, 500 μL of fresh SKM03 medium (MYOCEA, SKM-03) containing 1% Penicillin-Streptomycin was added to each 24-well cell. After 24 hours, the skeletal muscle cells in the 24-well plate were washed with 500 μl of D-PBS, and 500 μl of SKM03 medium containing 1% Penicillin-Streptomycin was added to each well, and the medium was changed every 2-3 days thereafter. After 7 days, the infected cell samples were harvested for related detection.

[0318] 4.3 gRNA-induced target gene exon skipping efficiency analysis

[0319] Total RNA was extracted from the cells according to the E.Z.N.A. HP Total RNA Kit (Omega bio-tek, R6812) kit instructions, and after passing the test, the next step was reverse transcription. The reverse transcription system is shown in Table 2, the PCR system and reaction conditions are shown in Table 3, the exon skipping detection primer sequences are shown in SEQ ID NO. 125-126, the electrophoresis results are shown in FIG. 13, and the electrophoresis band sequencing results are shown in FIG. 14.

[0320] Results analysis: Figure 13A shows that the products after combination editing with AAV9-TAM / AAV9-E53-S18 or AAV9-TAM / AAV9-E53-2 / AAV9-E53-4 can be observed in the electrophoretogram, and the skipping ratio is shown in Figure 13B. After recovering the gel of each band, Sanger sequencing was performed, and the whole E53 skipping can be induced after two kinds of combination editing (Figure 13A bands 2, 4, 6, 8), and the band skipping type and ratio are shown in Figure 13C. Figure 13D shows that the products after combination editing with AAV9-TAM / AAV9-E53-S26 can be observed in the electrophoretogram, and the skipping ratio is shown in Figure 13E. After recovering the gel of each band, Sanger sequencing was performed, and the results showed that E53-S26 can induce the whole E53 skipping (Figure 13D bands 2, 4), and the band skipping type and ratio are shown in Figure 13F.

[0321] Conclusion: E53-S18, E53-S26 and E53-2 / 4 combination can all induce DMD exon 52 deletion skeletal muscle to skip exon 53 under the delivery system of AAV, and restore the whole DMD reading frame at the mRNA level.

[0322] Example 5 Verification of gRNA affecting DMD by gene editing on DMD model mice using adeno-associated virus

[0323] Effect of gene splicing

[0324] This example uses adeno-associated virus AAV to deliver TAM base editor and gRNA E53-S26 to verify the effect of gRNA affecting DMD gene splicing in DmddelE52hE53 model mice again. The DmddelE52hE53 mouse model was prepared by Guangzhou Mingxun Biotechnology Co., Ltd. First, the mouse embryonic stem cells (C57BL / 6N background) were genetically targeted and modified, including replacing the mouse Dmd gene exon 53 (E53) and its flanking intron sequence (about 1400+454bp) with humanized sequence, knocking out mouse Dmd gene exon 52 and intron 52 synchronously by homologous recombination, and inserting PGK-BSD selection marker gene; then the FLP-FRT system was used to delete the PGK-BSD selection marker gene. Finally, the targeted modified embryonic stem cells were used to generate DmddelE52hE53 mice by TurboMice technology (Beijing Bocon Biotechnology Co., Ltd.). TM Technically cultivate humanized mouse model DmddelE52hE53. All newborn mice are derived from the same clone, and the genetic structure is shown in Figure 15.

[0325] 6.1 Packaging operation of AAV virus as described in Example 4

[0326] 6.2 Intravenous administration of AAV to DMD humanized mice

[0327] AAV9-TAM and AAV9-E53-S26 (genome ratio of 1:3, total dose of 5E13 vg / kg) were administered by tail vein injection to 3-week-old DmddelE52hE53 model mice. Heart tissue collection was performed 13 weeks after administration for DNA editing efficiency, exon skipping efficiency and dystrophin protein expression analysis.

[0328] 6.3 DNA editing efficiency, exon skipping efficiency and dystrophin protein expression analysis.

[0329] DNA of cells was extracted according to the instructions of the tissue genomic DNA extraction kit (TIANGEN, DP304) for amplicon sequencing analysis, and the primer sequences are shown in SEQ ID NO. 176-177, and the PCR reaction is shown in Table 3. RNA was extracted by Trizol-chloroform extraction method, and reverse transcription was performed according to the reaction of Table 2, and then fluorescence quantitative PCR was performed. The reaction of fluorescence quantitative PCR is shown in Table 6, and the primer sequences are shown in SEQ ID NO. 178-183. The E53 skipping efficiency was obtained by calculating the ratio of E53 skipping mRNA to total mRNA. Western Blot analysis was performed after the tissue was treated with RIPA protein lysis buffer (Bi Yun Tian, P0013B) (primary anti-Dystrophin mouse monoclonal antibody, D8168, Sigma; primary anti-vinculin rabbit monoclonal antibody, ab129002, Abeam). The editing efficiency of the target gene is shown in Figure 16, the exon skipping efficiency is shown in Figure 17, and the dystrophin protein expression is shown in Figure 18.

[0330] Table 6. Fluorescence quantitative PCR reaction system

[0331] Result analysis: Figure 16 shows that after intravenous delivery of DMD model mice for 13 weeks, the AAV9-TAM / AAV9-E53-S26 combination can induce about 9.46% target gene editing in heart tissue. Figure 17 shows that after editing of the target point, 29.8% of the exons produce E53 skipping. Figure 18A shows that the dystrophin protein expression of AAV9-TAM / AAV9-E53-S26 treated mice is restored to about 10% of the wild type mouse level, and the quantitative results are shown in Figure 18B.

[0332] Conclusion: In the AAV delivery system, AAV9-TAM combined with AAV9-E53-S26 can successfully edit the E53 target region of the DMD gene in the myocardium of model mice, induce E53 skipping of mRNA, restore the reading frame of the entire DMD gene, and then successfully restore the expression of dystrophin protein.

[0333] Example 6 Verification of gRNAs that can edit DMD exons by HEK293T cells

[0334] According to the rules of mini-gene screening, editing multiple bases of SA and SD can achieve DMD exon skipping. TAM BE designed sgRNAs targeting other exons in order to obtain effective editing and possibly obtain effective targeted exon skipping.

[0335] The method described in Reference Example 1 and Example 2 was used to design additional gRNAs targeting exon 6, exon 7, exon 16, exon 46 and exon 52 of the human DMD gene, and the editing effects of these gRNAs were verified in HEK293T cells. The sequences of gRNAs E6-2, E7-1, E7-2, E7-3, E7-4, E16-1, E16-2, E16-3, E46-1 and E52-1 are shown in SEQ ID NO. 127-136.

[0336] 5.1 Passage and culture of HEK293T cells

[0337] For example, when the cell confluence reaches 80-90%, discard the culture medium, slowly add 3 mL of D-PBS along the wall of the culture dish, and gently shake it. Discard the D-PBS and add 1 mL of trypsin containing EDTA, and incubate at 37°C in a carbon dioxide incubator for 3 minutes. Stop the reaction with 3 mL of DMEM complete medium containing 10% FBS and 1% Penicillin-Streptomycin (Cytiva, SH30243.01), collect the cells, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend with 1 mL of complete medium, and count under a microscope. 1x10 6 HEK293T cells were plated in a 100mm culture dish containing 10 mL of complete medium, cultured in a 37°C cell incubator for 2 to 3 days, and the next passage was performed when the cell confluence was 80-90%. The HEK293T cells used in the following examples were cells with a passage number of P8-P15.

[0338] 5.2 Plasmid transfection of HEK293T cells

[0339] One day before transfection, HEK293T cells were passaged once and plated at 6x10 5 cells per 1 mL of medium in each well of a 12-well plate and incubated in a CO2 incubator. About 24 h later, when the cell density reached about 70%, plasmid transfection was performed using PEI Max transfection reagent (Polyscience, 24765-100). 100 μL of serum-free medium Opti-MEM was used to dilute 1 μg of gRNA plasmid, while 8 μL of PEI was used to dilute 1 μg of oTAM plasmid. The two diluted plasmids were then mixed well and incubated at room temperature for 15 min. The plasmid-PEI complex was then added to the HEK293T cells. After 24 h of transfection, the medium was discarded and 1 mL of DMEM complete medium was added. After 72 h of transfection, the genomic DNA of the cells was extracted for analysis.

[0340] 5. Editing efficiency of gRNA on target genes

[0341] Genomic DNA of the cells was extracted according to the instructions of the TIANamp Genomic DNA kit (TIANGEN, DP304-03), and after passing the test, PCR amplification was performed. The PCR system and reaction conditions are shown in Table 3, and the forward (F) and reverse (R) primer sequences for each exon detection are shown in SEQ ID NO. 137-146. The PCR products were subjected to Sanger sequencing, and the editing efficiency of each sample was analyzed by EditR 1.0.10. The results are shown in Figure 19.

[0342] Results analysis: Compared with the control group, each gRNA had effective editing on the corresponding target. The highest point editing efficiency of gRNA6-2, gRNA7-4, gRNA16-2, gRNA46-1, and gRNA52-1 was more than 25%.

[0343] Conclusion: gRNA6-2, gRNA7-1, gRNA7-2, gRNA7-3, gRNA7-4, gRNA16-1, gRNA16-2, gRNA16-3, gRNA4-16, and gRNA52-1 can successfully edit the corresponding regions, disrupt the splicing sites of the corresponding DMD exons, and according to the splicing mechanism, it is likely to cause effective target exon skipping.

[0344] Example 7: Optimization of gRNA promoter to optimize transfection and improve gene editing efficiency

[0345] The gene editing system is composed of two parts of Cas protein and gRNA, and the transcription level of gRNA directly determines the effect of gene editing. At present, the polIII promoters commonly used to initiate the transcription of gRNA mainly include U6, H1 and 7SK, among which the transcription ability of U6 is the most prominent. AAV is a commonly used delivery tool for gene therapy, and its transloading capacity is limited to 4.7 kb. Since the gene editing enzyme is large in size, in most cases, the editing enzyme and sgRNA cannot be delivered simultaneously by one vector, thereby the best editing efficiency cannot be obtained and the cost of the drug is increased. The polIII promoter commonly used for sgRNA is modified in the present application, aiming to improve the expression efficiency and shorten the sequence length, so as to better adapt to the construction of single-vector gene editing tool. According to the classification of promoter elements, as shown in FIG. 20, the elements are replaced and modified in the order of Oct, SPH, PSE, spacer, TATA box and DS. In the promoter activity detection link, the gRNA targets the-14~+6 region at the junction of the 50th exon and the 50th intron of the human DMD gene, and the specific target position is shown in FIG. 21, and the sequence of gRNA E50-1 is shown as SEQ ID NO. 147.

[0346] 6.1 Design and construction of different polIII promoters

[0347] Based on the structural analysis and literature research of polIII promoter, 21 mutants are designed and synthesized. The structure diagram of the constructed polIII promoter is shown in Table 7, and the sequences of U6 and the mutants are shown as SEQ ID NO. 148-169, wherein an additional G base is added between the promoter U6 and the gRNA to improve the transcription efficiency.

[0348] Table 7 Structure diagram of different polIII promoter mutants

[0349] 6.2 Screening of polIII promoter mutants

[0350] The subculture and culture of HEK293T, plasmid transfection, and the influence of different polIII promoter mutants on the editing efficiency of base editor are described in Example 5. The forward (F) and reverse (R) primer sequences for human DMD exon 50 editing detection are shown as SEQ ID NO. 170-171. The PCR products are subjected to Sanger sequencing, and the editing efficiency of each sample is analyzed by EditR1.0.10, and the results are shown in FIG. 22A, B and Table 8.

[0351] Table 8. Activity and length list of different polIII promoters

[0352] Results analysis: Fig. 22 and Table 8 show that the editing efficiency of P4, P5, P6, P7, P14 is increased by more than 10% compared with U6, P9 is increased by more than 15%, P8, P16, P17, P19, P24 is increased by more than 20%, and P20, P23 is increased by more than 30%. Except P5, all the promoters are shortened by more than 50% compared with U6.

[0353] Conclusion: The activity of promoters P4, P6, P7, P8, P9, P14, P16, P17, P19, P20, P23 and P24 is significantly higher than that of U6, and they show better adaptability in the application of single vector construction.

[0354] Gene sequence

[0355] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A method for screening potential gene editing sites affecting exon splicing based on mini-genes, characterized in that, include: Gene editing is performed on 1-20 (preferably 1-10, more preferably 1-5) base editing sites in introns before the splice acceptor (SA) of exons in mini-genes and / or 1-20 (preferably 1-10, more preferably 1-5) base editing sites in exons after the splice acceptor (SA) of exons. Gene editing is performed on 1-20 (preferably 1-10, more preferably 1-5) base editing sites in the exons before the splice donor (SD) and / or 1-20 (preferably 1-10, more preferably 1-5) base editing sites in the introns after the splice donor (SD) in mini-genes, thereby obtaining potential gene editing sites that affect exon splicing based on the proportion of exon skipping; The exons include exon 45 (E45), exon 51 (E51), exon 53 (E53), exon 48 (E48), exon 54 (E54), and / or exon 44 (E44).

2. The method as described in claim 1, characterized in that, When the proportion of exon skipping is ≥30%, it indicates that the edited site is a potential gene editing site that affects exon splicing.

3. The method as described in claim 1, characterized in that, The gene editing sites include one or more selected from the group consisting of: (a) One or more sites in exon 45 (E45) selected from the group consisting of: SA(-1), SA(-1,+9), SA(-1,+1,+9,+10), SA(-1,+1), SA(-2), SD(+1), SD(-1,+1), SD(+2), SA(-1,+1,+9,+10,+13,+14); (b) One or more sites in exon 51 selected from the following group: SD(+2), SA(-1,+1), SA(-1,+11), SD(-1,+1), SD(+1,+5,+7); (c) One or more sites in exon 53 selected from the following group: SA(-1), SD(+4), SD(+5), SD(-1, +1, +5), SA(-1, +3, +7), SA(-2, +4, +5, +6), SA(-2), SD(+1), SD(-1, +1), SA(-1, +3, +7, +14), SA(-1, +3, +7, +14, +20); (d) One or more sites in exon 48 selected from the following group: SD(+1), SD(-6, -1, +1, +4, +5, +6); (e) The following sites in exon 54: SD(+1); (f) One or more sites in exon 44 selected from the following group: SA(-1, +1), SA(-4, -1, +1, +3), SA(-4, -1, +1, +3, +8, +12), SD(-1, +1), SD(-1, +1, +5), SD(-11, -10, -1, +1, +5) 4. A gRNA that affects exon splicing, characterized in that, The gRNA targets potential gene editing sites obtained by the method of any one of claims 1-3.

5. The gRNA as described in claim 4, characterized in that, The target sequence of the gRNA is shown in any one of SEQ ID NO.111-117.

6. The gRNA as described in claim 4, characterized in that, The gRNA further comprises one or more target sequences selected from the group consisting of: SEQ ID NO.127-136.

7. A nucleic acid, characterized in that, The nucleic acid encodes the gRNA of claim 4, or a precursor of the gRNA of claim 4.

8. A carrier, characterized in that, The vector comprises the gRNA of claim 4 or the nucleic acid of claim 7.

9. The carrier as described in claim 8, characterized in that, The vector contains one or more promoters selected from the group consisting of: SEQ ID NO.149-169.

10. A gene editing system, characterized in that, The gene editing system includes the gRNA described in claim 2.

11. The gene editing system as described in claim 10, characterized in that, The gene editing system also contains one or more promoters selected from the group consisting of SEQ ID NO.149-169.

12. A cell, characterized in that, The cells were obtained by editing them using the gene editing system of claim 11.

13. A composition, characterized in that, It includes a protein component or an mRNA component encoding the protein component; and a gRNA component or an mRNA component encoding the gRNA component; wherein the gRNA or the mRNA component it encodes includes the gRNA of claim 4, and the protein component or the mRNA component encoding the protein component contains a base editor.

14. A composition, characterized in that, include: The system of claim 10 or the cell of claim 12; And pharmaceutically acceptable carriers.

15. A delivery composition, characterized in that, It comprises an active ingredient and a delivery medium, wherein the active ingredient comprises the gRNA of claim 4, the vector of claim 8, or the system of claim 10.

16. A host cell, characterized in that, The host cell comprises the gRNA of claim 4, the nucleic acid of claim 7, the vector of claim 8, the system of claim 10, the composition of claim 714, or the delivery composition of claim 15.

17. A CRISPR complex, characterized in that, The complex comprises the gRNA of claim 4, the base editor, and the potential gene editing site obtained by the method of claim 1.

18. A medicine box, characterized in that, include: A first container, and an active ingredient or a drug containing the active ingredient, the active ingredient or the drug being located in the first container, the active ingredient comprising the gRNA of claim 4, the nucleic acid of claim 7, the vector of claim 8, the system of claim 10, the cell of claim 12, the composition of any one of claim 13 or 14, or the delivery composition of claim 15, or the host cell of claim 16, or the CRISPR complex of claim 17.

19. A method for gene editing in cells, characterized in that, This includes contacting the cells with the gRNA of claim 4, the nucleic acid of claim 7, the vector of claim 8, the system of claim 10, the composition of any one of claim 13 or 14, or the delivery composition of claim 15, or the host cell of claim 16, or the CRISPR complex of claim 17, or the kit of claim 18.

20. A kit for gene editing, characterized in that, The kit comprises the gRNA of claim 4, the nucleic acid of claim 5, the vector of claim 7, the system of claim 10, the composition of any one of claims 13 or 14, or the delivery composition of claim 15, or the host cell of claim 16, or the CRISPR complex of claim 17, or the kit of claim 128.

21. The use of the gRNA of claim 4, the nucleic acid of claim 7, the vector of claim 8, the system of claim 10, the composition of any one of claims 13 or 14, or the delivery composition of claim 15, or the host cell of claim 16, or the CRISPR complex of claim 17, or the kit of claim 18, or the reagent kit of claim 20 in (i) influencing exon splicing, and / or (ii) in the prevention and / or treatment of DMD.

22. A gene editing system, characterized in that, The system targets potential gene editing sites obtained by the method of claim 1.

23. A gRNA that affects exon splicing, characterized in that, The sequence of the gRNA is shown in any one of SEQ ID NO.127-136.

24. A separate promoter, characterized in that, The sequence of the promoter is shown in any one of SEQ ID NO.149-169.

25. An expression box, characterized in that, The expression cassette comprises: the promoter of claim 24 and a target gene, wherein the target gene is operatively linked to the promoter.

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