Nucleic acid molecules for treating bietti crystalline dystrophy and uses thereof

Abstract

This document discloses a gRNA targeting cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2), a donor nucleic acid molecule containing a human CYP4V2 nucleotide sequence, and use of the gRNA and the donor nucleic acid molecule in the manufacture of a medicament for treating Vietti crystalline dystrophy.

Description

[Technical field]

[0001] The present application relates to the field of biomedicine, and more particularly to gRNA and donor nucleic acid molecules for gene editing treatment of Vietti crystalline dystrophy. [Background technology]

[0002] Bietti crystalline dystrophy (BCD), also known as crystalline retinitis pigmentosa, Bietti crystalline corneoretinal dystrophy, Bietti crystalline retinopathy, or Bietti's retinal dystrophy, is an autosomal recessively inherited retinal degenerative disease that leads to blindness. The CYP4V2 gene is one of the causative genes for BCD currently discovered (Li et al. Am J Hum Genet. 74:817-826, 200). CYP4V2 belongs to the cytochrome P450 superfamily and is a member of the heme-thiolate protein cytochrome P450 subfamily 4 (CYP4).

[0003] Currently, there are several treatments for this disease, such as gene replacement therapy, which uses a viral transfection system or other transfection systems (e.g., AAV, lentivirus, retrovirus) to transfect wild-type CYP4V2 genes into mutant cells, allowing the mutant cells to express wild-type CYP4V2. This method can only partially restore the function of mutant cells, and the effectiveness of the treatment is limited. This is because mutant gene products remain in the cells, and these mutant proteins competitively inhibit normal gene products. Therefore, the development of a safer and more effective treatment method is expected.

[0004] The information disclosed in the Background section is intended only to enhance understanding of the general background of the present application, and is not an admission or any form of suggestion that such information forms prior art publicly known to those skilled in the art. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application provides a gRNA that specifically targets cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2), the gRNA specifically targets the c.802-8_810del17bpinsGC mutation site, and the gRNA has a good cleavage effect on the mutation site, so that the original mutation CYP4V2 gene product is no longer present in the cell. The present application also provides a donor nucleic acid molecule, the donor nucleic acid molecule contains the exact nucleotide sequence of CYP4V2 that does not contain the c.802-8_810del17bpinsGC mutation site, and the donor nucleic acid molecule repairs the mutation site of CYP4V2 in the gene mutation cell after the endogenous CYP4V2 is cleaved by the gRNA, thereby producing a CYP4V2 protein with normal function, and has an excellent repair effect. The present application provides a vector containing the gRNA and / or the donor nucleic acid molecule, which enables CYP4V2 mutant cells to accurately express cytochrome P450 family 4 subfamily V polypeptide 2 and has good gene editing repair efficiency.

[0006] On the other hand, the present application provides a gRNA that specifically targets the cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene, wherein the gRNA specifically binds to a nucleotide sequence within 200 bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site. [Means for solving the problem]

[0007] In one embodiment, the gRNA comprises the nucleotide sequence of any of SEQ ID NOs: 74, 78-80, 82-84.

[0008] In one embodiment, the gRNA comprises 5'-(X)n-SEQ ID NO:74, 78-80, 82-84-backbone sequence-3', where X is any base selected from A, U, C and G, and n is any integer from 0 to 15.

[0009] In one embodiment, the gRNA is a single-stranded guide RNA (sgRNA).

[0010] On the other hand, the present application provides one or more isolated nucleic acid molecules encoding gRNAs that specifically target the CYP4V2 gene.

[0011] On the other hand, the present application provides a donor nucleic acid molecule containing a normal wild-type nucleotide sequence within 800 bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site of the CYP4V2 gene.

[0012] In one embodiment, the nucleic acid molecule comprises the nucleotide sequence of any of SEQ ID NOs:64-66.

[0013] On the other hand, the present application provides a vector containing the isolated nucleic acid molecule and / or the donor nucleic acid molecule.

[0014] In one embodiment, the isolated nucleic acid molecule and the donor nucleic acid molecule are present on the same vector.

[0015] In one embodiment, the vector is a viral vector.

[0016] On the other hand, the present application provides a cell containing the isolated nucleic acid molecule, the donor nucleic acid molecule and / or the vector.

[0017] In one embodiment, the cells comprise HEK293 cells, renal epithelial cells and / or induced pluripotent stem cells.

[0018] In one embodiment, the cells are modified to be differentiation potent.

[0019] In one embodiment, the cells are capable of differentiating into 3D-retinal organoids.

[0020] On the other hand, the present application provides a pharmaceutical composition comprising the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and / or the vector, and a pharma- ceutically acceptable carrier.

[0021] On the other hand, the present application provides the use of the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and / or the vector in the manufacture of a medicament for the treatment of a disease, including a disease caused by the c.802-8_810del17bpinsGC mutation in the CYP4V2 gene.

[0022] In one embodiment, the disease comprises Bietti crystalline dystrophy.

[0023] On the other hand, the present application provides a method for treating Vietti crystalline dystrophy, comprising the step of introducing the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and / or the vector into a subject in need thereof.

[0024] In one embodiment, the introduction results in a normally functional CYP4V2 protein.

[0025] In one embodiment, the introducing comprises an injection.

[0026] In one embodiment, the introducing comprises a subretinal injection.

[0027] On the other hand, the present application provides a method for regulating expression of the CYP4V2 gene in a cell, comprising introducing the gRNA, the one or more isolated nucleic acid molecules, and / or the vector into a cell.

[0028] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. Those skilled in the art will appreciate that, upon learning the contents of the present application, they may make changes to the specific embodiments disclosed without departing from the spirit and scope of the present invention. Therefore, the drawings and description of the present application are illustrative only and not restrictive. [Brief description of the drawings]

[0029] Particular features of the present invention are set forth in the appended claims and may be more clearly understood by reference to the following detailed description of exemplary embodiments and drawings, which are briefly described as follows:

[0030] [Figure 1A] FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA1-2 described in the present application. [Figure 1B] FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA3-5 described in the present application. [Figure 1C] FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA6-8 described in the present application. [Figure 1D] FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA9-11 described in the present application. [Figure 1E] FIG. 1 shows the results of dsDNA cleavage of the mutant genes by sgRNA12-14 described in the present application. [Figure 1F] FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA15 described in the present application. [Figure 1G]FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA16-19 described in the present application. [Figure 1H] FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA20-22 described in the present application. [Figure 1I] FIG. 1 shows the results of dsDNA cleavage of the mutant gene by sgRNA23 described in the present application. [Diagram 2] FIG. 1 shows the plasmid map of the AAV-saCas9-puro vector described in the present application. [Diagram 3] FIG. 1 shows the results of a T7E1 enzyme cleavage experiment of fragments after cleavage of sgRNA8, sgRNA12, sgRNA13, sgRNA14, sgRNA16, sgRNA17, and sgRNA18. [Figure 4A] FIG. 13 shows the results of gene sequencing of a PCR fragment containing the genomic DNA cleaved by sgRNA13. [Figure 4B] FIG. 1 shows the results of gene sequencing of PCR fragments containing the cleavage site of genomic DNA after sgRNA17 cleavage. [Figure 4C] FIG. 1 shows the results of gene sequencing of a PCR fragment containing the genomic DNA cleaved by sgRNA18. [Diagram 5] FIG. 1 shows histograms of cleavage efficiencies in 293T cells of sgRNA13, sgRNA17, sgRNA18, sgRNA13+17, and sgRNA13+18. [Figure 6] FIG. 1 shows the design of the donor sequence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Hereinafter, embodiments of the present invention will be described with reference to certain specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the contents disclosed herein.

[0032] Definition of Terms

[0033] In the present application, the term "CYP4V2" generally refers to a protein that is a member 2 of cytochrome P450 family 4 subfamily V. The term "cytochrome P450", which may also be called cytochrome P450 or CYP450, generally refers to a family of hemoproteins, a type of monooxygenase, involved in the metabolism of endogenous or exogenous substances, including drugs and environmental compounds. According to the homology of amino acid sequences, the members are classified into three groups: family, subfamily, and individual enzyme. The cytochrome P450 enzyme system can be abbreviated as CYP, where family is represented by Arabic numerals, subfamily is represented by capital letters, and individual enzyme is represented by Arabic numerals, for example, CYP4V2 in the present application. The human CYP4V2 gene (HGNC:23198; NCBI ID:285440) is usually 19.28 kb in length, located at 4q35, has 11 exons, and plays an important role in fatty acid metabolism (Kumar S., Bioinformation,2011,7:360-365). The CYP4V2 described in this application may include its functional variants, fragments, homologs, etc. CYP4V2 is expressed in almost all tissues, but is expressed at high levels in the retina and retinal pigment epithelium and at somewhat lower levels in corneal tissue. Mutations in the CYP4V2 gene may be associated with Vietti crystalline dystrophy and / or retinitis pigmentosa.

[0034] In the present application, the term "c.802-8_810del17bpinsGC" refers to a relatively common CYP4V2 gene mutation, which refers to the removal of 17 base pairs at the 3' end of intron 6 and the 5' end of exon 7, thereby resulting in the deletion of exon 7, leading to changes in the structure and activity of the transcription protein. An exemplary sequence between intron 6 and exon 7 of wild-type CYP4V2 can be represented as caaacagaagcatgtgattatcattcaaaTCATACAGGTCATCGCTgaacgggccaatgaaatgaacgccaatga (SEQ ID NO:90). An exemplary c.802-8_810dell7insGC mutation sequence can be represented as follows: In the sequence between intron 6 and exon 7, 17 bp was deleted (sequence represented in uppercase in wild type) and GC (represented in uppercase) was inserted: caaacagaagcatgtgattatcattcaaaGCgaacgggccaatgaaatgaacgccaatga (SEQ ID NO:91). (Xiao et al.Biochem Biophys Res Commun. 409:181-6, 2011; Meng et al.2014, Mol. Vis., 20:1806-14; Wada et al.Am J Ophthalmol. 139:894-9, 2005; Jiao et al.European Journal of Human Genetics (2017) 25, 461-471.). In the present application, the "c.802-8_810del17bpinsGC mutation site" may be present in a portion that is deleted in the nucleotide sequence shown in SEQ ID NO:91, or in a position where CG has been increased."Within 800 bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site" can include nucleotides within about 800 bp in the 5' direction from the base bound to the N-terminus of the nucleotide sequence written in capital letters (i.e., "a"), or nucleotides within about 800 bp in the 3' direction from the base bound to the C-terminus of the nucleotide sequence written in capital letters (i.e., "g") in caaacagaagcatgtgattatcattcaaaTCATACAGGTCATCGCTgaacgggccaatgaaatgaacgccaatga (SEQ ID NO:90).

[0035] In this application, the term "mutation" generally refers to a difference in the amino acid or nucleic acid sequence of a particular protein or nucleic acid (gene, RNA) relative to the wild-type protein or nucleic acid. The mutated protein or nucleic acid may be expressed by or found in one allele (heterozygous) or two alleles (homozygous) of a gene. Mutations include deletion mutations, truncations, inactivations, disruptions, substitution mutations or rearrangements. These types of mutations are well known in the art. In this application, CYP4V2-related mutations include, but are not limited to, missense, repeats, splice sites, frameshifts, deletions, insertions, indels, nonsense, polymorphisms (e.g., single nucleotide polymorphisms), and premature terminations of the CYP4V2 gene, and deletions of the entire CYP4V2 gene.

[0036] As used herein, the term "isolated nucleic acid molecule" refers to a molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. Such an isolated nucleic acid molecule is one that has been removed or separated from its normal or natural environment, or has been produced in a manner that does not cause the molecule to be present in its normal or natural environment, and is separated from polypeptides, peptides, lipids, carbohydrates, other polynucleotides, and other substances in the normal or natural environment. The isolated nucleic acid molecule of the present application can encode an RNA, for example, a gRNA that specifically targets the CYP4V2 gene.

[0037] As used herein, the term "donor nucleic acid molecule" generally refers to a nucleic acid molecule that provides a heterologous nucleic acid sequence to a recipient (eg, a recipient nucleic acid molecule).

[0038] In the present application, the term "backbone sequence" generally refers to a portion of a gRNA other than the portion that recognizes or hybridizes to a target sequence, and may include a sequence between the gRNA pairing sequence and the transcription terminator in an sgRNA. The backbone sequence generally does not change with changes in the target sequence, and does not affect the recognition of the target sequence by the gRNA. Therefore, the backbone sequence may be any sequence available in the prior art. The structure of the backbone sequence may refer to portions other than the spacer sequence described in Nowak et al. Nucleic Acids Research 2016. 44:9555-9564, A and B in Figure 1, A, B, C in Figure 3, and A, B, C, D, and E in Figure 4.

[0039] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of autonomous replication in a suitable host, which can transfer an inserted nucleic acid molecule (e.g., an exogenous sequence) into and / or between host cells. The vectors can include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors primarily for transcriptional and / or translational expression of DNA or RNA. The vectors can also include vectors having multiple of the above functions. The vectors can be polynucleotides that can be transcribed and translated into a polypeptide upon introduction into a suitable host cell. Typically, the vectors can produce a desired expression product by culturing a suitable host cell containing the vector. The vectors described in the present application can include, for example, expression vectors, viral vectors (lentiviral and / or retroviral vectors), phage vectors, phagemids, mucosal particles, cosmids, artificial chromosomes (yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)), and / or plasmids.

[0040] In the present application, the term "gRNA" generally refers to a guide RNA, which is an RNA molecule. In nature, crRNA and tracrRNA usually exist as two separate RNA molecules, constituting gRNA. The term "crRNA", which may be referred to as CRISPR RNA, generally refers to a stretch of nucleotide sequence complementary to the targeted target DNA, and the term "tracrRNA" generally refers to a scaffold-type RNA that can bind to Cas nuclease. The crRNA and tracRNA may also be fused into a single strand, in which case the gRNA is also referred to as a single guide RNA (sgRNA). Since sgRNA has become the most common form of gRNA used by those skilled in the art in CRISPR technology, the terms "sgRNA" and "gRNA" may have the same meaning in the present application. sgRNA can be artificially synthesized or produced in vitro or in vivo from a DNA template. The sgRNA can bind to a Cas nuclease and target a target DNA, directing the Cas nuclease to cleave the DNA site complementary to the gRNA.

[0041] As used herein, the term "specifically targeting" generally refers to an interaction between two molecules (e.g., molecule A and molecule B) (e.g., molecule A specifically recognizes and / or binds (e.g., targets) molecule B). Molecule A interacts with molecule B to a statistically significant extent compared to interactions with other non-B molecules. The interaction may be covalent or non-covalent. In the context of polynucleotides, specifically targeting may refer to molecule A (or a single strand thereof) and molecule B (or a single strand thereof) having a base-complementary pairing relationship. As used herein, specifically targeting may refer to the process by which a gRNA recognizes and / or binds a target sequence.

[0042] In this application, the terms "target nucleic acid", "target nucleic acid", "target sequence" and "target region" may be used interchangeably and generally refer to a nucleic acid sequence that may be recognized by a gRNA, and the target nucleic acid may refer to a double-stranded or single-stranded nucleic acid.

[0043] In this application, the term "3D-retinal organoid" generally refers to the artificially cultured retina that has a three-dimensional structure, can self-renew, self-organize, and shows the basic functions of the retina (e.g., photosensitivity).3D-retinal organoid can be differentiated from primary tissue or stem cells (e.g., pluripotent stem cells), and has all the cells of the retina that are necessary to respond to light and send signals to the brain.

[0044] In this application, the term "Vietti crystalline dystrophy" generally refers to a group of autosomal recessive genetic eye diseases. The main symptoms include crystals in the cornea (transparent membrane), fine yellow or white crystalline deposits deposited in the light-sensitive tissue of the retina, and progressive atrophy of the retina, choriocapillaris, and choroid. Vietti crystalline dystrophy can include diseases caused by CYP4V2 gene mutations.

[0045] In the present application, the term "subretinal injection" generally refers to the introduction of a substance between photoreceptor cells and the retinal pigment epithelium (RPE) layer. During subretinal injection, a substance (e.g., a gRNA described herein, one or more isolated nucleic acid molecules described herein, a donor nucleic acid molecule described herein, a vector described herein, and a pharma- ceutically acceptable carrier) is introduced between the photoreceptor cells and the retinal pigment epithelium (RPE) layer, forming a space therebetween.

[0046] In this application, the term "cell" has its commonly accepted meaning in the art. The term is used in its normal biological sense and does not refer to an intact multicellular organism, such as a human specifically. Cells can be present in avian, plant and mammalian organisms, such as humans, cows, sheep, apes, monkeys, pigs, dogs and cats. Cells can be pronuclear (e.g., bacterial cells) or eukaryotic (e.g., mammalian or plant cells). Cells can be of somatic or germ cell origin, totipotent or pluripotent, dividing or non-dividing. Cells can be derived from or include gametes or embryos, stem cells, or fully differentiated cells.

[0047] In the present application, the term "induced pluripotent stem cells" generally refers to cells that have been converted under certain conditions from somatic cells to a totipotent state. The totipotency refers to the ability to differentiate into any type of cell in the body, to form a complete embryo, or to develop into a new individual. For example, in the present application, the induced pluripotent stem cells include those that have the ability to differentiate into retinal cells, which can be obtained by culturing renal epithelial cells.

[0048] In this application, the term "HEK293 cells" generally refers to "human embryonic kidney cells 293", which are cells derived from human embryonic kidney cells, are easy to culture, have high transfection efficiency, and are a cell line commonly used in the art for research on exogenous genes. In this application, the term "293T cells" or "HEK293T cells" refers to cells derived from "HEK293 cells", which are a cell line commonly used in the art.

[0049] In this application, the term "renal epithelial cells" generally refers to kidney epithelial cells collected from human urine. In this application, it may be a source of induced pluripotent stem cells. In the art, the induction of pluripotent stem cells using renal epithelial cells in urine is cost-effective, versatile, and can be adapted to various ages, genders, and races. With this technology, it is much easier and more economical to obtain a large number of patient samples than other existing methods.

[0050] As used herein, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a subject in need thereof. For example, a pharmaceutical composition described herein may contain a gRNA described herein, one or more isolated nucleic acid molecules described herein, a donor nucleic acid molecule described herein, and / or a vector described herein, and a pharmaceutically acceptable carrier. The terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein to refer to a subject, e.g., a mammalian subject, in need of administration of a pharmaceutical composition of the present application. Animal subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, yellow cattle, dairy cows, etc., e.g., mice. In one embodiment, the pharmaceutical composition includes a composition for subretinal, parenteral, transdermal, intracavitary, intraarterial, intramembrane, and / or intranasal administration, or for injection directly into a tissue. For example, the pharmaceutical composition is administered to the subject by subretinal injection.

[0051] In addition to the specific proteins and nucleotides described herein, the present application can include the use of variants, derivatives, analogs, homologs and fragments thereof.

[0052] In the context of this application, a variant of any given sequence refers to a sequence in which a specific sequence of residues (either amino acid residues or nucleotide residues) has been modified such that said polypeptide or polynucleotide essentially retains at least one endogenous function. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one amino acid residue and / or nucleotide residue present in the naturally occurring protein and / or polynucleotide.

[0053] As used herein, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application that includes any substitution, mutation, modification, substitution, deletion and / or addition of one (or more) amino acid residues from / to the sequence, so long as the resulting polypeptide or polynucleotide essentially retains at least one endogenous function thereof.

[0054] As used herein, the term "analog", generally with respect to a polypeptide or polynucleotide, includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that has at least one endogenous function of the polypeptide or polynucleotide that it mimics.

[0055] Typically, the modified sequence can have, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, so long as the modified sequence essentially retains the desired activity or ability. The amino acid substitutions can be non-naturally occurring analogs.

[0056] Proteins or polypeptides used herein may also have deletions, insertions or substitutions of amino acid residues that result in non-phenotypic changes to said amino acid residues and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues, so long as the intrinsic function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with similar hydrophilicity values ​​that do not have an electrically polar head group include asparagine, glutamine, serine, threonine and tyrosine.

[0057] In the present application, the term "homolog" generally refers to an amino acid sequence or a nucleotide sequence that has a certain degree of homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be understood equivalently to "identity". A homologous sequence may include an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% homologous to a subject amino acid sequence. Typically, a homolog contains an active site, etc. that is homologous to the subject amino acid sequence. Homology may be considered in terms of similarity (i.e., amino acid residues that have similar chemical properties / functions) or may be expressed in terms of sequence identity. In this application, a sequence having a percent identity to any one of the SEQ ID NOs of a referenced amino acid or nucleotide sequence refers to a sequence having said percent identity over the entire length of the referenced SEQ ID NO.

[0058] To determine sequence identity, sequence alignment can be performed. This can be done in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment over the entire length of the sequences being compared.

[0059] In this application, the term "and / or" should be understood to mean either one or both of the alternatives.

[0060] In this application, the term "comprising" generally refers to the inclusion of the explicitly specified features, but is not intended to exclude other elements.

[0061] In this application, the term "about" generally refers to a variation within 0.5%-10% above or below the specified numerical value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0062] gRNA

[0063] On the other hand, the present application provides a gRNA that specifically targets the cytochrome P450 family 4 subfamily V polypeptide 2 gene (CYP4V2 gene), wherein the gRNA specifically targets a mutation site in the CYP4V2 gene.

[0064] In certain embodiments, one or more introns and exons of CYP4V2 are modified with a different number (e.g., 1 pair, 2 pairs, 3 pairs, 4 pairs, 10 pairs, 20 pairs, 50 pairs, 100 pairs) of base pairs removed or gained, nucleotide mutations or substitutions, thereby resulting in a change in CYP4V2 protein structure and loss of function. For example, the mutation sites of the CYP4V2 gene can include the mutation sites shown in Table 1.

[0065] [Table 1-1] [Table 1-2] [Table 1-3]

[0066] In certain embodiments, the mutation site of the CYP4V2 gene comprises c.802-8_810del17bpinsGC. In the present application, the term "c.802-8_810del17bpinsGC" refers to a relatively common CYP4V2 gene mutation, which refers to the removal of 17 base pairs at the 3' end of intron 6 and the 5' end of exon 7, thereby resulting in the deletion of exon 7, leading to changes in the structure and activity of transcription protein. In certain embodiments, the c.802-8_810del17bpinsGC can also be combined with other types of mutations, such as c.992A>C (p.H331P), c.1091-2A>G (Exon 9del).

[0067] In certain embodiments, the gRNA can specifically bind to a nucleotide sequence 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 250bp, 300bp, 350bp, 400bp or 500bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site. In certain embodiments, the gRNA can specifically bind to a nucleotide sequence 200bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site. In certain embodiments, the gRNA is capable of specifically binding to a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to the nucleotide sequence 200 bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site.

[0068] In certain embodiments, the gRNA can specifically bind to a nucleotide sequence complementary to a nucleotide sequence 100bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 250bp, 300bp, 350bp, 400bp, 500bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site. In certain embodiments, the gRNA can specifically bind to a nucleotide sequence complementary to a 200bp sequence upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site. In certain embodiments, the gRNA is capable of specifically binding to a nucleotide sequence complementary to a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to an amino acid sequence complementary to the 200 bp sequence upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site.

[0069] The gRNA described herein can bind to a sequence in a target nucleic acid of interest (e.g., a mutation site in the CYP4V2 gene). The gRNA can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the gRNA can vary depending on the sequence of the target nucleic acid of interest.

[0070] In the present application, the gRNA can include any of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84. In the present application, the gRNA can include any of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84 that have at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to any of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.

[0071] In the present application, the gRNA can include any of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:83, and SEQ ID NO:84. In the present application, the gRNA can include any of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:83, and SEQ ID NO:84 that have at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to any of the nucleotide sequences of SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:83, and SEQ ID NO:84.

[0072] In the present application, the gRNA can include any of the nucleotide sequences of SEQ ID NO: 79, SEQ ID NO: 83, and SEQ ID NO: 84. In the present application, the gRNA can include a nucleotide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to any of the nucleotide sequences of SEQ ID NO: 79, SEQ ID NO: 83, and SEQ ID NO: 84.

[0073] In the present application, the gRNA may contain, from the 5' to 3' end, any of the nucleotide sequences of SEQ ID NOs: 74, 78-80, 82-84 and a backbone sequence, where X is any base selected from A, U, C, and G, and n is any integer from 0 to 15. In the present application, the gRNA may contain 5'-(X)n-any of the nucleotide sequences of SEQ ID NOs: 74, 78-80, 82-84-backbone sequence-3', where X is any base selected from A, U, C, and G, and n is any integer from 0 to 15. For example, the backbone sequence described in the present application may include a backbone sequence derived from AAV-saCas9-puro.

[0074] In certain embodiments, the gRNA may be a single-stranded guide RNA (sgRNA).

[0075] The present application also provides sgRNA combinations, where the sgRNA combinations can include sgRNAs described herein. For example, the sgRNAs can include two or more sgRNAs described herein. For example, the sgRNA combinations can include sgRNA13 and sgRNA17, or for example, the sgRNA combinations can include sgRNA13 and sgRNA18, or for example, the sgRNA combinations can include sgRNA17 and sgRNA18, or for example, the sgRNA combinations can include sgRNA13, sgRNA17, and sgRNA18.

[0076] The present application provides one or more isolated nucleic acid molecules, which can encode a gRNA that specifically targets the above-mentioned CYP4V2 variant gene. For example, the isolated nucleic acid molecule can contain any of the nucleotide sequences of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0077] In the present application, the gRNA sequence can be designed to hybridize to a target nucleic acid near a PAM sequence recognizable by a Cas nuclease. The gRNA can be fully or incompletely complementary to the target sequence. The degree of complementarity between the gRNA and its corresponding target sequence is at least 50% (e.g., at least about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or more). The "Cas nuclease" generally refers to an enzyme that has the ability to recognize and cleave a specific DNA strand using a CRISPR sequence (e.g., gRNA) as a guide. Examples include Cas9 nuclease, Csn1, or Csx12. Cas9 nuclease usually contains a RuvC nuclease domain and an HNH nuclease domain, each of which cleaves two different strands of a double-stranded DNA molecule. Cas9 nuclease has been identified in different bacterial species, such as S. thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and S. pyogenes (Deltcheva, Chylinski et al. 2011).For example, the amino acid sequence of the Cas9 protein from Streptococcus pyogenes is shown in SwissProt database accession number Q99ZW2; the amino acid sequence of the Cas9 protein from Neisseria meningitides is shown in UniProt database accession number A1IQ68; the amino acid sequence of the Cas9 protein from Streptococcus thermophilus is shown in UniProt database accession number Q03LF7; and the amino acid sequence of the Cas9 protein from Staphylococcus aureus (e.g., SaCas in the vectors described herein) is shown in UniProt database accession number J7RUA5. Cas nucleases are typically capable of recognizing specific PAM sequences from DNA. The "PAM" refers to a protospacer adjacent motif, which is recognized as a cleavage site by a CRISPR nuclease, and the PAM is altered by the nuclease (e.g., Cas9, Cpfl, etc.). A protospacer element sequence is usually located directly upstream of the PAM site. For example, the PAM can contain a nucleotide sequence set forth in any of SEQ ID NOs:24-38.

[0078] The gRNA and / or isolated nucleic acid molecule described herein can be delivered using a vector. In the present application, the vector (e.g., pX601) may or may not contain a nucleic acid encoding a Cas nuclease. In the present application, the Cas nuclease can be delivered separately as one or more polypeptides. Alternatively, the nucleic acid molecule encoding the Cas nuclease can be delivered separately or in a complex with one or more guide RNAs, or one or more crRNAs and tracrRNAs. For example, the nucleic acid molecule of the present application (e.g., an isolated nucleic acid molecule encoding an sgRNA that specifically targets the CYP4V2 gene) and the nucleic acid molecule encoding the Cas9 nuclease can be present in the same vector (e.g., a plasmid). The vector can include viral or non-viral vectors known in the art.

[0079] Non-viral delivery vectors include, but are not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras and RNA fusion protein complexes.

[0080] In the present application, the isolated nucleic acid molecule and / or the nucleic acid molecule encoding the DNA endonuclease can be delivered via a plasmid.

[0081] In certain embodiments, the vector may be a viral vector, including, for example, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus. Methods for producing viral vectors containing a nucleic acid molecule (e.g., an isolated nucleic acid molecule described herein) as part of the vector genome are known in the art and can be performed by one of ordinary skill in the art without undue experimentation. In other embodiments, the vector may be a recombinant AAV viral particle that packages a nucleic acid molecule described herein. The method for producing the recombinant AAV may include introducing a nucleic acid molecule described herein into a packaging cell line, introducing a packaging plasmid of rep and cap genes expressing AAV into the cell line, and collecting the recombinant AAV from the packaging cell line supernatant. The packaging cell line may be various types of cells. For example, packaging cell lines that can be used include, but are not limited to, HEK 293 cells, HeLa cells, and Vero cells.

[0082] In certain embodiments, the vector may be an adeno-associated virus (AAV). In this application, the term "adeno-associated virus" generally refers to vectors derived from naturally occurring and useful adenoviruses and artificial AAVs. The AAVs can include different serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, and any AAV variants or mixtures. Both ends of the AAV genome generally have inverted terminal repeats (ITRs), and the term "ITR" or "inverted terminal repeats" refers to nucleic acid sequence segments present in AAV and / or recombinant AAVs that can form T-shaped palindrome structures necessary to complete the lytic and latent life cycle of AAV. The technique of producing AAV vectors is a standard technique in the art and includes providing cells with the polynucleotide to be carried, the rep and cap genes, and the packaged AAV genome that supports viral functions. Production of an AAV vector typically requires the presence of the following components in a single cell (referred to herein as a packaging cell): a rAAV genome, the AAV rep and cap genes isolated from (e.g., not present in) the rAAV genome, and an accessory virus. The AAV rep and cap genes may be from any AAV serotype, including but not limited to the AAV serotypes described herein, and may be from an AAV serotype different from the AAV genomic ITRs.

[0083] Donor nucleic acid molecules and vectors

[0084] On the other hand, the present application provides a donor nucleic acid molecule. In the present application, the term "donor nucleic acid molecule" generally refers to a nucleic acid molecule that provides a heterologous nucleic acid sequence to a recipient (e.g., a recipient nucleic acid molecule). In certain embodiments, the donor nucleic acid molecule can be introduced into a recipient cell to repair a DNA fragment (e.g., a fragmented double-stranded DNA) cleaved by the isolated nucleic acid molecule. In other specific embodiments, the fragmented DNA can be repaired through the donor nucleic acid molecule. Repair methods include, but are not limited to, repair by homologous recombination (HR) that depends on DNA homology and repair by non-homologous end joining (NHEJ).

[0085] In certain embodiments, HDR can be used to insert an exogenous polynucleotide sequence into a target nucleic acid cleavage site. The exogenous polynucleotide sequence is referred to as a donor polynucleotide (or donor, or donor sequence, or polynucleotide donor template). A donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide can be inserted into a target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, rather than a sequence that is naturally present at the target nucleic acid cleavage site. HDR is a method of inserting a specific DNA sequence into a cleavage site using a homologous sequence or a donor sequence (e.g., the donor nucleic acid molecule) as a template. The homologous sequence can be present in an endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, a double-stranded oligonucleotide, or a virus. These exogenous nucleic acids can contain regions of high homology to the locus cleaved by the donor DNA nuclease, and can also contain additional sequences or sequence changes, including deletions that can be integrated into the cleaved target locus. For example, in the present application, a homolog arm can be used to insert an exogenous gene fragment (e.g., a donor nucleic acid molecule described herein) attached to the homolog arm into a target site (e.g., a site targeted by a gRNA described herein). As used herein, a "homolog arm" generally refers to a nucleotide sequence that has homology to the nucleotide sequence of the DNA cleavage site.

[0086] An exogenous donor template can be used to introduce additional nucleic acid sequences (e.g., the targeting vector) or modifications (e.g., single or multiple base modifications or deletions) between the homologous flanking regions to integrate additional or modified nucleic acid sequences into the target locus. The exogenous donor can be carried by a plasmid vector, e.g., an AAV vector and / or a TA cloning vector (e.g., a ZT4 vector).

[0087] With respect to the donor nucleic acid molecule described herein, the donor nucleic acid molecule may be a nucleotide sequence, gene fragment, or gene fragment of wild-type human CYP4V2. For example, the donor nucleic acid molecule may contain a nucleotide sequence within 500bp, 700bp, 750bp, 800bp, 850bp, 900bp, 1000bp, 2000bp, 3000bp, 5000bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site. In a particular embodiment, the donor nucleic acid molecule may contain 800bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site. In certain embodiments, the donor nucleic acid can contain a nucleotide sequence having at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity with the upstream and / or downstream 800bp of the c.802-8_810del17bpinsGC mutation site. The "nucleic acid molecule" that can contain a CYP4V2 nucleotide sequence described herein is distinct from the gRNA or "isolated nucleic acid molecule" that specifically targets CYP4V2 described herein.

[0088] For example, in the present application, the donor nucleic acid molecule can contain the nucleotide sequence of any of SEQ ID NOs: 64-66. Also, for example, the donor nucleic acid molecule can include a nucleotide sequence having at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to the nucleotide sequence of any of SEQ ID NOs: 64-66.

[0089] On the other hand, the present application provides a vector containing the isolated nucleic acid molecule (e.g., an isolated nucleic acid molecule encoding an sgRNA that specifically targets the CYP4V2 gene) and / or the donor nucleic acid molecule (e.g., a nucleotide molecule encoding the human CYP4V2 gene).

[0090] In certain embodiments, the isolated nucleic acid molecule and the donor nucleic acid molecule can be present on different vectors.In other specific embodiments, the isolated nucleic acid molecule and the donor nucleic acid molecule can be present on the same vector.In the present application, when the vector containing the isolated nucleic acid molecule and the vector containing the donor nucleic acid molecule are simultaneously introduced into a cell, Cas nuclease cleaves the donor nucleic acid molecule and the cell genome DNA, and the donor nucleic acid molecule is inserted into the cell genome at a precise position (e.g., CYP4V2 gene fragment).

[0091] In the present application, the vector may contain one or more of the sgRNAs. For example, it may contain one, two, three, four, five, six, seven or more of the sgRNAs described in the present application. In certain embodiments, when multiple sgRNAs are used, the multiple sgRNAs may be present in one or more vectors and may be administered simultaneously or one after the other. For example, the sgRNAs may be present in different vectors and administered, or may be present in the same vector and administered. For example, the sgRNAs may be present in the same AAV vector and administered. For example, the sgRNAs may be located in different vectors and administered simultaneously. For example, the sgRNAs may be located in the same vector and administered one after the other.

[0092] In one embodiment, the vector is a viral vector, such as AAV, lentivirus, retrovirus, adenovirus, herpesvirus and hepatitis virus. Methods for producing viral vectors that contain a nucleic acid molecule (e.g., an isolated nucleic acid molecule described herein) as part of the vector genome are known in the art and can be performed by one of ordinary skill in the art without undue experimentation.

[0093] Cells, pharmaceutical compositions, uses and methods

[0094] The present application provides a cell, which may contain the isolated nucleic acid molecule and / or the donor nucleic acid molecule. The cell described herein may express sgRNA and Cas nuclease and have excellent DNA cleavage effect. The cell described herein may express CYP4V2 protein with normal function. The cell may include a mammalian cell, for example, a cell derived from a human. For example, the cell may include a COS cell, a COS-1 cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a NS0 cell, or a myeloma cell, a stem cell (for example, a pluripotent stem cell and / or a totipotent stem cell), and / or an epithelial cell (for example, a renal epithelial cell and / or a retinal epithelial cell). In the present application, the cell may include a HEK293 cell and / or a urinary renal epithelial cell. In the present application, the cell may have differentiation potential by modification. The differentiation ability can include the ability to differentiate into any type of cell in the body (neurons, astrocytes, oligodendrocytes, retinal epithelial cells, epidermis, hair and keratinocytes, hepatocytes, beta cells, intestinal epithelial cells, lung foam cells, hematopoietic cells, endothelial cells, cardiac myocytes, smooth muscle cells, skeletal muscle cells, kidney cells, adipocytes, chondrocytes and / or bone cells). For example, the cells can be reprogrammed into induced pluripotent stem cells (iPSCs) that overexpress important reprogramming genes (e.g., OCT4, KLF4, SOX2, cMYC, NANOG and / or LIN28).

[0095] The cells described herein can be used to evaluate the efficacy and safety of substances (e.g., sgRNA and donor nucleic acid molecules) required for gene editing therapy. The present application also provides a tissue model that can include a 3D-retinal organoid containing accurate human CYP4V2 cDNA. The cells and tissue models can be used to evaluate the efficacy and safety of substances (e.g., sgRNA and donor nucleic acid molecules) required for gene editing therapy. For example, after introducing the polynucleotide encoding the gRNA, the donor nucleic acid molecule and / or the vector into the cells and / or the tissue model, the expression of the gRNA, accurate CYP4V2 protein can be tested, for example, using PCR sequencing or gel electrophoresis, or the cell and / or tissue model does not produce immune rejection, toxicity, and / or the introduced substance does not affect other functions of the cell and / or the tissue model. For example, repair efficiency testing can be used as an index to evaluate the efficacy of gene editing, and an exemplary method is shown in Example 4. For example, detargeting efficiency can be tested as an index to evaluate the safety of gene editing, and detargeting efficiency can be tested, for example, using whole genome sequencing.

[0096] The present application provides a use of the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, and / or the vector in the manufacture of a medicament for the treatment of a disease, the disease including a disease caused by a CYP4V2 gene mutation. For example, a disease caused by the c.802-8_810del17bpinsGC mutation in the CYP4V2 gene. For example, the disease can include retinitis pigmentosa. For example, the disease can include crystalline retinitis pigmentosa.

[0097] The present application provides a pharmaceutical composition, comprising the gRNA, the one or more isolated nucleic acid molecules, the donor nucleic acid molecule, the vector, and a pharma- ceutically acceptable carrier, which should be non-toxic and should not interfere with the efficacy of the active ingredient.

[0098] The pharmaceutical compositions described herein can be administered locally to the eye by various methods, including, but not limited to, intravitreal injection (e.g., anterior, medial or posterior intravitreal injection), subconjunctival injection, intracameral injection, injection into the anterior chamber via the temporal limbus, intrastromal injection, injection into the subchoroidal space, intracorneal injection, subretinal injection and intraocular injection. The administration can include subretinal injection, which is an injection into the subretinal space, i.e., under the neurosensory retina. During subretinal injection, the injected (e.g., the targeting vector, the gRNA, and / or the plasmid) is introduced directly between the photoreceptor cells and the retinal pigment epithelium (RPE) layer, forming a space between them.

[0099] The present application provides a method of treating Vietti crystalline dystrophy, comprising introducing into a subject in need thereof the gRNA (e.g., an sgRNA specifically targeting the CYP4V2 gene), the one or more isolated nucleic acid molecules (an isolated nucleic acid molecule encoding an sgRNA specifically targeting the CYP4V2 gene), the donor nucleic acid molecule (a nucleotide molecule encoding the human CYP4V2 gene), and / or the vector, wherein said introduction results in the subject obtaining a normally functioning CYP4V2 protein.

[0100] The methods described herein may include ex vivo methods. In certain embodiments, subject-specific induced pluripotent stem cells (iPSCs) may be obtained. The methods described herein may then be used to edit the genomic DNA of these iPSCs. For example, the methods may include editing the CYP4V2 gene of the iPSCs at or near the mutation site so that the gene does not encode a CYP4V2 protein having the mutation. The gene-edited iPSCs may then be differentiated into other cells, such as photoreceptor cells or retinal progenitor cells. Finally, the differentiated cells (e.g., photoreceptor cells or retinal progenitor cells) may be transplanted into the subject.

[0101] In certain embodiments, subject-specific induced pluripotent stem cells (iPSCs) can be obtained. Furthermore, induced pluripotent stem cells can be differentiated into any kind of cell, such as photoreceptor cells or retinal progenitor cells. In the present application, it may be 3D retinal organoids. Then, the genomic DNA of these 3D retinal organoid cells can be edited using the method described in the present application. For example, the method can include editing the 3D retinal organoid cells in or near the mutation site of the CYP4V2 gene so that they do not code for the CYP4V2 protein having the mutation. Finally, the 3D retinal organoid cells can be transplanted into the subject's body.

[0102] In other specific embodiments, photoreceptor cells or retinal progenitor cells can be isolated from a subject. The methods described herein can then be used to edit the genomic DNA of these photoreceptor cells or retinal progenitor cells. For example, the methods can include editing the CYP4V2 gene of photoreceptor cells or retinal progenitor cells at or near the mutation site so that they do not encode CYP4V2 with the mutation. Finally, the gene-edited photoreceptor cells or retinal progenitor cells can be transplanted into the subject.

[0103] The method can include performing a full analysis of the therapeutic agent prior to administration. For example, whole genome sequencing can be performed on the calibration cells to ensure that there are no off-target effects (if any) at genomic locations associated with minimal risk to the subject. Additionally, specific populations of cells, including clonal cell populations, can be isolated prior to transplantation.

[0104] Methods described herein include using site-specific nucleases to cleave DNA at precise target locations in the genome, thereby generating single- or double-stranded DNA breaks at specific locations in the genome, which can be periodically repaired by endogenous cellular processes, such as homologous recombination and non-homologous end joining.

[0105] The method described herein can include generating one or two DNA breaks at a position adjacent to a target sequence in a target locus, and the two DNA breaks can be double-stranded breaks or two single-stranded breaks. The breaks can be achieved by a site-directed polypeptide. The site-directed polypeptide (e.g., a DNA endonuclease) can introduce a double-stranded break or a single-stranded break into a nucleic acid (e.g., genomic DNA). The double-stranded break can stimulate the cell's endogenous DNA repair pathway, e.g., HR, NHEJ.

[0106] An exogenous donor template can be used to introduce additional nucleic acid sequences (e.g., the targeting vector) or modifications (e.g., single or multiple base modifications or deletions) between the homologous flanking regions to integrate additional or modified nucleic acid sequences into the target locus. The exogenous donor can be carried by a plasmid vector, e.g., an AAV vector and / or a TA cloning vector (e.g., a ZT4 vector).

[0107] The present application provides a method for regulating expression of the CYP4V2 gene in a cell, comprising introducing into the cell the gRNA, the one or more isolated nucleic acid molecules, and / or the vector.

[0108] In the present application, the method may be to introduce the gRNA into the cell.For example, the gRNA targets the CYP4V2 gene fragment in the genome of the recipient cell and cleaves it with nuclease, which reduces the chance that the CYP4V2 gene is translated into protein, and the translated protein cannot exert its normal function.

[0109] In the present application, the method may be to introduce the one or more isolated nucleic acid molecules into the cell. For example, the isolated nucleic acid molecule encoding sgRNA specifically targeting the CYP4V2 gene disrupts the CYP4V2 gene fragment, thereby reducing the chance of the CYP4V2 gene being translated into protein, and the translated protein cannot exert its normal function.

[0110] In the present application, the method may be to introduce the vector into the cell. The vector contains the isolated nucleic acid molecule and / or the donor nucleic acid molecule. In a specific embodiment, the vector contains the isolated nucleic acid molecule and the donor nucleic acid molecule, so that the CYP4V2 gene in the cell is replaced by the donor nucleic acid molecule, resulting in a change in the chance of the CYP4V2 gene being translated into protein (e.g., the expression of CYP4V2 protein changes from no expression to a normal amount of expression), and the function of the translated protein changes from abnormal to normal. In a specific embodiment, the vector contains the isolated nucleic acid molecule, so that the CYP4V2 gene fragment is disrupted, so that the chance of the CYP4V2 gene being translated into protein decreases, and the translated protein cannot perform its normal function. In another specific embodiment, the vector contains the donor nucleic acid molecule, so that the vector contains more CYP4V2 gene fragments in the cell, and can be transcribed and translated into more CYP4V2 proteins.

[0111] Without wishing to be bound by a particular theory, the following examples are intended only to illustrate the nucleic acid molecules, proteins, production methods, uses, and the like of the present invention, and are not intended to limit the scope of the present invention. EXAMPLES

[0112] Example 1 Synthesis of sgRNA 1. sgRNA target site Design sgRNA for the mutation site c.802-8_810del17bpinsGC of the CYP4V2 mutant gene. Using the CRISPR-Cas9 system, the CYP4V2 mutant gene in one chromosome can be knocked out from mutant cells, and if the mutations in the two chromosomes are the same, the mutations in the two chromosomes can be knocked out simultaneously.

[0113] 2. sgRNA Design In the mutation site region of CYP4V2, sgRNA sequences with PAM sequences of NNGRRT and NNGRR (Staphylococcus aureus (SA; SaCas9) and a length of 21 bp were designed. The designed 23 sgRNA sequences are set forth in SEQ ID NOs: 67 to 89, respectively. The nucleic acid sequences encoding the sgRNAs are shown in Table 2.

[0114] [Table 2-1] [Table 2-2]

[0115] 3. sgRNA in vitro editing efficacy test (1) In vitro transcription of sgRNA:

[0116] 1) Primer design for in vitro transcription of sgRNA is shown in Table 3. [Table 3]

[0117] 2) Construction of in vitro transcription template for sgRNA The PCR reaction system was as follows. [Table 4]

[0118] The PCR reaction program was as follows: [Table 5]

[0119] 3) Recover the gRNA in vitro transcription template gel using 2.0% DNA gel electrophoresis using the OMEGA gel recovery kit as follows:

[0120] a) An equal volume of membrane binding solution was added to the PCR reaction product, and the recovery from the gel cut required the addition of 1 μL of membrane binding solution per 1 mg, which was then heated at 50-60°C for 7 min until all the gel was completely dissolved, mixed by vortexing, and recovered on a column. b) The above liquid was placed into a recovery column and centrifuged at 10,000×g for 1 minute, and the filtrate was removed. c) 700 μL of washing buffer was added, centrifuged at >13,000×g for 1 minute, and the filtrate was removed. d) Step c) was repeated.

[0121] e) Centrifuge at >13,000 x g for 10 minutes using an empty tube. f) The centrifuge column was transferred to a new 1.5 mL EP tube, marked, and 20-30 μL of elution buffer or ddH2O was added, followed by leaving the tube to stand at room temperature for 2 minutes. g) The mixture was centrifuged at 13,000 x g for 1 min, the adsorption column was removed, and the DNA was stored at 2-8°C. The concentration was measured and recorded, and the DNA was stored long-term at -20°C.

[0122] 4) In vitro transcription of sgRNA (20 μL system): [Table 6]

[0123] Note: The T7 RNA polymerase mixture was added last, mixed, and then placed in a constant temperature incubator at 37°C to react. After the reaction was completed, 2 μL of DNA enzyme I was added, and the mixture was reacted at 37°C for 30 min, followed by electrophoresis.

[0124] 5) Recover gRNA according to the OMEGA gel recovery kit instructions, and the steps are the same as above.

[0125] (2) Preparation of CYP4V2 sgRNA cleavage template DNA was extracted from iPSCs derived from a patient homozygous for CYP4V2 c.802-8_810del17bpinsGC, and used as a template to produce CYP4V2 sgRNA cleaved template dsDNA.

[0126] 1) Genomic DNA extraction: a) Cells were collected by centrifugation at 400×g for 5 min, and the supernatant was removed. 220 μL of PBS (phosphate buffer), 10 mL of RNase solution, and 20 μL of PK working solution were added to the sample, and the cells were resuspended. The sample was left to stand at room temperature for 15 min or more.

[0127] b) 250 μL of GB buffer was added to the cell resuspension, mixed by vortexing, incubated in a water bath at 65° C. for 15-30 min, and purified on a column. c) 250 μL of absolute ethanol was added to the digestion solution and mixed by vortexing for 15-20 s.

[0128] d) A gDNA adsorption column was set in a 2 mL collection tube. The mixture (including precipitate) obtained in the previous step was transferred to the adsorption column. It was centrifuged at 12,000 × g for 1 min. If the column was clogged, it was centrifuged again at 14,000 × g for 3-5 min. If the mixture exceeded 750 μL, it was applied to the column in several aliquots.

[0129] e) The filtrate was discarded, and the adsorption column was placed in a collection tube. 500 μL of washing buffer A was added to the adsorption column. Centrifugation was performed at 12,000 × g for 1 minute. f) The filtrate was discarded, and the adsorption column was placed in a collection tube. 650 μL of washing buffer B was added to the adsorption column. Centrifugation was performed at 12,000 × g for 1 min. g) Step 4 was repeated. h) The filtrate was discarded, and the adsorption column was placed in the collection tube. Centrifugation was performed for 2 minutes at 12,000×g in an empty tube.

[0130] i) The adsorption column was placed in a new 1.5 mL centrifuge tube. 30-100 μL of elution buffer preheated to 70° C. was added to the center of the membrane of the adsorption column and left at room temperature for 3 minutes. Centrifugation was performed at 12,000×g for 1 minute. NOTE: For tissues with high DNA content, the elution can be repeated by adding an additional 30-100 µL of elution buffer. The adsorption column was removed, and the DNA was stored at 2-8°C, the concentration was measured and recorded, and it was stored long-term at -20°C.

[0131] 2) The primers used in the PCR process are shown in Table 4: [Table 7]

[0132] 3) The PCR reaction system is as follows: [Table 8]

[0133] The PCR reaction program was as follows: [Table 9]

[0134] 4) Using 1.5% DNA gel electrophoresis, recover the PCR product using the OMEGA gel recovery kit, and the process is the same as above.

[0135] (3) SaCas9-SgRNA in vitro enzymatic cleavage reaction: The reaction system is as follows: [Table 10]

[0136] After thorough mixing, the mixture was reacted at 37°C for 30 minutes, 3 μL of DNA loading buffer was added and mixed, and the mixture was boiled at 65°C for 5 minutes, and the results of the enzyme cleavage were analyzed by 2% agarose gel analysis.

[0137] The theoretical cleavage fragment lengths are shown in Table 5, and the experimental results are shown in Figure 1A-1I. The results show that sgRNA8, sgRNA12, sgRNA13, sgRNA14, sgRNA16, sgRNA17, and sgRNA18 all have good cleavage effects on the target fragments and can cleave dsDNA into fragments of corresponding lengths. However, other sgRNAs cannot cleave dsDNA into corresponding fragments.

[0138] [Table 11]

[0139] Example 2 Cellular level sgRNA target site validation 1. Synthesis of sgRNA Based on in vitro cleavage experiments, effective sgRNAs were selected: sgRNA8; sgRNA12; sgRNA13; sgRNA14; sgRNA16; sgRNA17; sgRNA18. Based on the sequence of the enzyme cleavage site Bbs1, Bbs1 enzyme cleavage sites (uppercase letters) were added upstream and downstream of the designed sgRNAs, and the nucleic acid molecules encoding the sgRNAs are shown in Table 6.

[0140] [Table 12]

[0141] 2. Construction of sgRNA Vector and Extraction of Plasmid (1) Annealing of sgRNA Each of the sgRNAs (F, R) synthesized above was diluted to 50 μmol, and 5 μL of each of the sgRNA FRs was taken to prepare sgRNA mixtures 1-7. T4 PNK and 10x T4 ligation buffer were thawed on ice until ready for use. The following reactions were prepared: [Table 13]

[0142] The reaction system prepared in the fifth step was placed in a PCR device, and the following reaction program was started. [Table 14] The reaction product was collected.

[0143] (2) Vectorase enzyme cleavage The sgRNA vector was constructed and the plasmid used was the AAV-saCas9-puro vector. The plasmid map is shown in Figure 2.

[0144] The binding site of sgRNA was released using BbsI enzyme cleavage, and the following enzyme cleavage reaction system was prepared in a 1.5 mL PCR tube. [Table 15]

[0145] After 1-2 hours of enzymatic digestion (or overnight at 4°C), the product was collected and purified, its concentration was measured, and it was diluted to 50 ng / μL.

[0146] (3) Ligation Using the vector recovered in the previous step and the annealed sgRNA, the following ligation system (200 μL PCR tube) was prepared. [Table 16]

[0147] The ligation reaction system from the previous step was placed at 37°C for approximately 1-2 hours for ligation to complete the construction of the sgRNA vector.

[0148] (4) Plasmid transformation

[0149] (1) TransStbl3 chemically competent cells (CD-521-02, Beijing Global Gold Biotechnology Co., Ltd.) were taken out and thawed on ice. (2) 1 μL of the ligation product was added to 50 μL of competent cells, incubated on ice for 30 minutes, heat shocked at 42° C. for 90 s, and allowed to stand on ice for 2 minutes. (3) 500 μL of non-antibacterial culture medium was added and shaken at 37°C and 200 rpm for 1 hour. (4) After centrifugation at 800 rpm / min for 5 minutes, the supernatant was removed to obtain approximately 100 μL, and positive clones were screened using ampicillin-containing LB agar medium. (5) The next day, the screened positive clone bacteria (500 μL of culture medium) were shaken for 3-4 hours, and a 200 μL sample was sequenced.

[0150] 5. Extraction of plasmid (using Omega's Endofree Plasmid Maxi Kit)

[0151] 1) Plasmids that were determined to be accurate by sequencing were shaken overnight (50-200 mL), then shaken cultured at 37°C for 12-16 hours to amplify the plasmids, and extracted the next day (shaking time was less than 16 hours).

[0152] 2) 50-200 mL of the bacterial liquid was taken out and centrifuged at 4000 x g for 10 minutes at room temperature to collect the bacterial cells.

[0153] 3) The medium was discarded. 10 mL of Solution I / RNA enzyme mixture was added to the pellet, and the cells were completely resuspended by pipetting or vortexing.

[0154] 4) Add 10 mL of Solution II, close the lid, and gently invert the centrifuge tube upside down 8 to 10 times to obtain a clear lysate.

[0155] 5) Add 5 mL of pre-chilled N3 buffer, close the lid, and gently invert the centrifuge tube upside down 10 times until a white aggregate precipitate forms. You can leave it at room temperature for 2 minutes to incubate.

[0156] 6) Prepare a syringe filter, pull out the piston from the syringe, place the syringe vertically in a suitable test tube rack, and place a collection tube at the outlet of the lower end of the injector with the opening of the syringe facing upwards. The lysate was immediately poured into the filter syringe. The cell lysate was left to stand in the syringe for 5 min. Then, white aggregates began to float on the surface of the lysate. The cell lysate may have flowed out from the outlet of the filtration injector. Collect the cell lysate in a new 50 mL test tube. Carefully insert the injector piston lightly into the syringe and slowly push the piston so that the lysate flows into the collection tube.

[0157] 7) 0.1 volume of ETR solution (blue color) was added to the flow-through filtered lysate, the test tube was inverted 10 times, and the tube was placed in an ice bath for 10 minutes.

[0158] 8) The above lysate was placed in a water bath at 42℃ for 5 minutes. The lysate became cloudy again. Then, it was centrifuged at 25℃ and 4000×g for 5 minutes, and the ETR buffer formed a blue layer at the bottom of the test tube.

[0159] 9) The supernatant was transferred to another new 50 mL test tube, and 0.5 volumes of room temperature absolute ethanol was added. The test tube was gently inverted 6-7 times and allowed to stand at room temperature for 1-2 minutes.

[0160] 10) A HiBind® DNA Maxi Binding column was placed in a 50 mL collection tube, and 20 mL of the filtrate was added to the HiBind® DNA Maxi Binding column. Centrifugation was performed at room temperature for 3 minutes at 4000×g. The filtrate was discarded.

[0161] 11) Place a HiBind® DNA Maxi Binding column in the same collection tube and repeat step 10 and centrifuge under the same conditions until all the remaining filtrate is bound to the HiBind® DNA Maxi Binding column.

[0162] 12) A HiBind® DNA Maxi binding column was placed in the same collection tube, 10 mL of HBC buffer was added to the HiBind® DNA Maxi binding column, and the column was centrifuged at room temperature at 4000×g for 3 minutes, and the filtrate was discarded.

[0163] 13) A HiBind® DNA Maxi Binding column was placed in the same collection tube, and 15 mL of DNA washing solution (diluted with absolute ethanol) was added to the HiBind® DNA Maxi Binding column. The column was centrifuged at room temperature at 4000×g for 3 minutes, and the filtrate was discarded.

[0164] 14) A HiBind® DNA Maxi Binding column was placed in the same collection tube, 10 mL of DNA washing solution (diluted with absolute ethanol) was added to the HiBind® DNA Maxi Binding column, and the column was centrifuged at room temperature at 4000×g for 3 minutes, and the filtrate was discarded.

[0165] 15) Spin at maximum speed (<6000 x g) to dry the matrix of the HiBind® DNA Maxi Binding Column for 10 min.

[0166] 16) (Optional) Further air-dry the HiBind® DNA Maxi Binding Column. (Optional) Further dry the HiBind® DNA Maxi Binding Column before eluting the DNA (if required) by one of the following methods:

[0167] a) Place the HiBind® DNA Maxi Binding column in a vacuum chamber for 15 min to dry off the ethanol: At room temperature, transfer the column to a vacuum chamber and connect all the devices in the vacuum chamber. Seal the vacuum chamber and apply vacuum for 15 min. Transfer the HiBind® DNA Maxi Binding column to the next step. b) Dry the column in a vacuum oven or at 65°C for 10-15 min. Transfer the HiBind® DNA Maxi Binding column to the next step.

[0168] 17) Place the HiBind® DNA Maxi Binding Column in a clean 50 mL centrifuge tube and add 1-3 mL of endotoxin-free elution buffer directly to the matrix of the HiBind® DNA Maxi Binding Column (the amount depends on the concentration of the final product) and let stand at room temperature for 5 minutes.

[0169] 18) The DNA was eluted by centrifugation at 4000×g for 5 minutes. 19) The column was discarded and the DNA product was stored at -20°C. 20) To ensure that the constructed plasmids were accurate, the extracted plasmids were sequenced again.

[0170] 3. Verification of the efficacy of sgRNA in 293T cells (1) Cultivation of 293T cells 1) Thawing of cryopreserved cells

[0171] a) The temperature of the thermostatic water bath was adjusted to 37°C, and the frozen preserved cells were removed from the liquid nitrogen, and the lids were held with tweezers and quickly shaken in water.

[0172] b) The frozen storage solution was transferred to a 15 mL graduated centrifuge tube, 10 mL of cell culture solution was slowly added, and the tube was gently shaken to mix the liquid. The tube was then closed, heated, and centrifuged at 1000 rpm / min for 3 minutes.

[0173] c) After heating, an appropriate amount of culture medium was added, and the cell precipitate at the bottom was gently pipetted, after which the cells were transferred to a culture flask and cultured in an incubator. The medium used for 293T cells was high glucose DMEM supplemented with 10% fetal bovine serum and 100U / mL double antibody, and the cells were cultured at 37℃ and 5% CO2.

[0174] 2) Passaging of cells a) The morphology and density of the cells were observed under an inverted microscope, and when the confluency of the cells in the culture flask reached 80%-90%, the cells were subcultured.

[0175] b) Wash out the old culture medium from the cell culture bottle and wash it with PBS three times. Add 500μL of EDTA-containing trypsin to the culture bottle and incubate it in an incubator for about 1min. When the gaps between the cells become larger and the cells become round, immediately add 1mL of culture medium to the culture bottle to stop digestion. Gently pipette the cells with a suction tube until all the cells float from the bottom of the bottle. Transfer the liquid in the culture bottle to a centrifuge tube and centrifuge at 1000rpm / min for 2min.

[0176] c) The supernatant was removed, 2 mL of medium was added to the centrifuge tube, and the precipitated cells were resuspended. The cell suspension was placed in four new culture bottles, 4 mL of culture medium was added to each, and the culture bottles were gently shaken to mix the cells and spread them evenly in the culture bottles, and then the bottles were placed in a cell incubator for culture.

[0177] d) One day before transfection, 100w of 293T cells with 80% density, cell spreading, and uniform cell spacing were added per well, and the next day, when the cells reached 80-90% confluency, plasmid transfection was performed.

[0178] (2) Transfection of 293T cells using the PEI (polyethyleneimine) method 1) Take 1.5mL EP tubes, number them in the above order, add 250μL of DMEM medium (without serum) to each tube, add 1.5μg of AAV-CYP4V2- sgRNA8, sgRNA12, sgRNA13, sgRNA14, sgRNA16, sgRNA17, sgRNA18 plasmid and empty plasmid, mix thoroughly by vortexing, add 7.5μL of PEI transfection reagent to each tube, mix by vortexing, leave at room temperature for 20min, and then perform transfection. A negative control was also placed.

[0179] 2) Drop the medium (6-well plate medium, serum-free DMEM = 2 mL / well) and culture in an incubator, replacing half of the medium every 12-18 hours. The next day, observe the GFP expression status under a fluorescent microscope to evaluate the transfection efficiency, and if the transfection efficiency was good, continue culturing the transfected plasmid.

[0180] 3) Screening for resistant cells a) Preparation of solution: Puromycin 10 mg / mL (mother solution) diluted to 1 μg / mL, screened cells.

[0181] b) Fluid replacement: Two days after transfection, add 3mL of puromycin-containing 293T cell medium per well (including the negative control), and begin screening for positively transfected cells. After that, observe cell viability every day, replace the fluid every two days, and add the corresponding amount of puromycin when replacing the fluid. If the cells in the negative control wells are completely dead, while the cells in the experimental and control groups are alive (indicating successful transfection), discontinue antibiotic screening and use normal medium instead.

[0182] (3) Extraction of genomic DNA from 293T cells

[0183] (1) After the cells in the 6-well plate reached 80-90% confluence, they were subcultured in a 6 cm culture dish.

[0184] (2) After the cells reach 80-90% confluency, harvest the cells and prepare for genomic DNA extraction. The whole process takes about 7-10 days. For genome extraction, we use a cell extraction kit from Nanjing Vazyme Biotech. The experimental steps are as follows:

[0185] a) Cells were collected by centrifugation at 400×g for 5 min and the supernatant was removed. 220 μL of PBS, 10 mL of RNase buffer and 20 μL of PK working solution were added to the sample to resuspend the cells. The sample was left to stand at room temperature for 15 min or more.

[0186] b) 250 μL of GB buffer was added to the cell resuspension, mixed by vortexing, incubated in a water bath at 65° C. for 15-30 min, and purified on a column. c) 250 μL of absolute ethanol was added to the digestion solution and mixed by vortexing for 15-20 s.

[0187] d) A gDNA adsorption column was set in a 2 mL collection tube. The mixture (including precipitate) obtained in the previous step was transferred to the adsorption column. It was centrifuged at 12,000 × g for 1 min. If the column was clogged, it was centrifuged again at 14,000 × g for 3-5 min. If the mixture exceeded 750 μL, it was applied to the column in several aliquots.

[0188] e) The filtrate was discarded, and the adsorption column was placed in a collection tube. 500 μL of washing buffer A was added to the adsorption column. Centrifugation was performed at 12,000 × g for 1 minute. f) The filtrate was discarded, and the adsorption column was placed in a collection tube. 650 μL of washing buffer B was added to the adsorption column. Centrifugation was performed at 12,000 × g for 1 min. g) Step 4 was repeated. h) The filtrate was discarded, and the adsorption column was placed in the collection tube. Centrifugation was performed for 2 minutes at 12,000×g in an empty tube.

[0189] i) The adsorption column was placed in a new 1.5 mL centrifuge tube. 30-100 μL of elution buffer preheated to 70° C. was added to the center of the membrane of the adsorption column and left at room temperature for 3 minutes. Centrifugation was performed at 12,000×g for 1 minute. NOTE: For tissues with high DNA content, the elution can be repeated by adding an additional 30-100 µL of elution buffer.

[0190] j) The adsorption column was removed, and the DNA was stored at 2-8°C, the concentration was measured and recorded, and then stored long-term at -20°C.

[0191] (4) T7E1 enzyme cleavage experiment 1) Using genomic DNA extracted from 293T cells transfected with the above AAV-CYP4V2- sgRNA8, sgRNA12, sgRNA13, sgRNA14, sgRNA16, sgRNA17, and sgRNA18 plasmids as a template, the extracted genomic DNA was amplified using upstream and downstream primers near the target site, and PCR of the DNA fragment was performed. The primer sequences are shown in Table 7.

[0192] [Table 17]

[0193] 2) Liquid DNA was extracted from the PCR products using a liquid extraction kit (OMEGA Gel Extraction Kit (200) D2500-02). The DNA extraction process is as follows:

[0194] a) An equal volume of membrane binding solution was added to the PCR reaction product, and the recovery from the gel cut required the addition of 1 μL of membrane binding solution per 1 mg, which was then heated at 50-60°C for 7 min until all the gel was completely dissolved, mixed by vortexing, and recovered on a column. b) The above liquid was placed into a recovery column and centrifuged at 10,000×g for 1 minute, and the filtrate was removed. c) 700 μL of washing buffer was added, centrifuged at >13,000×g for 1 minute, and the filtrate was removed. d) Step c) was repeated. e) Centrifuge at >13,000 x g for 10 minutes using an empty tube. f) The centrifuge column was transferred to a new 1.5 mL EP tube, marked, and 20-30 μL of elution buffer or ddH2O was added, followed by leaving the tube to stand at room temperature for 2 minutes. g) The mixture was centrifuged at 13,000 x g for 1 min, the adsorption column was removed, and the DNA was stored at 2-8°C. The concentration was measured and recorded, and the DNA was stored long-term at -20°C.

[0195] 3) Verification of sgRNA efficiency by T7E1 enzyme cleavage experiment The PCR or gel cut recovered products obtained above were subjected to T7E1 enzyme digestion reaction.

[0196] [Table 18]

[0197] [Table 19]

[0198] [Table 20] The reaction was incubated at 37° C. for 20 min.

[0199] d) Electrophoresis of enzyme cleavage products Gel preparation: 2.5% gel, 2x dye Electrophoresis program: 140V, 20min to 30min

[0200] e) Confirmation of electrophoresis results The results of the enzymatic digestion are shown in FIG. 3, and the fragment lengths after each digestion are shown in Table 8.

[0201] [Table 21]

[0202] As a result, sgRNA8, sgRNA13, sgRNA17, and sgRNA18 all had the cleavage effect on the target site, and the cleaved fragments were consistent with those shown in Table 8.

[0203] (5) Gene sequencing of PCR fragments containing cleavage sites in genomic DNA, multi-peak test The above amplified DNA fragments were sequenced, and the sequencing spectra are shown in Figures 4A-C. As a result, the cleavage efficiency of sgRNA13, sgRNA17, and sgRNA18 was high, and multiple peaks appeared near the sgRNA cleavage site.

[0204] 4. Verification of double sgRNA cleavage efficiency (1) Construction and extraction of double sgRNA-AAV-saCas9-puro plasmid

[0205] According to the above experimental results, the cleavage efficiency of sgRNA13.17.18 is high. In the AAV-saCas9-sgRNA13 plasmid constructed in the above experiment, the region between the sa-gRNA Scaffold and the AAV ITR region was cleaved with NotI enzyme, and then the U6 promoter-sgRNA17-saScaffold fragment and the U6 promoter-sgRNA18-sa-gRNA Scaffold were inserted, respectively, to construct the AAV-saCas9-sgRNA13-sgRNA17 and AAV-saCas9-sgRNA13-sgRNA18 plasmids.

[0206] 1) The PCR primer sequences for the U6 promoter-sgRNA17-saScaffold fragment and the U6 promoter-sgRNA18-sa-gRNA Scaffold fragment are shown in Table 9.

[0207] [Table 22]

[0208] The PCR reaction system was as follows. [Table 23]

[0209] The PCR reaction program was as follows: [Table 24]

[0210] 2) Recover the gRNA in vitro transcription template gel using 2.0% DNA gel electrophoresis using the OMEGA gel recovery kit as follows:

[0211] a) An equal volume of membrane binding solution was added to the PCR reaction product, and the recovery from the gel cut required the addition of 1 μL of membrane binding solution per 1 mg, which was then heated at 50-60°C for 7 min until all the gel was completely dissolved, mixed by vortexing, and recovered on a column. b) The above liquid was placed into a recovery column and centrifuged at 10,000×g for 1 minute, and the filtrate was removed. c) 700 μL of washing buffer was added, centrifuged at >13,000×g for 1 minute, and the filtrate was removed. d) Step c) was repeated.

[0212] e) Centrifuge at >13,000 x g for 10 minutes using an empty tube. f) The centrifuge column was transferred to a new 1.5 mL EP tube, marked, and 20-30 μL of elution buffer or ddH2O was added, followed by leaving the tube to stand at room temperature for 2 minutes. g) The mixture was centrifuged at 13,000 x g for 1 min, the adsorption column was removed, and the DNA was stored at 2-8°C. The concentration was measured and recorded, and the DNA was stored long-term at -20°C.

[0213] 3) Enzyme-cleavable vector The site was released using NotI enzyme digestion, and the following enzyme digestion reaction system was prepared in a 1.5 mL PCR tube. [Table 25]

[0214] After 1-2 hours of enzymatic digestion (or overnight enzymatic digestion), the product was collected and purified, its concentration was measured, and it was diluted to 50 ng / μL.

[0215] 4) Ligation Using the vector recovered in the previous step and the annealed sgRNA, the following ligation system (200 μL PCR tube) was prepared. [Table 26]

[0216] The ligation reaction system from the previous step was placed at 37°C for approximately 1-2 hours for ligation to complete the construction of the sgRNA vector.

[0217] 5) Plasmid transformation a) TransStbl3 chemically competent cells were removed and thawed on ice. b) 1 μL of the ligation product was added to 50 μL of competent cells, incubated on ice for 30 minutes, heat shocked at 42° C. for 90 seconds, and then allowed to stand on ice for 2 minutes. c) 500 μL of non-antibacterial culture medium was added and shaken at 37°C and 200 rpm for 1 hour. d) The mixture was centrifuged at 800 rpm / min for 5 minutes, and the supernatant was removed to obtain approximately 100 μL. Positive clones were screened using ampicillin-containing LB agar medium. e) The next day, the screened positive clone bacteria (500 μL of medium) were shaken for 3-4 hours, and a 200 μL sample was subjected to sequencing.

[0218] 6) Extraction of plasmid (using Omega's Endofree Plasmid Maxi Kit) was the same as above.

[0219] (2) Verification of the efficacy of double sgRNA and single sgRNA in 293T cells 1) Cultivation of 293T cells a) Thawing of cryopreserved cells i) The temperature of the thermostatic water bath was adjusted to 37°C, and the frozen preserved cells were removed from the liquid nitrogen, and, holding the lid with tweezers, were quickly shaken in water. ii) The frozen storage solution was transferred to a 15 mL graduated centrifuge tube, 10 mL of cell culture solution was slowly added, and the tube was gently shaken to mix the liquid. The tube was then closed, heated, and centrifuged at 1000 rpm / min for 3 minutes. iii) After heating, an appropriate amount of culture medium was added, and the cell precipitate at the bottom was gently pipetted, after which the cells were transferred to a culture flask and cultured in an incubator. The medium used for 293T cells was high glucose DMEM supplemented with 10% fetal bovine serum and 100U / mL double antibody, and the cells were cultured at 37℃ and 5% CO2.

[0220] b) Cell Passage i) The morphology and density of the cells were observed under an inverted microscope, and when the confluence of the cells in the culture flask reached 80%-90%, subculture of the cells was started.

[0221] ii) Wash out the old culture medium from the cell culture bottle and wash it with PBS three times. Add 500μL of EDTA-containing trypsin to the culture bottle and incubate it in an incubator for about 1min. When the gaps between the cells become larger and the cells become round, immediately add 1mL of culture medium to the culture bottle to stop digestion. Gently pipette the cells with an aspirator until all the cells float from the bottom of the bottle. Transfer the liquid in the culture bottle to a centrifuge tube and centrifuge at 1000rpm / min for 2min.

[0222] iii) The supernatant was removed, 2 mL of medium was added to the centrifuge tube, and the precipitated cells were resuspended. The cell suspension was placed in four new culture bottles, 4 mL of culture medium was added to each, the culture bottles were gently shaken to mix the cells, and the cells were evenly spread in the culture bottles, and then the bottles were placed in a cell incubator for culture.

[0223] iv) One day before transfection, 100w of 293T cells with 80% density, cell spreading, and uniform cell spacing were added per well, and the next day, when the cells reached 80-90% confluency, plasmid transfection was performed.

[0224] (2) Transfection of 293T cells using the PEI (polyethyleneimine) method a) Take 1.5mL EP tubes, number them in the above order, add 250μL of DMEM medium (without serum) to each tube, add 1.5mg of AAV-CYP4V2-sgRNA13 plasmid, AAV-CYP4V2-sgRNA17 plasmid, AAV-CYP4V2-sgRNA18 plasmid, AAV-CYP4V2-sgRNA 13-sgRNA17 plasmid, AAV-CYP4V2-sgRNA 13-sgRNA18 plasmid and empty plasmid in order, mix thoroughly by vortex, add 7.5μL of PEI transfection reagent to each tube, mix by vortex, and leave at room temperature for 20min before transfection. A negative control was also installed.

[0225] b) Drop it into the medium (6-well plate medium, serum-free DMEM = 2mL / well), culture it in an incubator, and replace half of the medium every 12-18 hours. The next day, observe the GFP expression status under a fluorescent microscope to evaluate the transfection efficiency, and if the transfection efficiency was good, continue culturing the transfected plasmid.

[0226] c) Screening for resistant cells i) Preparation of solution: Puromycin diluted from 10 mg / mL (mother solution) to 1 μg / mL, screened cells ii) Fluid replacement: Two days after transfection, 3mL of 293T cell medium containing puromycin was added per well (including the negative control), and screening for positively transfected cells was started, and the cell viability was observed every day thereafter, and the fluid was replaced every two days, with the corresponding amount of puromycin added when replacing the fluid. If the cells in the negative control wells were completely dead, and the cells in the experimental and control groups were viable (indicating successful transfection), antibiotic screening was discontinued, and normal medium was used instead.

[0227] 3) Extraction of genomic DNA from 293T cells a) After the cells in the 6-well plate reached 80-90% confluency, they were subcultured in a 6 cm culture dish.

[0228] b) After the cells reach 80-90% confluency, harvest the cells and prepare for genomic DNA extraction. The whole process takes about 7-10 days. For genome extraction, we use a cell extraction kit from Nanjing Vazyme Biotech, and the experimental steps are as follows:

[0229] i) Cells were collected by centrifugation at 400×g for 5 min and the supernatant was removed. 220 μL of PBS, 10 mL of RNase solution and 20 μL of PK working solution were added to the sample to resuspend the cells. The sample was left to stand at room temperature for 15 min or more. ii) 250 μL of GB buffer was added to the cell resuspension, mixed by vortexing, incubated in a water bath at 65° C. for 15-30 min, and purified on a column. iii) 250 μL of absolute ethanol was added to the digestion solution and mixed by vortexing for 15-20 s. iv) A gDNA adsorption column was set in a 2 mL collection tube. The mixture (including precipitate) obtained in the previous step was transferred to the adsorption column. It was centrifuged at 12,000 × g for 1 min. If the column was clogged, it was centrifuged again at 14,000 × g for 3-5 min. If the mixture exceeded 750 μL, it was applied to the column in several aliquots. v) The filtrate was discarded, and the adsorption column was placed in a collection tube. 500 μL of washing buffer A was added to the adsorption column. Centrifugation was performed at 12,000 × g for 1 min.

[0230] vi) The filtrate was discarded, and the adsorption column was placed in a collection tube. 650 μL of washing buffer B was added to the adsorption column. Centrifugation was performed at 12,000 × g for 1 min. vii) Step 4 was repeated. viii) The filtrate was discarded, and the adsorption column was placed in the collection tube. Centrifugation was performed at 12,000×g for 2 min. ix) The adsorption column was placed in a new 1.5 mL centrifuge tube. 30-100 μL of elution buffer preheated to 70° C. was added to the center of the membrane of the adsorption column and left at room temperature for 3 minutes. Centrifugation was performed at 12,000×g for 1 minute. NOTE: For tissues with high DNA content, the elution can be repeated by adding an additional 30-100 µL of elution buffer. x) The adsorption column was removed, and the DNA was stored at 2-8°C, the concentration was measured and recorded, and then stored long-term at -20°C.

[0231] 4) Gene sequencing of PCR fragments containing cleavage sites in genomic DNA, multi-peak test a) Based on the cleavage target site of the DNA, primers were designed to amplify the fragment. The primer sequences are shown in Table 10.

[0232] [Table 27]

[0233] b) Vector Linearization and Ligation The plasmid vector is PMD-19T-MCS vector, the vector linearization method is the same as above, the enzymes used are MluI enzyme and KpnI enzyme, which are not detailed here. The PCR product was collected, purified, sequenced, and ligated into the PMD-19T-MCS linearized vector, the method is the same as above, which is not detailed here.

[0234] c) Plasmid transformation i) TransStbl3 chemically competent cells were removed and thawed on ice. ii) 1 μL of the ligation product was added to 50 μL of competent cells, incubated on ice for 30 minutes, heat shocked at 42° C. for 90 seconds, and then allowed to stand on ice for 2 minutes. iii) 500 μL of non-antibacterial culture medium was added and shaken at 37°C and 200 rpm for 1 hour. iv) The mixture was centrifuged at 800 rpm / min for 5 minutes, and about 100 μL of the supernatant was removed. Positive clones were screened using ampicillin-containing LB agar medium. v) The next day, 80 of each of the screened positive clones were picked and subjected to sequencing.

[0235] d)Positive rate analysis The sequencing spectrum was calculated and the positive rates were as follows: The sgRNA13 positive rate was 54%, the sgRNA17 positive rate was 48.5%, the sgRNA18 positive rate was 62.5%, the sgRNA13+17 positive rate was 64%, and the sgRNA13+18 positive rate was 51.2%. It was found that the double sgRNA positivity rate increased to some extent. The experimental results are shown in Figure 5.

[0236] Example 3 Screening of Donor (Donor Nucleic Acid Molecule) 1, Donor design The donor design used the HDR method. Considering that the cleavage site of sgRNA13 is in intron 6 and the cleavage sites of sgRNA17 and sgRNA18 are in exon 7, the EGFP gene was selected and inserted between the cleavage sites of sgRNA17 and sgRNA18 (specifically, exon 7: c.802+42 site), and 800 bp homolog arms were added to the left and right of the EGFP gene. The Donor design drawing is shown in Figure 6. The donor sequence is set forth in any of SEQ ID NOs:64-66.

[0237] 2. The construction and extraction procedures of PMD19-T-donor plasmid were the same as above, and will not be described here.

[0238] 3. Verification of donor efficacy using 293T cells (1) Cultivation of 293T cells The process is the same as above, so I won't go into detail here.

[0239] (2) Transfection of 293T cells using the PEI (polyethyleneimine) method 1) Take 1.5mL EP tubes, number them in the above order, add 250μL of DMEM medium (without serum) to each tube, add 1.5μg of AAV-CYP4V2-sgRNA13 plasmid, AAV-CYP4V2-sgRNA17 plasmid, AAV-CYP4V2-sgRNA18 plasmid, AAV-CYP4V2-sgRNA13-sgRNA17 plasmid, AAV-CYP4V2-sgRNA13-sgRNA18 plasmid and empty plasmid in order, mix thoroughly by vortex, add 1.5μg of PMD19-T-donor plasmid (see table below for correspondence) corresponding to each tube, mix, add 7.5μL of PEI transfection reagent, mix by vortex, leave at room temperature for 20min, and then perform transfection. A negative control was also installed.

[0240] [Table 28]

[0241] 2) Drop the medium (6-well plate medium, serum-free DMEM = 2 mL / well) and culture in an incubator, replacing half of the medium every 12-18 hours. The next day, observe the GFP expression status under a fluorescent microscope to evaluate the transfection efficiency, and if the transfection efficiency was good, continue culturing the transfected plasmid.

[0242] 3) Screening for resistant cells a) Preparation of solution: Puromycin 10 mg / mL (mother solution) diluted to 1 μg / mL, screened cells.

[0243] b) Fluid replacement: Two days after transfection, add 3mL of puromycin-containing 293T cell medium per well (including the negative control), and begin screening for positively transfected cells. After that, observe cell viability every day, replace the fluid every two days, and add the corresponding amount of puromycin when replacing the fluid. If the cells in the negative control wells are completely dead, while the cells in the experimental and control groups are alive (indicating successful transfection), discontinue antibiotic screening and use normal medium instead.

[0244] 4) Flow cytometry screening The cells after antibiotic screening (about 7 days) were screened by flow cytometry in the following steps.

[0245] a) The medium in the 6-well plate was removed by suction, and the plate was washed twice with DPBS (Dulbecco's phosphate buffer). b) 500 μL of 0.05% trypsin was added, and digestion was incubated at 37° C. for 4 minutes. c) 3-5 volumes of DMEM neutralized trypsin were added and centrifuged at 800 r / min for 2 min. d) Aspirate the supernatant, add PBS heavy suspension cells, centrifuge at 800 r / min for 2 min, and repeat once; e) The supernatant was aspirated and 200 μL of PBS containing 2% FBS was added and the cells resuspended. f) The liquid obtained in the above step was added to the filtration tube and allowed to pass entirely through the filtration mesh. g) After the incubation, the positive cells were transferred to 12-well or 6-well plates and cultured.

[0246] 5) Extraction of genomic DNA from 293T cells a) After the cells in the 6-well plate reached 80-90% confluency, they were subcultured in a 6 cm culture dish. b) After the cells reach 80-90% confluency, harvest the cells and prepare for genomic DNA extraction. The whole process takes about 7-10 days. The genome extraction is performed using a cell extraction kit from Nanjing Vazyme Biotech. The experimental process is the same as above, so we will not go into detail here.

[0247] 6) Sequencing of target fragment a) Primers were designed upstream and downstream of the donor, and enzyme cleavage sites were added upstream and downstream of the primers.

[0248] b) Vector Linearization and Ligation The plasmid vector is PMD-19T-MCS vector, the vector linearization method is the same as above, the enzymes used are MluI enzyme and KpnI enzyme, which are not detailed here. The PCR product was collected, purified, sequenced, and ligated into the PMD-19T-MCS linearized vector, the method is the same as above, which is not detailed here.

[0249] c) Plasmid transformation The method is the same as above.

[0250] d) Sequencing Each plasmid was picked and subjected to sequencing, and the sequencing results were tabulated.

[0251] Example 4 In vitro validation of gene editing efficiency of sgRNA using 3D retinal tissue from patients 1. Extraction and culture of renal epithelial cells from patients The experiment was carried out using a renal epithelial cell isolation and culture kit provided by Beijing Speil. The experimental steps are as follows:

[0252] 1) UrinEasy complete isolation medium, additives, gelatin, and washing solution were brought into the cell culture room. 2) Turn on the ultraviolet lamp and prepare a 12-well plate, a 50 mL centrifuge tube, a 15 mL centrifuge tube, an electric pipette, a pipette, an aspirator, a 5 mL micropipette and tips, and a 1 mL micropipette and tips. Open the 37°C water bath. 3) Urine collection: Wearing gloves, disinfecting, midstream urine was collected and sealed in sealing film. 4) Gelatin was applied to the bottom of the dish (3 wells) at 750 μL / well and allowed to stand at 37° C. for more than 30 minutes. 5) The outer surface of the urinal was disinfected with 75% alcohol, and the sample was divided into 50 mL conical-bottom centrifuge tubes, sealed, and centrifuged at 400 x g for 10 minutes.

[0253] 6) Prepare UrinEasy Isolation Complete Medium and additives by removing them (mix 5 mL of basal medium per 0.5 mL of additive). 7) Move the pipette along the upper liquid surface at minimal speed to slowly aspirate the supernatant until 1 mL is remaining. 8) The cells were resuspended and placed in a 15 mL centrifuge tube, 10 mL of washing solution was added and mixed, and the tube was centrifuged at 200×g for 10 minutes. 9) The 12-well plate was taken out, the gelatin was removed by suction, and the plate was washed once with washing solution (500 μL). 750 μL of UrinEasy complete isolation medium was added per well, and the plate was allowed to stand at 37°C. 10) The 15 mL centrifuge tube was removed, leaving a 0.2 mL cell pellet. 11) Resuspend the cell pellet in UrinEasy Isolation Complete Medium: 1 well for males and 2 wells for females, designated as D0 (day 0).

[0254] 12) Observation: Day 1: Observe for the presence or absence of contamination; Day 2: Replenishment of isolation medium - female: 500 μL / well; male: 250 μL / well; Day 4: If no attachment occurs: Replace half the volume of medium, changing the liquid every 2 days and adding 1 mL of isolation complete medium along the surface.

[0255] 13) After attachment: Once the cells had attached to the surface (3–7 days or 9–10 days), they were cultured in UrinEasy Amplification Complete Medium, and after 2 days of culture, the entire volume was replaced with 500 μL of culture medium. After attachment to the surface, approximately 9-12 days (but not more than 14 days) the cells were passaged when they reached 80-90% confluence, then passaged sequentially into 6-well plates, 6 cm culture dishes, and 10 cm culture dishes, and frozen for later use.

[0256] (2) Induction of iPSCs Patient-derived (c.802-8_810del17bpinsGC) kidney epithelial cells were induced into iPSCs by the following steps:

[0257] 1) When the somatic cells reach 70-90% confluence, digestion and subculture can be performed. The cells are seeded into a 96-well plate; the seeding density is controlled to be 5000-15000 cells / well, and three density gradients can be set according to the cell conditions, with triple wells set up for each gradient. The day of cell seeding is designated as day -1.

[0258] 2) Day 0: The cells were observed under a microscope for confluence and their condition. Digestion and counting were performed on the multi-wells with different gradients, and initialization was performed on the wells where the number of cells reached 10,000-20,000. The composition of the initialization medium A is as follows: [Table 29]

[0259] 3) After centrifuging the initialization additive II, 97 μL of initialization medium A was added to the test tube of initialization additive II and mixed evenly to prepare initialization medium B. 100 μL of initialization medium B was added to wells of the 96-well plate that met the selected conditions, and the culture plate was returned to the incubator.

[0260] 4) Days 1-2: Observation was performed under a microscope, and changes in cell morphology were recorded by photographs. If the cell morphology clearly changed, the reprogramming medium B was removed, and instead, the culture was continued with the reprogramming medium A. If no changes in morphology were evident, there was no need to change the liquid.

[0261] 5) Day 3: If the morphology of the cells has obviously changed in the first two days and the cell proliferation rate is fast, they can be subcultured by trypsin digestion. According to the state of the cells and their quantity, the cells are subcultured into two to six wells of a 6-well plate, and the initialization medium C is added to form single cell attachment as much as possible. The formulation of the initialization medium C is as follows: [Table 30]

[0262] 6) Day 4: The adhesion of the cells to the vessel was observed. If most of the cells were well attached to the vessel, the medium was replaced with fresh somatic cell medium and the culture was continued.

[0263] 7) Day 5: Observe under the microscope and if small cluster clones (a clonal group of four or more cells) are formed, change the somatic cell medium to Reproeasy human somatic cell reprogramming medium. If small cluster clones have not yet formed, you can continue observation for 1-2 days and then change to Reproeasy human somatic cell reprogramming medium.

[0264] 8) Day 6-8: Under microscope observation, if the small cluster clones grow and one clonal cell population has more than 10 cells, the Reproeasy human somatic cell reprogramming medium can be changed directly to PSCeasy human pluripotent stem cell medium (or PGM1 human pluripotent stem cell medium). If there are many dead cells in the observation before changing the liquid, the liquid can be changed after washing with PBS equilibrated at room temperature.

[0265] 9) Days 9-20: Cell morphological changes were observed under a microscope and photographed. The medium was replaced with fresh PSCeasy human pluripotent stem cell medium equilibrated at room temperature every day.

[0266] 10) Day 21: Under microscope observation, if a single cell clone filled the entire 10x field of view, cut the clone with a 1mL injection needle (or an instrument such as a glass needle) and pick it into a 48-well plate pre-coated with Matrigel (if the clone is in good condition, the cells are thick and grow quickly, it can be picked directly into a 24-well plate).

[0267] 11) After the clones were picked, they were seeded into PSCeasy human pluripotent stem cell thawing medium. After the cells attached, the medium was replaced with PSCeasy human pluripotent stem cell medium and cultured until the required number of generations had been reached.

[0268] (3) Induction of patient-derived 3D retinal cells The specific steps are shown in Table 12. [Table 31]

[0269] (4) Construction and coating of AAV8 virus 1) Plasmid amplification. The constructed AAV vector (sgRNA and donor are both modified from plasmid pX601-AAV-CMV-SaCas9 (Addgene Plasmid #61591), for convenience of description, the sgRNA plasmid is hereinafter named AAV-CYP4V2-sgRNA, and the donor plasmid is hereinafter named AAV-CYP4V2-donor, where AAV-CYP4V2-donor contains a part of EGFP sequence and is the same as the above-mentioned PMD-19T-donor sequence), packaging plasmid and auxiliary plasmid must undergo large-scale endotoxin-free extraction, and the Qiagen Maxi Kit is used to perform large-scale extraction of plasmids, and the process is the same as above.

[0270] 2) Transfection of AAV8-293T cells. Observe the cell density on the day of transfection. When the cell density reaches 80-90%, the vector plasmid, packaging plasmid and auxiliary plasmid can be transfected. 3) Harvesting AAV8 virus: Viral particles are co-present in packaging cells and culture supernatant. To obtain the best yield, cells and culture supernatant can be harvested. 4) AAV purification and long-term storage at -80°C.

[0271] (5) Validation of sgRNA gene editing efficiency in vitro using 3D retinal tissue 1) AAV8 was used to infect 3D retina tissue. 2) Gene editing efficiency was verified.

[0272] 1) T7E1 enzyme cleavage experiment The process is the same as above.

[0273] 2) TOPO PCR cloning To quantify the editing efficiency of AAV-CYP4V2-sgRNA and AAV-CYP4V2-donor and perform statistical analysis of the cleavage efficiencies of the experimental and control groups, experiments were performed using Invitrogen's Zero Blunt TOPO PCR Cloning Kit, and sequencing was performed using Sanger after cell selection.

[0274] a) PCR reactions were performed using DNA obtained after infection of 3D retinal tissue with the AAV virus as an amplification template, using Invitrogen's Platinum SuperFi (registered trademark) series DNA polymerase. [Table 32]

[0275] b) Touch down PCR program: The process is the same as above.

[0276] c) The reaction system for TOPO PCR cloning is as follows: [Table 33]

[0277] The above reaction system was prepared, mixed gently, and allowed to stand at room temperature for 5 minutes. The mixture was placed on ice, the competent cells were removed, and the mixture was prepared for transformation.

[0278] d) Transformation of competent cells by TOPO PCR cloning reaction Take 50 or 100 μL of competent cells, add 2 μL of the above TOPO PCR cloning reaction solution, and leave on ice for 5-30 min; heat shock at 42°C for 30 s without shaking; quickly transfer the reaction system to ice and add 250 μL of SOC medium; shake and thaw at 200 rpm at 37°C for 1 h; take a corresponding amount of LB culture plate (containing 25 μg / mL bleomycin (Zeocin)), spread 100 μL of the above bacterial solution on the plate, and culture overnight in a 37°C incubator.

[0279] e) Sanger sequencing of selected organisms. The next morning, the cell plates were removed and 80 cells were selected from each plate; shaken at 200 rpm at 37° C. for 3-4 h; and 200 μL of each cell sample was taken for Sanger sequencing.

[0280] Example 5: Verification of gene editing efficiency in a humanized mouse model 1. Verification of gene editing efficiency in humanized mouse models 1) Construction of humanized mice Construction of humanized mice was performed by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Regarding the human mutation site, we asked Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to construct a CYP4V2 humanized mouse model.

[0281] 2) Details of the technology: a) Design and construction of sgRNA to recognize the target sequence; b) Construction of CRISPR / Cas9 vectors that cause targeted gene cleavage; c) sgRNA / Cas9 activity test; d) Design and construction of a knockout targeting vector and replacement of exons 6-8 (including intron sequences) according to the design; in vitro transcribed sgRNA / Cas9 mRNA;

[0282] e) Injection of sgRNA / Cas9 mRNA and targeting vector into mouse fertilized eggs; f) Examination and propagation of CYP4V2 knock-in F0 mice; g) Obtaining CYP4V2 knock-in F1 heterozygous mice and genotyping (southern blotting (DNA hybridization) assay, including one exogenous and one endogenous probe).

[0283] 3)Technique methods: a) We amplified and sequenced the target sequence in the mouse genome, designed and constructed a CRISPR / Cas9 vector plasmid targeting the target sequence, and performed activity testing. b) Mouse genomic DNA was extracted and sgRNA that recognizes the target sequence was amplified. c) Designed and constructed a CRISPR / Cas9 vector plasmid that causes target gene cleavage.

[0284] d) Use: Activity testing was performed on sgRNA / Cas9 using a test kit independently developed by Biocytogen Pharmaceuticals (Beijing) Co., Ltd. e) Highly active sgRNA / Cas9 target site sequence information was selected, and a CYP4V2 knockout targeting vector was designed and constructed.

[0285] f) sgRNA / Cas9 mRNA and targeting vector were injected into mouse fertilized egg pronuclei to obtain F0 / F1 positive mice. This experiment mainly includes the following: In vitro transcription of sgRNA / Cas9 mRNA; Collection of mouse fertilized eggs; injection of RNA and targeting vectors into mouse fertilized eggs; Transfer of fertilized eggs into the oviducts of surrogate mice; Genotyping and propagation of F0 mice with point mutations in the CYP4V2 gene humanization; Obtaining and genotyping F1 mice carrying point mutations in the humanized CYP4V2 gene (including PCR and southern blotting hybridization assays).

[0286] (2) Rearing and breeding After obtaining both kinds of humanized mice, they were intercrossed with F1 heterozygous mice to obtain a sufficient number of F2 or F3 homozygous humanized mice for AAV virus injection as soon as possible.

[0287] (3) AAV virus injection into the subretinal space of mice The AAV-CYP4V2-sgRNA (SaCas9 donor) and AAV-CYP4V2-donor vectors were packaged into adeno-associated viruses and injected into the retina of CYP4V2 mutant model mice to verify the in vivo editing and repair efficiency of the designed sgRNA and Donor. Taking AAV-CYP4V2-sgRNA13+AAV-CYP4V2-donor13 as an example, the specific steps are as follows. The experimental methods for the sgRNA17-18 groups are the same.

[0288] 1) Experimental design a) Blank control group: saline. b) Experimental groups: AAV-CYP4V2-sgRNA13 (donor of SaCas9) and AAV-CYP4V2-donor13.

[0289] 2) Selection of AAV (adeno-associated virus) serotype The AAV2 / 8 serotype was chosen due to its better retinal preference.

[0290] 3) Viral packaging The AAV-CYP4V2-donor13 and AAV-CYP4V2-sgRNA13 vectors were packaged into adeno-associated virus (AAV) with AAV2 / 8 and AAV-helper, respectively.

[0291] 4) Injection experiment in mice The experiment was conducted by dividing 20 CYP4V2 mutant model mice into four groups of five mice each.

[0292] The experimental design is as follows. a) Blank control group: Mice were injected with 2 μL saline per eye. b) Experimental group: Packaged AAV-CYP4V2-sgRNA13 and AAV-CYP4V2-donor13 viruses were mixed in equal amounts at a 1:1 ratio and injected into each eye of mice at 2 μL (1E10 vg). c) The treatment effect was examined after one month.

[0293] The experimental steps are as follows: a) Thirty minutes before injection, pupils were dilated with 1% atropine; pupils were dilated again before anesthesia.

[0294] b) After anesthetizing the mouse by intraperitoneally injecting 80 mg / kg ketamine and 8 mg / kg xylazine, the mouse was placed in front of the animal experiment table of the ophthalmic surgery microscope, and a drop of 0.5% proparacaine was dropped into the mouse's eye for local anesthesia. AAV virus was added to the stock solution of sodium fluorescein in a ratio of 100:1, and mixed by centrifugation at low speed.

[0295] c) A small hole was made in advance in the ciliary plane of the mouse's eyeball with an insulin injection needle, and the needle of a microinjector was passed through the small hole and entered into the vitreous cavity of the mouse's eyeball. At this time, an appropriate amount of 2% hydroxymethylcellulose was dropped onto the mouse's eyeball so that the fundus of the mouse's eyeball could be clearly seen under a microscope. The needle was then inserted into the peripheral part of the retina on the opposite side, avoiding the vitreous body, and the AAV virus containing sodium fluorescein was slowly pushed out. The injection volume per eye was 1 μL, and sodium fluorescein was used as an indicator to determine whether it had been injected into the subretinal space.

[0296] d) After surgery, the mice were observed for any abnormalities and administered neomycin eye ointment to prevent infection.

[0297] 5) Evaluation of the efficacy of gene editing therapy The experimental results showed that the therapeutic effects of each experimental group were basically consistent, so the effects of gene editing therapy are explained using the AAV-CYP4V2-sgRNA13 and AAV-CYP4V2-donor13 virus groups as representatives.

[0298] (4) Evaluation of editing efficiency of AAV8- AAV-CYP4V2-sgRNA in humanized mice For the verification method, refer to “(5) Verification of sgRNA gene editing efficiency in vitro using 3D retinal tissue in Example 4”.

[0299] The experimental results showed that the therapeutic effects of each experimental group were basically consistent, so the effects of gene editing therapy are explained using the AAV-CYP4V2-sgRNA13 and AAV-CYP4V2-donor13 virus groups as representatives.

[0300] Functional alterations in mice after treatment were assessed by ERG (retinal electrophysiology), and retinal function of the mutant mouse model was improved after treatment.

[0301] After sectioning the samples, the morphological changes in the mouse retina were observed using HE staining, and the morphology was found to be close to that of the retina in wild-type mice.

[0302] Immunofluorescence staining was used to observe whether the gene editing therapeutic vector was expressed in the corresponding location of the retina, and the gene editing therapeutic vector was expressed in the corresponding location of the retina.

[0303] Morphological changes in retinal tissue were observed through a series of staining traces of a special retinal marker, and it was found that the morphology of the retinal tissue of the mice was improved after treatment.

[0304] Finally, it should be noted that the above examples are only used to explain the technical idea of ​​the present application, and are not intended to limit the same. Although the present application has been described in detail with reference to the above examples, those skilled in the art will understand that the technical idea described in the above examples can be modified or some technical features can be replaced equally without departing from the spirit and scope of the present invention.

Claims

1. A gRNA that specifically targets the cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene, specifically binding to a nucleotide sequence within 200 bp upstream and / or downstream of the c.802-8_810del17bpinsGC mutation site, wherein the gRNA contains any of the nucleotide sequences of SEQ ID NOs: 74, 78-80, and 82-84.

2. The gRNA of claim 1, comprising 5'-(X)n-SEQ ID NO: 74, 78-80, 82-84 backbone sequence-3', where X is any base selected from A, U, C and G, and n is any integer from 0 to 15.

3. The gRNA of claim 1 , wherein the gRNA is a single-stranded guide RNA (sgRNA).

4. One or more isolated nucleic acid molecules encoding gRNAs that specifically target the CYP4V2 gene of any one of claims 1 to 3.

5. A vector comprising the isolated nucleic acid molecule of claim 4.

6. The vector according to claim 5, further comprising a donor nucleic acid molecule, wherein the donor nucleic acid molecule comprises a normal wild-type nucleotide sequence within 800 bp upstream and / or downstream of the CYP4V2 gene c.802-8_810del17bpinsGC mutation site.

7. The vector described in claim 6, wherein the donor nucleic acid molecule contains a nucleotide sequence of any of SEQ ID NOs: 64-66.

8. The vector of claim 6 , wherein the isolated nucleic acid molecule and the donor nucleic acid molecule are present on the same vector.

9. The vector according to any one of claims 5 to 8, wherein the vector is a viral vector.

10. A cell containing the isolated nucleic acid molecule of claim 4 and / or the vector of any of claims 5 to 9.

11. The cell of claim 10 , wherein the cell comprises a HEK293 cell, a renal epithelial cell and / or an induced pluripotent stem cell.

12. The cell according to claim 10 , wherein the cell has differentiation potential due to modification.

13. The cell according to any one of claims 10 to 11, wherein the cell is capable of differentiating into a 3D-retinal organoid.

14. A pharmaceutical composition comprising: i) a gRNA described in any one of claims 1 to 3, one or more isolated nucleic acid molecules described in claim 4, and / or a vector described in claim 5; ii) a Cas nuclease, or a nucleic acid molecule or vector comprising a nucleotide sequence encoding the Cas nuclease; iii) a donor nucleic acid molecule defined in claim 6 or 7, or a vector comprising the donor nucleic acid molecule; and iv) a pharma- ceutically acceptable carrier.

15. 15. Use of the gRNA of any one of claims 1 to 3, the one or more isolated nucleic acid molecules of claim 4, and / or the vector of any one of claims 5 to 9 in the manufacture of a pharmaceutical composition for the treatment of a disease, wherein the disease comprises a disease caused by the c.802-8_810del17bpinsGC mutation in the CYP4V2 gene, and the pharmaceutical composition is the pharmaceutical composition of claim 14.

16. The use according to claim 15, wherein the disease comprises Bietti crystalline dystrophy.

17. A non-therapeutic method for regulating expression of the CYP4V2 gene in a cell, the method comprising introducing into the cell a gRNA described in any one of claims 1 to 3, one or more isolated nucleic acid molecules described in claim 4, and / or a vector described in any one of claims 5 to 9.

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