CN109266648B

CN109266648B - Gene editing compositions or kits for in vivo gene therapy

Info

Publication number: CN109266648B
Application number: CN201811144025.3A
Authority: CN
Inventors: 薛天; 才源
Original assignee: University of Science and Technology of China USTC
Current assignee: Hefei Xingmou Biotechnology Co ltd
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2021-10-19
Anticipated expiration: 2038-09-26
Also published as: WO2020063178A1; CN109266648A

Abstract

本发明提供用于在体基因治疗的基因编辑组合物或试剂盒。本发明提供一种基因编辑组合物或试剂盒，其包含1)靶向目的突变基因的与结合蛋白的核酸分子连接的sgRNA或其编码序列，2)修复目的突变基因的模板核酸或其编码序列，3)sgRNA引导的核酸酶或其编码序列，4)与核酸结合蛋白融合的促进同源重组的蛋白或其编码序列，其中所述核酸结合蛋白能够与上述1)中的核酸分子结合。本发明还涉及所述sgRNA、组合物或试剂盒治疗基因突变导致的疾病如遗传性疾病的用途。本发明的sgRNA、组合物或试剂盒能够有效的提高同源重组修复的效率，并且在非分裂细胞内也能实现同源重组修复。The present invention provides gene editing compositions or kits for in vivo gene therapy. The present invention provides a gene editing composition or kit, which comprises 1) a sgRNA targeting a target mutant gene linked to a nucleic acid molecule of a binding protein or its coding sequence, 2) a template nucleic acid or its coding sequence for repairing the target mutant gene , 3) a sgRNA-guided nuclease or its coding sequence, 4) a homologous recombination-promoting protein or its coding sequence fused to a nucleic acid-binding protein, wherein the nucleic acid-binding protein is capable of binding to the nucleic acid molecule in 1) above. The present invention also relates to the use of the sgRNA, composition or kit for treating diseases caused by gene mutation, such as hereditary diseases. The sgRNA, composition or kit of the present invention can effectively improve the efficiency of homologous recombination repair, and can also realize homologous recombination repair in non-dividing cells.

Description

Gene editing composition or kit for in vivo gene therapy

Technical Field

The present invention relates to the field of molecular biological gene editing. Specifically, the present invention provides a gene editing composition for efficiently and accurately performing gene repair of in vivo gene mutation caused by gene mutation, such as hereditary diseases, e.g., retinitis pigmentosa, hereditary muscular sclerosis, hereditary tyrosinemia, etc.

Background

In recent years, due to the rapid advance of genetic engineering technology, the CRISPR/Cas9 technology has become one of the hot spots of the scientific community, and is widely applied to various in vivo and in vitro genetic modification, construction of transgenic model animals, gene therapy and other fields. In 2013, Science is loaded with two CRISPR technical papers with important significance, a research group of Zhang Feng, the massachusetts institute of Science, and a type II prokaryotic CRISPR adaptive immune system has been shown to promote RNA-guided site-specific DNA cleavage. Researchers designed two different type II CRISPR systems and demonstrated that Cas9 nuclease could induce precise cleavage at endogenous genomic loci in human and mouse cells via short RNAs. Cas9 can also be converted to a nickase to facilitate homology directed repair with minimal mutagenic activity.

The basic principle of current genome site-directed editing/modification is to utilize spontaneous or induced DNA double-strand gaps (DSBs) in target regions, which activate DNA repair mechanisms in cells to perform genome modification, such as Non-homologous region end joining (NHEJ) or Homologous Recombination (HR). In mammalian cells, the probability of spontaneous generation of DSB is about less than 1/104, if the DSBs are induced by nucleases such as spCas9 and SaCas9 through a genetic engineering method, the efficiency can be improved to more than 10%, and the site specificity is realized, so that the gene repair process of the endogenous gene target site is facilitated to be smoothly carried out in the next step. After the DSBs activate intracellular DNA repair pathways, two different repair mechanisms competitively participate in the repair of the DSBs, namely the end of a non-homologous region is connected with NHEJ, and the end of a homologous recombination HR. Accurate editing of genomic target sites requires reliance on intracellular homologous recombination repair mechanisms. Therefore, directly increasing the frequency of homologous recombination during DSBs repair or suppressing the end joining (NHEJ) of non-homologous regions all contribute to the efficiency of genome site-directed editing/modification. Both end joining of non-homologous regions (NHEJ) and Homologous Recombination (HR) occur with a variety of proteins involved.

Retinitis pigmentosa is a group of inherited diseases in which abnormalities in the photoreceptor cells of the retina (rods or cones) or the retinal pigment epithelium lead to progressive vision loss.In the pathogenesis of retinitis pigmentosa, chronic death of rods in the retina is mainly caused, and apoptosis of cones is caused. Whereas rod cells are responsible for vision when the light is low, apoptosis of the cells can make dark vision worse. The primary symptoms of retinitis pigmentosa usually begin early in childhood, with progressive loss of peripheral vision that ensues over time. In the later stages of the disease, people have a smaller area of central vision and a smaller peripheral vision retention (a narrow visual field). Phosphodiesterase (PDE) plays an important role in the light conduction process, and stimulation of external photons activates rhodopsin (rhodopsin), which activates downstream light transduction protein (transducin), which in turn activates PDE in the rod cells, which degrades cyclic guanosine monophosphate (cGMP) in the photoreceptor cells to reduce its concentration. cGMP is a specific receptor of photoreceptor ion channel, and its degradation causes the closing of rod cell membrane cation channel, the decrease of Na +, Ca2+ influx, and the hyperpolarization of photoreceptor cell, thus causing the nerve impulse generated by visual signal to transmit to visual center step by step, and people feel the stimulation of light. It has now been found that abnormalities of PDEs are widespread in many patients with retinitis pigmentosa disease. The Pde6b gene encodes a Pde protein beta subunit consisting of 856 amino acid residues, and its mutation often causes Pde dysfunction, and the light signaling pathway is cut off, which results in excessive oxidative stress and ultimately leads to apoptosis of photoreceptor cells. As the first discovered retinal degeneration (rd), rd1 mouse is a retinal pigment degeneration mouse model widely studied, and the pathogenic gene is identified as pd 6b, and a nonsense mutation (TAC → TAA) in exon 7 is responsible for the disease. Pigment retinitis model mouse pde6b^rd1/rd1The inner and outer nuclear layers of retina of the mouse begin to become thin 10 days after birth, the rod cells are rapidly degenerated and lost, and the rod cells are completely degenerated 30 days after birth.

In recent years, in vivo gene therapy has been successfully achieved in model animals with respect to genetic diseases such as muscular sclerosis, but since the method has some limitations and optimization, further progress in improving homologous recombination efficiency against genetic diseases caused by gene defects in nerve cells has been demanded.

Disclosure of Invention

In order to increase the efficiency of homologous recombination repair, which is critical in the context of in vivo gene therapy, and to achieve post-natal in vivo homologous recombination repair, it is further desirable to maintain the occurrence of homologous recombination repair in non-dividing cells such as the nervous system, thereby enabling in vivo gene therapy in post-natal individuals.

Aiming at the defects of the existing gene editing technology, the invention provides a gene editing composition, a preparation method thereof and application of the composition in treating diseases caused by gene mutation in vivo, such as hereditary diseases, such as retinitis pigmentosa, hereditary muscular sclerosis, hereditary tyrosinemia and the like, so as to improve the efficiency and treatment effect of site-specific gene repair.

In some embodiments, the present invention provides a gene editing composition or kit comprising 1) a sgRNA or coding sequence thereof linked to a nucleic acid molecule that binds to a protein that targets a mutant gene of interest, 2) a template nucleic acid or coding sequence thereof that repairs the mutant gene of interest, 3) a sgRNA-guided nuclease or coding sequence thereof, 4) a protein or coding sequence thereof that promotes homologous recombination fused to a nucleic acid binding protein, wherein the nucleic acid binding protein is capable of binding to the nucleic acid molecule of 1) above.

In some embodiments, the present invention provides a gene editing composition or kit comprising 1) a nucleic acid sequence of SEQ ID NO: 1, 2) a template nucleic acid that repairs the pde6b mutant gene, and 3) a sgRNA-guided nuclease or coding sequence thereof. In some embodiments, the sgRNA can be linked to a nucleic acid molecule that binds to a protein, as described above. In some embodiments, the composition or kit further comprises a protein or coding sequence thereof that promotes homologous recombination fused to a nucleic acid binding protein, wherein the nucleic acid binding protein is capable of specifically binding to a nucleic acid molecule as described above.

In some embodiments, the mutant gene of interest may be, for example, a gene that causes a disease, such as a genetic disease, due to a mutation thereof. In some embodiments, the mutant gene of interest can be, for example, a Pde6b gene, a Fah gene, a dystropin gene, and the like. In some embodiments, the sgRNA is one that targets the mutant gene of interest. In some embodiments, the sgRNA is one that targets the Pde6b gene, e.g., comprises SEQ ID NO:2, or a sgRNA having the sequence shown in figure 2.

In some embodiments, the protein that promotes homologous recombination comprises a protein that promotes homologous recombination in prokaryotic and eukaryotic cells. In some embodiments, the proteins that promote homologous recombination include, for example, RecA in prokaryotes, Rad51, Rad52, and Brca1 in eukaryotes, and the like. Rad51, Rad52 and Brcal are important proteins involved in the homologous recombination repair process, and RecA is a homologous protein of Rad51 in prokaryotes, and can promote the homologous recombination repair process. In some embodiments, the protein that promotes homologous recombination can be fused to a nucleic acid binding protein to form a fusion protein.

In some embodiments, the nucleic acid binding protein is not particularly limited so long as it is capable of specifically binding to a nucleic acid molecule attached to a sgRNA. In some embodiments, the nucleic acid binding protein can be, for example, MS2 protein, RPA-4 subunit, BRCA2, XRCC2, XRCC3, RadA, HNRNPA1, NABP2(SSB1), NABPI (SSB2), and UHRF 1. In some embodiments, the protein-binding nucleic acid molecule comprises, for example, a sequence that is specifically recognized by the above-mentioned proteins, such as a DNA and/or RNA sequence, e.g., a nucleic acid aptamer, such as a DNA and/or RNA aptamer. In some embodiments, proteins that specifically bind to a particular nucleic acid sequence are well known in the art, and may be, for example, proteins that have a higher affinity than a control sequence, thereby enabling binding and isolation of a sequence of interest from the control sequence. In some embodiments, the sequence specifically recognized by the above-described proteins may have a specific structure, such as a hairpin sequence. In some embodiments, the sequence specifically recognized by the above protein may be, for example, a sequence of SEQ id no:3, or a hairpin sequence shown in the specification.

In some embodiments, components 1), 2), 3), and 4) of the composition or kit are present in one or more carriers. For example, in some embodiments, the sgRNA can be present in one vector, while the template nucleic acid to repair the mutant gene can be contained in a separate vector, or provided as a separate polynucleotide. For example, in some embodiments, the sgRNA and the coding sequence of the sgRNA-guided nuclease can be contained in the same vector. In some embodiments, components 1), 2), 3) and optionally 4) of the composition or kit are present in a plurality of carriers, e.g., 2, 3, 4 carriers.

In some embodiments, the template nucleic acid for repairing the mutant gene of interest is a homologous complementary repair template for restoring the mutant gene of interest to the wild-type gene sequence or a desired sequence. In some embodiments, the desired sequence is a sequence that eliminates disease-causing mutations in the gene, which is not limited to the wild-type sequence. In some embodiments, the template sequence may be, for example, a single-stranded template nucleic acid or a double-stranded template nucleic acid, such as a single-stranded DNA template, a double-stranded DNA template, or a circular plasmid template. In some embodiments, the homologous complementary repair template is used to repair a mutation, such as a point mutation, deletion, insertion or other mutation, in the pde6b gene, e.g., exon 7. In some embodiments, a homologous complementary repair template is used to repair a mutation in the pde6b gene, e.g., exon 7, that results in loss of gene activity, e.g., a nonsense mutation. In some embodiments, a homologous complementary repair template is used to repair a mutation in the pde6b gene, e.g., exon 7, that results in retinitis pigmentosa.

In some embodiments, the sgRNA-guided nuclease is not particularly limited and can include, for example, a CRISPR enzyme, e.g., Cas9, e.g., spCas9, saCas9, e.g., SEQ ID NO: 11 encoded spCas 9.

In some embodiments, the present invention provides a sgRNA molecule, a nucleic acid sequence encoding the sgRNA sequence, a vector, such as a plasmid, comprising the sgRNA sequence or a coding sequence thereof, and/or a cell comprising the vector, wherein the sgRNA molecule can be a sgRNA described herein for gene editing. In some embodiments, the sgRNA molecule is linked to a nucleic acid molecule that specifically binds to a protein. In some embodiments, the sgRNA molecule can be, for example, a nucleic acid molecule comprising SEQ ID NO:2, sgRNA. In some embodiments, provided herein are cells comprising the vector. In some embodiments, the cell is a mammalian cell, such as a human cell.

In some embodiments, provided herein are methods of treating a disease caused by a mutation in a gene in a subject, such as a genetic disease, e.g., retinitis pigmentosa, hereditary muscular sclerosis, hereditary tyrosinemia, etc., the method comprising introducing into the subject, or introducing into a cell of the subject, the sgRNA sequence, the DNA sequence, the vector and/or the modified cell, such that the sgRNA directs a nuclease to the mutated gene, thereby restoring expression of the mutated gene. In some embodiments, wherein the method is performed in vivo (in vivo), in vitro (in vitro), and/or ex vivo (ex vivo).

In some embodiments, the invention provides the use of a composition or sgRNA molecule of the invention, a nucleic acid sequence encoding the sgRNA sequence, a vector comprising the sgRNA sequence or a coding sequence thereof, and/or a cell comprising the vector in the preparation of a medicament for the treatment of a disease caused by a genetic mutation, such as a genetic disease.

In some embodiments, the disease caused by a genetic mutation, such as a genetic disease, may be a disease caused, for example, by a genetic mutation. In some embodiments, genetic mutations include mutations such as substitutions, deletions, insertions, and the like. In some embodiments, the genetic mutation comprises a frameshift mutation, a missense mutation, a nonsense mutation, a stop codon mutation, or the like. In some embodiments, the disease can be, for example, hereditary retinitis pigmentosa, hereditary muscular sclerosis, hereditary tyrosinemia, and the like.

In some embodiments, provided herein is a composition or kit comprising any one or more selected from the sgRNA sequence, the DNA sequence, the vector, and/or the cell. In some embodiments, the compositions and/or kits comprise instructions for use, preferably the kits can be used for the uses and methods described herein. In some embodiments, the kit includes various reagents, such as buffers, etc., suitable for storing the sgRNA sequence, the vector, and/or the DNA molecule. In some embodiments, the kit includes various reagents suitable for performing the reaction of the sgRNA sequence, the vector, and/or the DNA molecule with a target gene, including enzymes, transformation or transfection reagents, and the like. In some embodiments, the kit includes a gene expression agent suitable for modulating a gene of interest by reaction with a target gene via the sgRNA sequence, the vector, and/or the DNA molecule.

In some embodiments, the RNA sequences provided herein, such as sgRNA sequences and/or DNA sequences, are isolated sequences or synthetic sequences. In some embodiments, the invention provides the use of sgRNA molecules and/or compositions and/or kits described herein for treating diseases caused by genetic mutations, such as genetic diseases. In some embodiments, the present invention provides a method of treating a disease caused by a genetic mutation, such as a genetic disease, comprising administering a sgRNA molecule and/or composition and/or kit described herein to a patient having the disease, thereby treating the disease. In some embodiments, the invention provides the use of a sgRNA molecule and/or composition and/or kit described herein in the preparation of a medicament for the repair of a disease caused by a genetic mutation, such as a genetic disease. In some embodiments, the invention provides sgRNA molecules and/or compositions and/or kits described herein for use in treating a disease caused by a gene mutation, such as a genetic disease.

In some embodiments, the invention provides an in vivo gene editing method and gene editing composition that targets a disease caused by a gene mutation, such as a genetic disease, comprising a nuclease, a sgRNA linked to a nucleic acid molecule that binds a protein, a homologous complementary repair template, a protein that promotes homologous recombination fused to a nucleic acid binding protein. The sgRNA molecules and/or compositions and/or kits of the invention have been found to achieve efficient, site-directed, and accurate repair of mutant genes.

In some embodiments, the nucleic acid molecule of the binding protein to which the sgRNA is linked can bear a specific RNA hairpin structure. In some embodiments, for example, an RNA hairpin, such as an MS2 hairpin (see, e.g., fig. 13), is accessed at a specific site of the sgRNA (SpCas9 and SaCas9), and such hairpin structure is specifically recognized by a corresponding nucleic acid binding protein, such as an MS2 binding protein. In some embodiments, the nucleic acid sequence of the MS2 hairpin may be as set forth in SEQ ID NO:3, and (b) is the sequence shown in the specification.

In some embodiments, the homologous repair template described herein can be an artificially synthesized single-stranded DNA sequence containing the normal sequence of pde6 b. In some embodiments, the homologous repair template may comprise SEQ ID NO: 5 or a fragment thereof.

In some embodiments, the invention includes constructing a protein that promotes homologous recombination, such as an MS2-recA fusion protein, that targets a nucleic acid binding protein, such as an MS2 protein, that recognizes the hairpin structure of a nucleic acid molecule, such as MS2, in a sgRNA, that precisely localizes the protein that promotes homologous recombination, such as recA, to a repaired genetic locus. In some embodiments, the sequence of the fusion protein may be expressed under a promoter capable of driving expression of the fusion protein, such as the EF1a promoter.

The inventors have found in experiments that the gene editing method of the present invention can greatly improve the efficiency of homologous recombination occurring in retinitis pigmentosa in vivo.

In some embodiments, the invention provides a fluorescently labeled retinitis pigmentosa model mouse and a method of constructing the same. In some embodiments, the method comprises crossing a pde6b gene mutant retinitis pigmentosa mouse with a fluorescent protein, such as GFP, and backcrossing the resulting F1 generation with the pde6b gene mutant retinitis pigmentosa mouse, screening mice homozygous for the pde6b gene mutation and having a fluorescent protein, such as GFP, to obtain a fluorescently labeled retinitis pigmentosa model mouse. In some embodiments, the optional further comprises introducing a composition or sgRNA molecule described herein into the constructed model mouse. In some embodiments, the invention uses retinitis pigmentosa mice with fluorescent labels. In order to realize the visual observation of rdl mouse rod cells in the whole experiment process, a Nrl-eGFP mouse is adopted to mark all rod cells in a mouse retina, a retinitis pigmentosa rd1 mouse and a Nrl-eGFP mouse are hybridized, then an obtained F1 generation backcross rd1 mouse is used, a mouse homozygous for rd1 gene mutation and with Nrl-eGFP is screened as a female parent of a subsequent experiment, the condition of the mouse is observed, and a P0 or P3 young mouse is selected to carry out an in vivo repair experiment after birth.

In some embodiments, the sgRNA molecules and/or compositions and/or kits can be introduced into a subject by in vivo electroporation. In some embodiments, the photoreceptor cells can be introduced into a subject and the efficiency of in vivo gene repair can be verified at six levels, genomic, transcriptional, protein, morphological, physiological, and behavioral.

The invention includes methods of in vivo gene editing. It has been found that through the interaction of a nucleic acid binding protein, such as the MS2 protein, and a corresponding binding sequence, such as the MS2 binding sequence, proteins that promote homologous recombination, such as the recA protein, can be directionally enriched at the Cas9 recognition cleavage site, effectively increasing the efficiency of homologous recombination repair, and also enabling homologous recombination repair in non-dividing cells.

Drawings

FIG. 1 is a schematic diagram of rdl mouse mutation sites and repair.

FIG. 2 shows the transfection method of neonatal rat subretinal space electroporation.

FIG. 3 is a schematic diagram of green fluorescent cell acquisition and subsequent detection of genome and transcription level of a single cell.

FIG. 4 shows the results of sequencing for genome level detection and repair.

FIG. 5 is off-target site prediction.

FIG. 6 shows the results of off-target site sequencing.

FIG. 7 shows the sequencing results of the transcript level detection repair.

FIG. 8 shows the results of protein level measurement.

FIG. 9 is a comparison of mouse retina morphology after 0 days postnatal electroporation transfection.

FIG. 10 is a graph showing the retinal cleavage status of mice 3 days after birth and the morphological comparison of the retinas of mice after electroporation transfection.

Figure 11 is an electrogram of mice ex vivo retinas after electroporation transfection.

Figure 12 is the pupillary light reflex function of mice after 0 days postnatal electroporation transfection.

Fig. 13 shows sgrnas with MS2 hairpin structures.

Figure 14 is a spCas9 expression vector plasmid map.

Fig. 15 is an expression vector plasmid map of sgRNA and MS2-recA fusion proteins with MS2 targeting hairpin structures.

Detailed Description

Some exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the specific embodiments set forth herein.

1.sgRNA

Generally, a sgRNA is a polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and direct specific binding of the CRISPR complex to the target sequence. In some embodiments, a sgRNA of the invention includes a sequence that is about 100%, about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to a specifically recited sequence, but still directs specific binding of the CRISPR complex to a target sequence.

In some embodiments, sgrnas shorter than 20nt in length (e.g., "truncated sgrnas" of 17-18 nt) can be employed without affecting their activity, and potentially with significantly reduced off-target risk. In some embodiments, shorter sgrnas, e.g., 17, 18, 19nt sgrnas, may be employed.

In some embodiments, for differences in sites of occurrence of different genetic diseases, surrounding sgRNA sequence design can be performed for the respective mutation sites to ensure targeting and efficiency of the entire gene editing composition.

2. Extended sequences comprising recognition sites for nucleic acid binding proteins

In some embodiments, an extension sequence that specifically binds to a nucleic acid binding protein is employed on the sgRNA that can interact with the nucleic acid binding protein, such as MS2 protein, to recruit the nucleic acid binding protein, such as MS2 protein, while in embodiments the nucleic acid binding protein, such as MS2 protein, is an associated fusion protein with a protein that promotes homologous recombination, such as RecA protein, thereby enriching a substantial amount of the protein that promotes homologous recombination, such as RecA protein, near the recognition site of the sgRNA to increase the efficiency of homologous recombination.

In some embodiments, the recognition sequence for the nucleic acid binding protein MS2 may be replaced with recognition sequences for the RNA binding proteins RPA-4 subunit, BRCA2, XRCC2, XRCC3, and the like, requiring the simultaneous replacement of the nucleic acid binding protein MS2 in the composition with the corresponding RNA binding protein RPA-4 subunit, BRCA2, XRCC2, XRCC3, and the like, fused to a homologous recombination protein. In some embodiments, it is preferred to use the MS2 sequence with MS2 protein.

3. Homologous recombination repair template

In some embodiments, homologous complementary repair templates are also provided. The cognate complementary repair template can be another single-stranded DNA component as described herein, or provided as a single double-stranded DNA component, or provided as a single copy or multiple copies of a vector comprising the sequence of interest.

In some embodiments, the homologous complementary repair template is designed to be used as a template in homologous recombination, such as within or near a target sequence that is cleaved or cleaved by a CRISPR enzyme that is part of a CRISPR complex. In some embodiments, the length of the cognate complementary repair template may be, for example, between 100bp and 10kb, such as 100bp, 120bp, 150bp, 180bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb or any length therebetween. In some embodiments, templates of 100bp to 500bp, such as homologous repair sequences of 100bp and 120bp, may be preferred.

CRISPR enzymes

In some embodiments, the invention relates to CRISPR enzymes, such as Cas 9. In some embodiments, the invention relates to nucleases, such as Cas9, that comprise at least one nuclear localization signal, at least one nuclease domain, and at least one domain that interacts with the sgRNA to target the nuclease to a specific nucleotide sequence for cleavage. In some embodiments, the nuclease may be modified to lack at least one functional nuclease domain. In some embodiments, the invention relates to nucleic acids encoding the nucleases to which the invention relates. In some embodiments, the nucleic acid is codon optimized for translation in a mammalian cell. In some embodiments, the nucleic acid is a coding sequence that is codon optimized for translation in a human cell. In some embodiments, the nucleic acid encoding the enzyme is operably linked to a promoter sequence.

Cas9, sgRNA vectors and delivery systems

In some embodiments, provided herein are sgRNA vectors. The sgRNA vector comprises a polynucleotide that can be transcribed into a sgRNA sequence that can edit a target gene.

In some embodiments, the vector is a plasmid vector. In some embodiments, the vector may be a viral vector, such as a lentivirus or baculovirus or preferably an adenovirus/adeno-associated viral vector.

In some embodiments, the vector includes, but is not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules, including nucleic acid molecules of DNA, RNA, or both. In some embodiments, the vector is a viral vector, e.g., a retroviral, replication defective retroviral, adenoviral, replication defective adenoviral, and adeno-associated viral vector. In some embodiments, the vector comprises one or more expression and regulatory elements selected for the host cell used for expression, operably linked to the nucleic acid sequence to be expressed, such as a sgRNA. Such expression and regulatory elements include promoters, enhancers, internal ribosome entry sites, and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). In some embodiments, provided herein are bicistronic vectors for sgrnas with Cas 9. In some embodiments, the sgRNA and Cas9 may be initiated by different promoters.

In some embodiments, the vector may be designed for expression of CRISPR transcripts or translations (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. In some embodiments, mammalian expression vectors are used, which are capable of driving expression of one or more sequences in mammalian cells. In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., expression of the nucleic acid using tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. In some embodiments, one or more vectors that drive expression of one or more elements of the CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system directs formation of a CRISPR complex at one or more target sites. In some embodiments, one vector comprises one or more insertion sites into which two or more sgrnas can be inserted.

In some embodiments, provided herein are isolated or recombinant polynucleotides comprising RNA or DNA sequences encoding sgrnas, various components of a sgRNA vector. The polynucleotide may be RNA or DNA, which may be single-or double-stranded, optionally comprising synthetic, non-natural or modified nucleotide bases. The polynucleotides of the present invention include, but are not limited to, single-stranded forms, double-stranded forms, hairpin structures, stem-loop structures, and the like. Recombinant polynucleotides comprising the sgRNA vectors and different components thereof are also provided. In some embodiments, a recombinant vector may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different than that found in nature. The carrier may be used alone or in combination with a carrier.

In some embodiments, one or more sgRNA vectors described herein can be provided in the form of expression cassettes for expression in different cell types. The expression cassette can include 5 'and 3' regulatory sequences operably linked to the polynucleotides provided herein.

6. Method of introduction

In some embodiments, methods are provided for introducing sgRNA vectors and corresponding repertoires into cells. The methods provided herein use electroporation for transfection. In some embodiments, transfection in vivo may be performed using a combination of electroporation and lipofection, but the transfection is not limited to a particular method as long as the polynucleotide is allowed to enter the interior of at least one cell of the host. Methods for introducing polynucleotides into host cells are known in the art and include, but are not limited to, virus-mediated methods. Introduction includes reference to the integration of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be integrated into the genome of the cell, and includes reference to the provision of a nucleic acid or protein to the cell. In some embodiments, introduction may be performed using a viral vector, such as a lentivirus or baculovirus or preferably an adenovirus/adeno-associated viral vector. In some embodiments, other delivery systems may be used, such as yeast systems, microvesicles, gene guns/attaching carriers to gold nanoparticles. In the vector, the sgRNA or encoding DNA can be operably linked to a promoter and direct nucleic acid delivery into the host cell. In some embodiments, the vector may be administered directly to the subject by microinjection. In some embodiments, for example, transfection of photoreceptor cells can be accomplished by injecting the plasmid mixture into the subretinal space of a subject and applying an electric field to cause DNA to enter the electroporated rods.

7. Composition and/or kit

Provided herein are methods of using sgrnas that are capable of gene editing a target gene when introduced into a cell and compositions and/or kits comprising the sgrnas.

The sgRNA, the vector and the cell can be used in pharmaceutical preparations and compositions, and can also be prepared into a kit convenient to apply. Suitably, the composition or kit comprises a pharmaceutically acceptable solvent, such as water or saline, a diluent, a carrier, a salt or an adjuvant.

The invention also includes pharmaceutical compositions and formulations comprising the nucleotides of the invention. The pharmaceutical compositions of the invention may be used for the treatment of diseases, for example for gene therapy.

In some embodiments, the compositions and/or kits provided herein comprise:

1) a polynucleotide sequence encoding a sgRNA and a nucleic acid-binding protein recognition sequence linked thereto, wherein the polynucleotide sequence comprises one or more sgRNAs which are capable of hybridizing to a target sequence in a eukaryotic cell, wherein the nucleic acid-binding protein recognition sequence can recruit a corresponding nucleic acid-binding protein enrichment,

2) a polynucleotide sequence encoding a CRISPR enzyme,

3) polynucleotide sequences encoding homologous recombinant proteins, which may be of prokaryotic or eukaryotic origin, one or more homologous recombinant proteins, and encoding fusion proteins formed from nucleic acid binding proteins corresponding to the components of 1), and

4) a polynucleotide sequence for homologous repair, wherein the polynucleotide sequence comprises a sequence at a site near a target sequence and wherein the site of a mutation in a genetic disease is to be repaired to a desired sequence.

Upon transcription, the sgRNA directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the sgRNA and CRISPR enzyme hybridized to the target sequence.

In some embodiments, the compositions and/or kits provided herein comprise a nuclease or coding sequence thereof, a sgRNA, a homologous complementary repair template, a nucleic acid binding protein such as MS2 or a coding sequence thereof, and a protein that facilitates homologous recombination such as a recA fusion protein or a coding sequence thereof. In some embodiments, the sgrnas can bear specific RNA hairpin structures. In some embodiments, for example, an RNA hairpin such as the MS2 hairpin to which a nucleic acid binding protein is capable of specifically binding is introduced into the sgRNA, such hairpin structure being specifically recognized by the nucleic acid binding protein such as the MS2 binding protein. In some embodiments, the nucleic acid sequence specifically recognized and bound by the nucleic acid binding protein is covalently linked to the sgRNA. The compositions and/or kits provided herein comprise an artificially synthesized single-stranded DNA sequence of the normal sequence of pde6 b. In some embodiments, the polynucleotide is comprised in a vector system comprising one or more vectors.

6. Application method

In some embodiments, provided herein are methods of treating retinitis pigmentosa, the methods comprising introducing the sgRNA sequence, the DNA sequence, and/or the vector into a cell, and culturing the cell such that the sgRNA directs a nuclease to a target gene. In some embodiments, the methods of the invention restore expression of a target gene, thereby treating retinitis pigmentosa. In some embodiments, the invention provides methods for modulating, e.g., restoring, the expression of a mutant pde6b gene. In some embodiments, the methods provided herein comprise: providing 1) a polynucleotide sequence encoding a sgRNA, wherein the polynucleotide sequence comprises one or more sgrnas that are capable of hybridizing to a target sequence, 2) a polynucleotide sequence encoding a CRISPR enzyme, the CRISPR enzyme optionally comprising at least one or more nuclear localization sequences. Upon transcription, the sgRNA directs sequence-specific binding of a CRISPR complex to a target sequence, wherein the CRISPR complex comprises the sgRNA and CRISPR enzyme hybridized to the target sequence. In some embodiments, the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA. In some embodiments, any or all of the polynucleotide sequence encoding a CRISPR enzyme, sgRNA, can be RNA. In some embodiments, the sequence encoding a CRISPR enzyme, sgRNA, can be RNA and can be delivered via a liposome, a nanoparticle, a microvesicle, or a gene gun. In some embodiments, the methods provided herein are performed in vivo (invivo), in vitro (in vitro), and/or ex vivo (ex vivo). In some embodiments, the method comprises inducing expression. In some embodiments, the vector is a viral vector, including an AAV or lentiviral vector. In some embodiments, the CRISPR enzyme is Cas 9.

In some embodiments, the methods of the invention comprise:

1) introducing into a cell (i) at least one CRISPR enzyme comprising at least one nuclear localization signal or a nucleic acid encoding at least one CRISPR enzyme comprising at least one nuclear localization signal, (ii) at least one RNA or DNA encoding at least one sgRNA, and

2) culturing the cell such that the sgRNA directs the CRISPR enzyme to a target site in the chromosomal sequence, wherein the CRISPR enzyme introduces a break into the target site, and the break is repaired by a DNA repair process such that the chromosomal sequence is modified. In some embodiments, the CRISPR enzyme is from Cas 9. In some embodiments, the CRISPR enzyme is a modified enzyme, e.g., a single nicking generating mutant. In some embodiments, the nucleic acid encoding the CRISPR enzyme is mRNA. In some embodiments, the nucleic acid encoding the CRISPR enzyme is DNA. In some embodiments, the DNA is part of a vector that further comprises a sequence encoding a sgRNA. In some embodiments, the cells include eukaryotic cells, such as human cells and non-human mammalian cells, stem cells. In some embodiments, the subject includes a mammal, e.g., human, monkey, horse, cow, dog, cat, mouse, rat, pig, and the like.

Example 1 design of sgrnas carrying a specifically targeted pde6b gene with an MS2 targeted hairpin structure

In order to repair a nonsense mutation (TAC → TAA) in the seventh exon of pde6b in vivo and to some extent restore function, a sgRNA specifically targeting the pde6b gene (Genbank NC — 000071.6) was designed near this mutation site, the sgRNA having the specific sequence SEQ ID NO 2.

Example 2 a homologous complementary repair template was designed, which was a single-stranded DNA template with the sequence of SEQ ID NO 5, with the mutation site pde6b being the normal sequence.

Example 3 a targeting MS2-recA fusion protein was constructed with the specific sequence of SEQ ID NO 10.

Example 4 construction of mouse lines for breeding fluorescent reporter genes

rd1 mouse rods will undergo apoptosis, and in order to visually observe this process, Nrl-eGFP mice (see 2006, PNAS, Targeting of GFP to newborns by Nrl promoter and temporal expression profiling of flow-targeted phosphor receptors) were used to label all rods in the mouse retina, and after crossing the two mice, the resulting F1 generation was used to backcross Rd1 mice (Rd1 mice are gene mutations found in nature and widely used in research on RP, see Invest Ophthalmol Vis Sci.2006, genomic-phenotype correlation of mouse pde6b variants. molecular Vision.2014Difference effects of genetic acid on phosphor analyzer in Rd1and Rd10 regeneration microorganism.) screening for Rd1 gene mutation homozygosity, and Nrl-eGFP-bearing mice are used as female parents for subsequent experiments, the conditions of vaginal suppository production after mating of the mice are observed, and in vivo repair experiments are selected after the birth of P0 or P3 young mice.

Example 5 transfection of mouse photoreceptor cells by in vivo electroporation

In the experimental group, spCas9 expression vector plasmids (the plasmid map is shown in figure 14) (the final concentration is about 3-5 ug/ul), sgRNA carrying a specific targeting gene pde6b and having an MS2 targeting hairpin structure and an expression vector plasmid of MS2-recA fusion protein (the plasmid map is shown in figure 15) (the final concentration is about 3-5 ug/ul), and a homologous repair template (50uM) are transformed; in a control group, spCas9 expression vector plasmids (the final concentration is about 3-5 ug/ul), sgRNA carrying a specific targeting gene pde6b and having an MS2 targeting hairpin structure, expression vector plasmids of MS2 protein (the final concentration is about 3-5 ug/ul) and a homologous repair template (50uM) are transferred. In the experiment, the plasmid mixture was injected into the subretinal space of a mouse through a hamilton microneedle, and a unidirectional electric pulse of 80V, 1Hz and 5% duty ratio was applied through an external electric field, so that negatively charged DNA entered into an electroporated rod cell (fig. 2), thereby completing the transfection of photoreceptor cells.

Example 6 detection of in vivo repair of pigmented retinal mice

1) Genome level detection: the experimental group and the control group are raised to P31 days, the mice are killed, eyeballs are picked up, the whole retina is peeled off under a body type microscope, the eyeballs are laid in PBS, green rod cells are absorbed by a glass electrode, 20-30 cells are collected, then oligo cell genome extraction is carried out, the genome is subjected to enzyme digestion of DdeI (the DdeI can cut rdl mutant sites), the genome is subjected to PCR amplification repair area around the repair sites (figure 3), and sequencing analysis effect is carried out (figure 4).

2) Detecting the transcription level: the mice of the experimental group and the control group are raised to P31 days, the mice are killed, eyeballs are picked up, the whole retina is peeled off under a body type microscope, the mice are laid in PBS, green rod cells are absorbed by a glass electrode, 20-30 cells are collected and then subjected to single-tube type single-cell reverse transcription (refer to smart seq2), the obtained cDNA is subjected to PCR (polymerase chain reaction) aiming at the cDNA near the mutation site, the PCR product is subjected to DdeI enzyme digestion treatment, and then secondary PCR (figure 3) is carried out and the sequencing analysis effect is sent (figure 5).

3) Protein level detection: mice in the experimental group and the control group were raised to P21 days, the mice were sacrificed to take out eyeballs and the whole retina was peeled off under a stereomicroscope, and the total retinal protein was obtained by cleavage of the retina to carry out western blot, and whether the intact pde6b protein was expressed in the restored retina was examined (fig. 6).

4) And (3) morphological detection: the mice of the experimental group and the control group are raised to P31 days, the mice are killed, the eyeballs are picked up and placed into 4% PFA for fixation for more than 2 hours, the eyeballs are cut off under a body type microscope after being cleaned by PBS, the cornea is peeled off, the crystalline lens is dehydrated and frozen and sliced, and the residual quantity of the green fluorescence labeled rod cells is observed and counted. Retinal sections were immunohistochemically stained for morphology and number of blue and green cone cells labeled with antibodies to sw/mwOpn (fig. 7, fig. 8).

5) And (3) detecting the physiological level: mice of the experimental group and the control group were raised to P14 days, sacrificed, the eyeballs were picked up, the whole retina was peeled off under a stereomicroscope, and the whole retina was laid flat in PBS, and after treatment with AP5, CNQX, and DL-AP4, an in vitro electroretinogram was recorded using an electrophysiological system (fig. 9).

6) And (3) behavior level detection: the mice of the experimental group and the control group are raised to P31 days, the head of the mouse is embedded with a cranial nail, dark adaptation is started in advance one day and night, the mouse is subjected to a photometric machine and the size of the pupil of the mouse is recorded by video recording when the head is adapted to ten points on the next day, and the pupil light reflex measurement is carried out (figure 10).

In conclusion, the gene editing composition used in the present invention efficiently realizes the repair of a nonsense mutation (TAC → TAA) in the seventh exon of the pde6b gene in a somatic retinitis mouse. The experimental group of mice retained about 6-fold amount of rod cells and also increased cone cell residues about 4-fold by increasing the efficiency of homologous recombination compared to the control group of mice developed to P31 days (fig. 9); the sequencing result shows that the sequence of the normal pde6b which appears on the gene level and the transcription level is really detected in the sample of the repair group; the results of western b1ot indicated that a small amount of the normal size pde6b protein was expressed in the repair group; the results of the in vitro electroretinogram also show that the photoreceptor cells of the mice in the repair group have certain photosensitive function, while the mice in the control group have no photosensitive function; the behavioral results of pupil light reflection show that the control group of mice does not respond to flash light stimulation, while the whole animal of the experimental group of mice has certain light sensitivity and can generate obvious behavior of pupil light reflection to light stimulation.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

SEQ ID NO: l position of sgRNA-targeted pde6b Gene of the invention

GCTGATCACTGGGCCCTGG

SEQ ID NO:2 sgRNA sequences of the invention (underlined PAM)

CCAGGGCCCAGUGAUCAGCCGG

SEQ ID NO: 3MS2 protein specificity recognition combined hairpin sequence

GCCAACAUGAGGAUCACCCAUGUCUGCAGGGCC

SEQ ID NO: exon 7 sequence of 4pde6b gene (rd1 mouse underlined sequence is A)

CACACCCCCGGCTGATCACTGGGCCCTGGCCAGTGGCCTTCCAACCTACGTAGCAGAAAGTGGCTTT

SEQ ID NO: 5 template DNA sequence

CTTTTCCCACAGCACACCCCCGGCTGATCACTGGGCCCTGGCCAGTGGCCTTCCAACCTACGTAGCAGAAAGTGGCTTTGTGAGTGTCCCTCTCCAGGCCTTGGCCTCTACTGGCCAGTG

SEQ ID NO: 6MS2 coding sequence

ATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATGTGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCCAACTCACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCCCAGAAgAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCC

SEQ ID NO: 7RecA coding sequence

TACCTGAACATGGAGCTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGGGGCTCCTCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTATCTACagcgctGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCggacctaagaaaaagaggaaggtggcggccgctgGATCTATGGTCCCAAAAAAGAAAAGGAAGGTGGCTATTGACGAGAACAAACAGAAAGCACTGGCAGCAGCACTGGGGCAGATTGAGAAACAGTTCGGGAAGGGATCCATTATGAGGCTGGGGGAAGACCGCTCAATGGATGTGGAGACTATCAGCACCGGATCTCTGAGTCTGGACATTGCTCTGGGAGCAGGAGGACTGCCAATGGGACGAATCGTGGAAATCTACGGACCTGAGAGCTCCGGCAAGACCACACTGACCCTGCAGGTCATCGCCGCTGCACAGAGAGAGGGCAAAACATGCGCCTTCATCGACGCCGAACACGCTCTGGATCCAATCTACGCTAGGAAGCTGGGCGTGGACATCGATAACCTGCTGTGCTCTCAGCCCGACACTGGGGAACAGGCCCTGGAGATTTGTGATGCACTGGCTAGGTCCGGAGCTGTGGACGTGATCGTGGTCGATTCTGTGGCCGCTCTGACCCCCAAAGCCGAGATCGAAGGGGAGATTGGAGACAGTCATATGGGCCTGGCAGCCCGAATGATGTCACAGGCAATGCGGAAGCTGGCCGGCAACCTGAAACAGAGCAATACACTGCTGATCTTCATTAACCAGATCCGCATGAAGATTGGCGTGATGTTTGGGAACCCCGAGACTACCACAGGAGGCAATGCTCTGAAGTTCTACGCAAGCGTGCGCCTGGACATCCGGAGAATTGGGGCCGTCAAAGAAGGAGAGAACGTGGTCGGCTCCGAGACCCGAGTGAAGGTGGTCAAGAACAAGATCGCTGCACCTTTCAAACAGGCCGAATTTCAGATCCTGTACGGCGAGGGCATCAACTTCTACGGGGAACTGGTGGACCTGGGAGTCAAGGAAAAACTGATTGAGAAGGCAGGAGCCTGGTACTCTTATAAGGGCGAGAAAATCGGACAGGGCAAAGCTAACGCAACAGCCTGGCTGAAGGATAATCCTGAGACTGCCAAAGAAATCGAGAAGAAAGTGAGAGAACTGCTGCTGAGTAACCCAAATTCAACCCCCGATTTTTCAGTGGATGATTCCGAAGGCGTCGCAGAGACTAATGAGGATTTTAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCGCTAGCAg

SEQ ID NO: 8EFla promoter sequence

AATTCctagcgtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaggtgtcgtgagcgCc

SEQ ID NO: 9PolyA sequence

Atcttagccactttctagagtcggggcggccggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatcgataaggatccg

SEQ ID NO: 10MS2-RecA complete sequence

AATTCctagcgtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaggtgtcgtgagcgCcATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATGTGACAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCCAACTCACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCCCAGAAgAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAGCTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGGGGCTCCTCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTATCTACagcgctGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCggacctaagaaaaagaggaaggtggcggccgctgGATCTATGGTCCCAAAAAAGAAAAGGAAGGTGGCTATTGACGAGAACAAACAGAAAGCACTGGCAGCAGCACTGGGGCAGATTGAGAAACAGTTCGGGAAGGGATCCATTATGAGGCTGGGGGAAGACCGCTCAATGGATGTGGAGACTATCAGCACCGGATCTCTGAGTCTGGACATTGCTCTGGGAGCAGGAGGACTGCCAATGGGACGAATCGTGGAAATCTACGGACCTGAGAGCTCCGGCAAGACCACACTGACCCTGCAGGTCATCGCCGCTGCACAGAGAGAGGGCAAAACATGCGCCTTCATCGACGCCGAACACGCTCTGGATCCAATCTACGCTAGGAAGCTGGGCGTGGACATCGATAACCTGCTGTGCTCTCAGCCCGACACTGGGGAACAGGCCCTGGAGATTTGTGATGCACTGGCTAGGTCCGGAGCTGTGGACGTGATCGTGGTCGATTCTGTGGCCGCTCTGACCCCCAAAGCCGAGATCGAAGGGGAGATTGGAGACAGTCATATGGGCCTGGCAGCCCGAATGATGTCACAGGCAATGCGGAAGCTGGCCGGCAACCTGAAACAGAGCAATACACTGCTGATCTTCATTAACCAGATCCGCATGAAGATTGGCGTGATGTTTGGGAACCCCGAGACTACCACAGGAGGCAATGCTCTGAAGTTCTACGCAAGCGTGCGCCTGGACATCCGGAGAATTGGGGCCGTCAAAGAAGGAGAGAACGTGGTCGGCTCCGAGACCCGAGTGAAGGTGGTCAAGAACAAGATCGCTGCACCTTTCAAACAGGCCGAATTTCAGATCCTGTACGGCGAGGGCATCAACTTCTACGGGGAACTGGTGGACCTGGGAGTCAAGGAAAAACTGATTGAGAAGGCAGGAGCCTGGTACTCTTATAAGGGCGAGAAAATCGGACAGGGCAAAGCTAACGCAACAGCCTGGCTGAAGGATAATCCTGAGACTGCCAAAGAAATCGAGAAGAAAGTGAGAGAACTGCTGCTGAGTAACCCAAATTCAACCCCCGATTTTTCAGTGGATGATTCCGAAGGCGTCGCAGAGACTAATGAGGATTTTAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCGCTAGCAgatcttagccactttctagagtcggggcggccggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatcgataaggatccg

SEQ ID NO: ll SpCas9 coding sequence

GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC

Claims

1. A gene editing composition or kit comprising 1) a sgRNA complex or coding sequence thereof linked to a nucleic acid molecule that binds to a protein targeting a mutant gene of interest, 2) a template nucleic acid that repairs the mutant gene of interest or coding sequence thereof, 3) a sgRNA complex-directed nuclease or coding sequence thereof, and 4) a protein that promotes homologous recombination or coding sequence thereof fused to a nucleic acid binding protein, wherein the nucleic acid binding protein is capable of binding to the nucleic acid molecule of 1) above, wherein the protein that promotes homologous recombination is RecA.

2. The composition or kit of claim 1, wherein the mutant gene of interest is a gene that causes disease due to its mutation.

3. The composition or kit of claim 1 or 2, wherein the mutant gene of interest is a gene that causes a genetic disease due to a mutation thereof.

4. The composition or kit of claim 1 or 2, wherein the mutant gene of interest is a Pde6b gene, a Fah gene, or a dystropin gene.

5. The composition or kit of claim 1 or 2, wherein the sgRNA complex is one that targets the mutant gene of interest.

6. The composition or kit of claim 1 or 2, wherein the sgRNA complex is a sgRNA complex comprising a sequence set forth in SEQ ID NO 2.

7. The composition or kit of claim 1 or 2, wherein the nucleic acid binding protein comprises MS2 protein, RPA-4 subunit, BRCA2, XRCC2, XRCC3, RadA, HNRNPA1, NABP2(SSB1), NABPI (SSB2), and UHRF1, and the nucleic acid molecule of the binding protein comprises a nucleic acid sequence specifically recognized by the above protein.

8. The composition or kit of claim 7, wherein the protein-binding nucleic acid molecule comprises a hairpin sequence.

9. The composition or kit of claim 7, wherein the protein-binding nucleic acid molecule comprises the hairpin sequence of SEQ ID NO. 3 specifically recognized by the MS2 protein.

10. The composition or kit of claim 1 or 2, wherein components 1), 2), and 3) of the composition or kit are present in one or more carriers.

11. The composition or kit of claim 10, wherein components 1), 2), 3) and 4) of the composition or kit are present in one or more carriers.

12. The composition or kit of claim 1 or 2, wherein the template nucleic acid for repairing a mutated gene of interest is a homologous complementary repair template for restoring the mutated gene of interest to a wild-type gene sequence or a desired sequence.

13. The composition or kit of claim 1 or 2, wherein the template nucleic acid for repairing the mutant gene of interest is a single-stranded template nucleic acid or a double-stranded template nucleic acid.

14. The composition or kit of claim 1 or 2, wherein the template nucleic acid for repairing the mutant gene of interest is a single-stranded DNA template, a double-stranded DNA template, or a circular plasmid template.

15. The composition or kit of claim 1 or 2, wherein the sgRNA complex-guided nuclease comprises a CRISPR enzyme.

16. The composition or kit of claim 1 or 2, wherein the sgRNA complex-guided nuclease comprises Cas 9.

17. The composition or kit of claim 1 or 2, wherein the sgRNA complex-guided nuclease comprises spCas9 and saCas 9.

18. Use of a composition or kit as defined in any one of claims 1 to 17 in the manufacture of a medicament for the treatment of a disease caused by a gene mutation.

19. The use of claim 18, wherein the disease is a genetic disease.

20. The use of claim 19, wherein the disease is hereditary retinitis pigmentosa, hereditary muscular sclerosis, or hereditary tyrosinemia.