CN111926044B - Nucleic acid molecules and kits incorporating mutant RHO genes - Google Patents

Abstract

The present application relates to a kit comprising 1) a vector, 2) a pharmaceutically acceptable carrier, wherein the vector comprises a nucleic acid molecule capable of encoding a sgRNA that specifically binds to a nucleic acid molecule comprising a mutation site selected from the group consisting of c.c50t and c.c403tRHOA gene. The application also relates to methods of treating retinitis pigmentosa using the kit.

Description

Nucleic acid molecules and kits incorporating mutant RHO genes

Technical Field

The application relates to the field of biological medicine, in particular to a Chinese-specific medicineRHOGene editing kits for adRP patients based on CRISPR/Cas9 technology and AAV technology.

Background

Currently, Retinitis Pigmentosa (RP) is a group of inherited blinding eye diseases that are primarily altered by progressive loss of photoreceptor and/or retinal pigment epithelial cell function. At present, no effective treatment method exists (the traditional medicine and the surgical treatment are basically ineffective). In recent years, the rapid development of the CRISPR/Cas9 technology brings about eosin for RP gene therapy, and the CRISPR/Cas9 technology is convenient and simple to operate, is the most common gene editing technology at present, and is also one of the main tools for hereditary retinal degeneration gene therapy.RHOIs the earliest discovered RP pathogenic gene, and about 30-40% of autosomal dominant RP (adRP) is caused by the gene and is the most main pathogenic gene of adRP.

In the usual case of the use of a magnetic tape,RHOone allele of an adRP patient carries a pathogenic mutation, while the other allele is normal;RHOthe pathogenic mechanism of the mutation is that of gain-of-function or dominant negative effects. Literature reportsRHOPro23His gene is in North AmericaRHOMutational hotspots of the adRP population, currently onRHOGene editing therapies based on CRISPR/Cas9 technology are mostly associated with this site. Aiming at Asian populationRHOThe research of mutation hot spots is almost not available and can not be used in ChinaRHO-gene editing therapy in the adRP population. Therefore, research on drug design aiming at gene mutation hotspots of Chinese population is required.

Adeno-associated virus (AAV) vectors are the most widely used vector for retinal gene therapy because they have the advantages of being non-pathogenic, low in immunogenicity, capable of effectively transferring target genes and long-term expression of therapeutic genes carried thereby, and the like, as gene transfer vectors. However, the AAV vector has a carrying capacity of 4.7kb at the maximum, and normally, only sgRNA and Cas9 can be packaged separately. Therefore, in order to improve targeting efficiency, it is necessary to select an appropriate vector.

Disclosure of Invention

The application provides a method for ChineseRHOThe gene editing kit of the adRP patient based on CRISPR/Cas9 technology and AAV technology can be used for ChineseRHODesigning gRNA with mutant allele specificity in the mutant hot spots of the population of adRP, so that the mutant allele can be knocked out, and the normal allele is retained, thereby achieving the purpose of treatment. In certain embodiments, the kits of the present application can use the CRISPR/SaCas9 system for gene editing, and can package the SaCas9 protein and grnas into a single AAV virus. For example, a pX601-SaCas9 plasmid vector can be used, and the gRNA and SaCas9 are included in one AAV vector (e.g., AAV8 vector) to improve targeting efficiency. In certain embodiments, the carrier can be injected by sub-retinal injectionRHOThe eyeball of an adRP patient for therapeutic purposes. The kit described in the present application is capable of specific cleavageRHOThe related mutation sites of the gene have certain cutting efficiency and safety.

The present application provides a kit comprising 1) a vector, 2) a pharmaceutically acceptable carrier, wherein the vector comprises a nucleic acid molecule capable of encoding a sgRNA that specifically binds to a mutant site-comprising sgRNARHOA gene, the mutation site being selected from one or more of the group consisting of: c.c50t and c.c 403t.

In certain embodiments, the sgRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 1, 2, and 4.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the vector comprises AAV.

In certain embodiments, the kit comprises a nucleic acid molecule encoding a Cas protein.

In certain embodiments, the nucleic acid molecule capable of encoding a sgRNA and the nucleic acid molecule encoding a Cas protein are located in the same vector.

In certain embodiments, the kit comprises a detection reagent that can be used to detect one or more RHO gene mutations selected from the group consisting of: c.c50t and c.c 403t.

In certain embodiments, the pharmaceutically acceptable carrier is suitable for subretinal administration.

In certain embodiments, the kit comprises one or more fine needles, one or more syringes, and/or a solvent.

In another aspect, the present application provides sgrnas that specifically bind to the RHO gene comprising a mutation site selected from one or more of the group consisting of: c.c50t and c.c 403t.

In certain embodiments, the sgRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs 1, 2, and 4 and/or a pharmaceutically acceptable carrier.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows a structural feature map of a gene editing vector used herein.

FIG. 2 is a schematic representation of aRHOThree gRNA-encoding sequences designed for p.thr17met.

FIG. 3 is a schematic representation of aRHOArg135trp two sequences encoding grnas.

FIG. 4 showsRHO17-SgAnd (5) detecting the in vitro efficiency of the RNA.

FIG. 5 showsRHO135-SgRNA in vitro efficiency detection results.

FIG. 6 shows in vitro assay using gRNA active fluorescence detection kitRHO17-SgRNA1 edit the results.

FIG. 7 shows in vitro assay using gRNA active fluorescence detection kitRHO17-SgRNA2 edit the results.

FIG. 8 shows in vitro assay using gRNA active fluorescence detection kitRHO17-SgRNA3 edit the results.

FIG. 9 shows in vitro assay using gRNA active fluorescence detection kitRHO135-SgRNA1 edit the results.

FIG. 10 shows in vitro assay using gRNA active fluorescence detection kitRHO135-SgRNA2 edit the results.

Fig. 11 shows flow sorting statistics for in vitro sgRNA editing efficiency detection using a gRNA active fluorescence detection kit.

Fig. 12 shows the results of running gel to verify sgRNA safety and specificity in vitro using 293T cells.

FIG. 13 showsRHO17-SgRNA1 andRHO17-SgRNA2 gene editing efficiency only for patient iPSCs.

Shown in FIG. 14RHO135-SgRNA1 only had gene editing efficiency on patient iPSCs.

FIG. 15 showsRHO17-SgRNA2 andRHOgene editing effects of 135-SgRNA1 on 3D retinal tissue in patients.

FIG. 16 showsRHOAnd (5) identifying the genotype of the humanized mouse.

FIG. 17 showsRHO17-SgRNA2 andRHOgene editing effect of 135-SgRNA1 on retinal tissue of humanized mice.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "c.c50t" generally refers to the wild typeRHOThe nucleotide sequences of the genes are compared with each other,RHOthe base at position 50 of the coding sequence of the gene (from 5 'end to 3' end, the "A" in the ATG starting from the coding sequence is position 1) is mutated from cytosine (C) to thymine (T). In this application, "c." generally refers to a coding sequence, i.e., a sequence from the start codon ATG to the stop codon, which may begin and end at any position in the mRNA. Thus, "c.c 50t" indicates that the 50 th nucleotide is mutated from C to T starting from a of the coding sequence ATG. The mutation of the base can result inRHOThe gene encodes an amino acid that is altered, for example, such that the amino acid is mutated from threonine (Thr) to methionine (Met). In the present application, the term "p.thr17met" generally means that the amino acid at position 17 of the RHO protein is mutated from threonine (Thr) to methionine (Met).

In the present application, the term "c.c 403t" generally refers to the wild typeRHOThe nucleotide sequences of the genes are compared with each other,RHOthe base at 403 th position (from 5 'end to 3' end, the 'A' in the initial ATG of the coding sequence is the 1 st position) of the gene coding sequence is mutated from cytosine (C) to thymine (T), and the mutation of the base can causeRHOThe amino acid encoded by the gene is mutated, for example, such that the amino acid is mutated from arginine (Arg) to tryptophan (Trp). In the present application, the term "p.arg135trp" generally means that the amino acid at position 135 of the RHO protein is mutated from arginine (Arg) to tryptophan (Trp).

In this application, the term " RHO Exon 1 "of an allele generally refers toRHOExon 1 in the gene. For example, in Ensembl databaseRHOThe allele has exon 1 ID of ENSE00001079597, which may include the nucleotide sequence at position 129,528,639-129,529,094 of homo sapiens chromosome 3.

In this application, the term " RHO Exon 5 "of an allele is generally referred to asRHOExon 5 in the gene. For example, in Ensembl databaseRHOOf allelesExon 5 has the ID ENSE00001079599, which may comprise the nucleotide sequence at positions 129,533,608-129,535,344 of homo sapiens chromosome 3.

In the present application, the term "double-strand breaks (DSBs)" generally refers to the phenomenon that occurs when two single strands of a double-stranded DNA molecule are cleaved at the same position. Double strand breaks may induce DNA repair, possibly resulting in genetic recombination, and cells also have some systems acting on double strand breaks otherwise caused. Double strand breaks can occur periodically during the normal cell replication cycle, and can also be enhanced in certain circumstances, such as ultraviolet light, inducers of DNA breaks (e.g., various chemical inducers). Many inducers can cause DSBs to occur indiscriminately in the genome, and DSBs can be regularly induced and repaired in normal cells. During repair, the original sequence can be reconstructed with full fidelity, but in some cases small insertions or deletions (called "indels") are introduced at the DSB site. In some cases, double strand breaks may also be specifically induced at specific locations, which may be used to cause targeted or preferential genetic modification at selected chromosomal locations. In many cases, the tendency of homologous sequences to readily recombine during DNA repair (and replication) can be exploited, which is the basis for the application of gene editing systems such as CRISPR. This homology-directed repair is used to insert the sequence of interest provided by the use of a "donor" polynucleotide into the desired chromosomal location.

The term "knock-out" refers to a change in the nucleic acid sequence of a gene that reduces the biological activity of a polypeptide normally encoded by the gene by at least 80% as compared to the unaltered gene. For example, the alteration may be an insertion, substitution, deletion, frameshift mutation, or missense mutation of one or more nucleotides.

In the present application, the term "complementary" generally refers to a nucleic acid (e.g., RNA) comprising a nucleotide sequence (e.g., Watson-Crick base pairing) that enables it to bind non-covalently to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid) "hybridize" or "complement" under appropriate in vitro and/or in vivo temperature and solution ionic strength conditions. As known in the art, standard Watson-Crick base pairing includes: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C).

In the present application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may comprise: disulfide bond formation, glycosylation, lipidation (acetylation), acetylation, phosphorylation, or any other manipulation (e.g., binding to a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine as well as D and L optical isomers, as well as amino acid analogs and peptidomimetics.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (one locus) defined according to ligation analysis, exons, introns, messenger RNA (mrna), transfer RNA, ribosomal RNA, short interfering RNA (sirna), short hairpin RNA (shrna), micro-RNA (mirna), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeled moiety.

In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, for transferring the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly for inserting a DNA or RNA into a cell, a vector mainly for replicating a DNA or RNA, and a vector mainly for expression of transcription and/or translation of a DNA or RNA. The vector also includes vectors having a plurality of the above-described functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell containing the vector.

In the present application, the term "plasmid" generally refers to a DNA molecule other than chromosome or karyoid in organisms such as bacteria, yeast, etc., which exists in the cytoplasm and has the ability to autonomously replicate so that it can maintain a constant copy number in progeny cells and express the genetic information carried thereby. Plasmids are used as vectors for genes in genetic engineering studies.

In the present application, the term "retroviral vector" generally refers to a viral particle that can control and express a foreign gene, but that cannot self-package into a proliferative capacity. Such viruses mostly have reverse transcriptase. Retroviruses contain at least three genes: gag, a gene comprising a protein constituting the viral center and structure; pol, a gene comprising reverse transcriptase; and env, which contains the genes that make up the viral coat. Through retroviral transfection, a retroviral vector can integrate its genome and the foreign gene it carries randomly and stably into the host cell genome, e.g., the CAR molecule can be integrated into the host cell.

In the present application, the term "lentiviral vector" generally refers to a diploid RNA viral vector belonging to a retrovirus. The lentivirus vector is based on lentivirus genome, removes a plurality of sequence structures related to virus activity to ensure that the lentivirus vector has biological safety, introduces the sequence and expression structure of a target gene required by an experiment into the genome framework, and prepares the lentivirus vector. Through transfection with lentiviral vectors, retroviral vectors can randomly and stably integrate their own genome and the foreign gene they carry into the host cell genome, e.g., the CAR molecule can be integrated into the host cell.

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

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 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 stated value.

Detailed Description

Method of producing a composite material

In one aspect, the present application provides a method of treating retinitis pigmentosa.

In the present application, the term "Retinitis Pigmentosa (RP)" generally refers to a genetic disease that causes retinal degeneration. There are approximately 80 genes identified that are associated with RP, and these genes are involved in autosomal recessive inheritance (50-60%), autosomal dominant inheritance (AD, 30-40%), and X-linked inheritance (5-15%). RP is characterized by progressive loss of vision due to dysfunction of the receptor cells of the retina (cones and rods) and/or the retinal pigment epithelium. Clinical manifestations of RP may include night blindness, progressive visual field loss, central vision loss after macular involvement, and eventually blindness, Electroretinograms (ERGs) show decreased or even extinguished rod cell (rows) function. RP changes mainly in the fundus oculi into retinal pigment disorder in the equatorial region, with osteocyte-like pigmentation, progressing gradually toward the posterior polar region and jagged edge. RPE, photoreceptor cells and choroidal capillary layers gradually atrophy, see choroidal great vessels, the retina appears grayish, the retinal arteries become thinner, and the disc is atrophied by cercoscence, with retinal angiostenosis, cercoscopy and osteocyte-like pigmentation being the typical triple features of RP (Hartong DT et al, 2006). Methods for assessing retinal function and morphology may include Visual Acuity by Best Corrected (BCVA), fundus autofluorescence, Visual field examination, ERG, fundus colorography, Optical Coherence Tomography (OCT), and fluorescein angiography (FFA), among others. Methods in which visual functionality is assessed may include methods of BCVA, visual field, and the like.

The methods described herein can include providing functionality to a subject in need thereofRHOA gene.

In some cases, the RP described herein may be a mixture ofRHOCaused by mutation of the gene. Associated with RPRHOThere are many kinds of gene mutations which can causeRHOThe gene encodes a dysfunctional rhodopsin protein (rhodopsin), and the mutation may include, but is not limited to, missense, nonsense, insertion, deletion, etc., of the gene. For example, the mutation site may comprise a mutation site selected from the group consisting of: c.c50t and c.c 403t. For another example, the mutation site may result in an amino acid change, which may include the following changes: p.thr17met and/or p.arg135trp. In the present application, the method may comprise rendering a subject in need thereof free of heterozygous mutation sitesRHOA gene, the mutation site may be selected from the group consisting of: c.c50t and c.c 403t.

Capable of repairing any one or more mutations to render functional a subject in need thereofRHOA gene. For example, pathological variations c.c50t and/or c.c403t may be removed, restored or corrected.

In this application, the term "RHOThe gene "may also be referred to as Rhodopsin 2 (Rhodopsin 2), Opsin-2, Opsin 2, OPN2, CSNBAD1, or RP 4. Rhodopsin proteins are localized to the Rod Outer Segment (ROS) and are essential for normal vision, particularly for the perception of weak light stimuli, and ROS is a scotopic vision of the photoreceptor cells of the retina. In the retinaAnother photoreceptor cell of (a) is cone cells (cons), sermingmy vision and color vision. In ROS, rhodopsin is commonly associated with 11-cis retinal (11-cisretinal, 11 cRAL), which is a derivative form of vitamin a. ROS absorbs photons to make Rhodopsin active Rhodopsin (R), 11cRAL isomerizes to all-trans retinaldehyde (atRAL), atRAL is reduced to all-trans retinol (atROL) shortly after separation from R, intercellular vitamin a binding protein (IRBP) is responsible for transferring atROL into RPE cells, where atROL is converted to all-trans retinol ester by lecithin retinol acyltransferase (irat), further converted to 11-cis retinol ester, and then isomerized to 11-cis retinol (11-cis retinol, 11 cRAL) by lre 65, 11cRAL is oxidized to rdl and bp is re-utilized in photoreceptor cells after binding; r converts GDP on alpha subunit of transducin G (Gt) in downstream membrane disc into GTP, separates alpha subunit from beta gamma subunit, activates cyclic guanosine monophosphate-phosphodiesterase 6 (cGMP-PDE 6), hydrolyzes cGMP, reduces cellular cGMP concentration, closes cGMP gated cation channel of OS, and makes Ca in photoreceptor cell²⁺The concentration is reduced, the cell membrane is hyperpolarized, and the optical signal is converted into a visual electric signal; after the light conduction is stopped, the photoreceptor cells return to the state of non-light through a series of chemical reactions, at the moment, R is phosphorylated, is combined with the arrestin and inhibits a downstream signal path, PDE6 is in a non-activated state, meanwhile, cGMP is synthesized in Rods, the concentration of cGMP is increased, a cation channel is opened, and then Ca is added²⁺Influx, depolarization of the cell membrane. The nerve impulse caused by the opening and closing of the cGMP gated cation channel is transmitted to the visual center of cerebral cortex through the connection of synaptic terminals of photoreceptor cells and neurons at all levels of retina and optic nerve conduction to form vision.

Of a human beingRHOThe gene is located at position 22.1 of the long arm of chromosome 3 (3 q 22.1), and the molecule is from 129,528,639 bp to 129,535,344 bp on chromosome 3 (Chile update notes issue: (Chile update notesHomo sapiens，Annotation release) 109.20200228 th edition, grch38.p13, NCBI).RHOThe nucleotide sequence of the gene can be found in NCBI GenBank Accession number NG-009115.1.RHOThe gene has 5 exons. Table 1 shows the Ensembl databaseRHOThe exon identifier of the gene and the start/stop site of the exon.

TABLE 1RHOExon of gene

The term "functionalRHOThe term "gene" generally refers to a gene that encodes a normally functioning rhodopsin protein. In the present application, the term denotes a compound which does not contain a mutation siteRHOGenes, for example, the mutation site may be selected from the group consisting of: c.c50t and c.c 403t. In some cases, one can include a mutation site (e.g., c.c50t and c.c403t)RHOThe gene being functionalRHOA gene. For example, the mutation sites (e.g., c.c50t and c.c403t) may be specifically removed, and for example, the mutation sites (e.g., c.c50t and c.c403t) may be removed and the mutant sites (e.g., c.c50t and c.c403t) may be included by gene knock-out in combination with Homology-dependent Repair (HDR) methodsRHOThe gene being functionalRHOA gene.

Make itRHOThe gene dysfunction condition can be gene mutation, and the mutation can include but is not limited to nucleotide insertion, deletion, missense, nonsense, frame shift and/or other mutation. In some cases, any one or more mutations may be repaired to recoverRHONormal function of the gene. For example, can be removedRHOThe site of mutation in the gene.

In some cases, the method may include the steps of: removing a subject in need thereofRHOThe mutation site of the gene. The method may comprise exon deletion. Targeted deletion of specific exons may be a strategy to treat a large number of patients with a single therapeutic cocktail. The exon deletion may be a single exon deletion or a multiple exon deletion. Although much moreAn exon deletion can cover more subjects, but for deletions of more nucleotides, the efficiency of the deletion decreases greatly with increasing nucleotide size. Thus, the range of removal can be 40 to 10,000 base pairs (bp). For example, the removal range can be 40-100, 100-300, 300-500, 500-1,000, 1,000-2,000, 2,000-3,000, 3,000-5,000, or 5,000-10,000 base pairs.

As has been described in the foregoing, the present invention,RHOthe gene contains 5 exons. Any one or more of the 5 exons may contain a mutation. Any one or more of the 5 mutated exons or aberrant intronic splice acceptor or donor sites may be removed to render the functionalRHOGenes do not contain mutation sites (e.g., influenceRHOMutations in gene function). In some cases, the mutation site may be fromRHOAny one or more of the following groups of genes: exon 1, exon 2, exon 3, exon 4, exon 5, or any combination thereof. For example, the genetic mutation may be a mutation selected from the group consisting of: c.c50t and c.c 403t. The gene mutation may result in amino acid mutation, ultimately resulting inRHOProtein dysfunction, such as mutant proteins, may interfere with the function of normal proteins or may not target ROS, etc.

The methods described herein can include knocking out the mutation site and/or reducing the expression level of the mutation site. Methods of knocking out a gene or reducing the expression level of a gene may include performing gene knock-out, conditional gene knock-out (e.g., using Cre/loxP and/or FLP-frt systems), inducible gene knock-out (e.g., knock-out based on Cre/loxP systems, including tetracycline induction, interferon induction, hormone induction, adenovirus induction, etc.), gene knock-out using random insertion mutation (e.g., gene trapping), gene knock-out using random insertion mutation, zinc finger endonuclease (ZNF) mediated gene editing techniques, transcription activator-like effector nucleases (TALEN) mediated gene editing techniques, clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short nucleotide sequences) (CRISPR/CRISPR) mediated gene editing techniques, and/or CRISPR/CRISPR-mediated gene editing systems And (4) editing technology. For any genome editing strategy, gene editing can be confirmed by sequencing or PCR analysis.

In certain instances, the methods described herein can be in vivo cell-based methods. In certain instances, the method comprises editing genomic DNA of a cell of the subject. For example, editing in a cell (e.g., a photoreceptor cell and/or a retinal progenitor cell) of a subject may be includedRHOA mutation in a gene. For example, the genetic mutation may be a mutation selected from the group consisting of: c.c50t and c.c 403t. While certain cells may be ideal targets for ex vivo methods or ex vivo therapies, the use of effective delivery methods may also allow for the delivery of desired agents directly to such cells in vivo. In certain instances, the methods can include targeting and editing to the relevant cells. Lysis of other cells can also be prevented by using promoters which are active only in certain cells and/or developmental stages.

The added promoter is inducible and thus, if the nucleic acid molecule is delivered in a plasmid vector, the delivery time can be controlled. The time that the delivered nucleic acid or protein is resident in the cell can also be adjusted by methods that alter the half-life. The in vivo approach may save some processing steps but requires higher editing efficiency. In vivo treatment can eliminate problems and losses associated with ex vivo treatment and implantation.

In vivo methods may facilitate the production and administration of therapeutic products. The same treatment or therapy will likely be used to treat more than one subject, e.g., many subjects with the same or similar genotype or allele.

The methods described herein may include ex vivo methods. In certain cases, subject-specific induced pluripotent stem cells (ipscs) may be obtained. The genomic DNA of these iPSC cells can then be edited using the methods described herein. For example, the method may be included in the iPSCRHOWithin or near the site of mutation of the geneEditing such that it does not have an amino acid mutation of p.thr17met and/or p.arg135trp, for example, the genetic mutation may be a mutation selected from the group consisting of: c.c50t and c.c 403t. Next, the gene-edited ipscs can 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) can be implanted into a subject.

In other cases, photoreceptor cells or retinal progenitor cells can be isolated from a subject. Next, the genomic DNA of these photoreceptor or retinal progenitor cells can be edited using the methods described herein. For example, the method may comprise administration of photoreceptor cells or retinal progenitor cellsRHOThe gene is edited within or near the mutation site so that it does not have an amino acid mutation of p.thr17met and/or p.arg135trp, for example, the gene mutation may be a mutation selected from the group consisting of: c.c50t and c.c 403t. Finally, the genetically edited photoreceptor or retinal progenitor cells can be implanted into a subject.

In other cases, mesenchymal stem cells may be isolated from in vivo in other cases, or from bone marrow or peripheral blood in other cases. Next, genomic DNA of these mesenchymal stem cells may be edited using the methods described herein. For example, the method may comprise the administration of mesenchymal stem cellsRHOThe gene is edited within or near the mutation site so that it does not have an amino acid mutation of p.thr17met and/or p.arg135trp, for example, the gene mutation may be a mutation selected from the group consisting of: c.c50t and c.c 403t. Next, the genetically edited mesenchymal stem cells may be differentiated into any type of cell, such as photoreceptor cells or retinal progenitor cells. Finally, the differentiated cells, e.g., photoreceptor cells or retinal progenitor cells, can be implanted into a subject.

The method may include a comprehensive analysis of the therapeutic agent prior to administration. For example, the entire genome of the calibration cell is sequenced to ensure that no off-target effects (if any) can be at the genomic location associated with minimal risk to the subject. In addition, populations of specific cells, including clonal cell populations, can be isolated prior to implantation.

Wild-type in the subject may not be affected using the methods described hereinRHOThe level of expression and/or function of the gene.

Gene editing

Methods described herein can include methods of cleaving DNA at a precise target location in a genome using a site-directed nuclease, thereby generating a single-stranded or double-stranded DNA break at a specific location within the genome. Such breaks can be repaired regularly by endogenous cellular processes, such as HDR and Non-Homologous End Joining (NHEJ). These two major DNA repair processes consist of a series of alternative pathways. NHEJ directly links DNA ends resulting from double strand breaks, sometimes losing or adding nucleotide sequences, which may disrupt or enhance gene expression. HDR uses homologous or donor sequences as templates to insert specific DNA sequences at breakpoints. Homologous sequences may be in an endogenous genome, such as sister chromatids (sister chromatids). Alternatively, the donor may be an exogenous nucleic acid, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a virus. These exogenous nucleic acids may comprise regions of high homology to the locus cleaved by the nuclease, and may additionally comprise additional sequences or sequence changes (including deletions that may be incorporated into the cleaved target locus). A third repair mechanism may be Microhomology-Mediated End Joining (MMEJ), also known as "substitutional NHEJ (ANHEJ)", where small deletions and insertions may occur at the cleavage site with genetic consequences similar to NHEJ. MMEJ can use several base pairs of homologous sequences flanking a DNA break to drive more favorable DNA end-joining repair results. In some cases, it is possible to predict the likely repair outcome based on analysis of potential microscopic homology to the site of DNA fragmentation.

These gene editing mechanisms can be used for the removal of the gene mutation sites required in the present application. The methods described herein can include creating one or two DNA breaks at a location in the target locus near the site of the intended mutation, twoThe DNA break may be a double strand break or two single strand breaks. In some cases, the removing can include causing the mutant to be includedRHOThe allele is double-stranded broken. The cleavage may be effected by site-directed polypeptides. Site-directed polypeptides (e.g., DNA endonucleases) can introduce double-stranded breaks or single-stranded breaks in nucleic acids (e.g., genomic DNA). The double-strand break may stimulate an endogenous DNA repair pathway of the cell, e.g., HDR, NHEJ, or MMEJ. NHEJ can repair cleaved target nucleic acid, without the need for homologous template.

In some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into a target nucleic acid cleavage site. The exogenous polynucleotide sequence may be referred to as a donor polynucleotide (or donor, or donor sequence, or polynucleotide donor template). The donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that is not naturally present at the target nucleic acid cleavage site.

HDR occurs when a homologous repair template or donor is available. The homologous donor template may comprise wild typeRHOAt least a portion of a gene or cDNA. Wild typeRHOAt least a portion of the gene or cDNA may be exon 1, exon 2, exon 3, exon 4, exon 5, intron regions, fragments or combinations thereof, or the entire gene or cDNARHOA gene or a cDNA. The donor template may be a single-stranded or double-stranded polynucleotide. The donor template can be delivered by AAV. The homologous donor template can comprise sequences homologous to sequences flanking the target nucleic acid cleavage site. For example, the donor template may have arms homologous to a 3q22.l region. The donor template may also have arms homologous to the pathological variations c.c50t and/or c.c 403t. Sister chromatids can be used by cells as repair templates. However, for gene editing purposes, the repair template may be provided as an exogenous nucleic acid, such as a plasmid, double-stranded oligonucleotide, single-stranded oligonucleotide, or viral nucleic acid. Using exogenous donor templates, additional nucleic acid sequences (e.g., transgenes) can be introduced between homologous flanking regionsOr modifications (e.g., single or multiple base changes or deletions) such that additional or altered nucleic acid sequences can also be incorporated into the locus of interest. MMEJ can use several base pairs of homologous sequences flanking the cleavage site to drive favorable end-ligated DNA repair results. In some cases, possible repair outcomes can be predicted based on analysis of potential micro-homology in the nuclease target regions.

CRISPR/Cas system

In the present application, the term "CRISPR/Cas system" or "CRISPR-Cas system" generally refers to a nuclease system consisting of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and a CRISPR associated protein (i.e. Cas protein) capable of cleaving almost all genomic sequences adjacent to a pro-spacer adjacent motif (PAM) in eukaryotic cells. The "CRISPR/Cas system" may be used to collectively refer to transcripts involved in a CRISPR-associated ("Cas") gene, as well as other elements involved in its expression or directing its activity, which may include sequences encoding the Cas gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or an active portion thereof), tracr mate sequences (encompassing "direct repeats" and partial direct repeats of processing in the context of an endogenous CRISPR/Cas system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR/Cas system), or other sequences and transcripts from the CRISPR locus. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

In the present application, the term "Cas protein", also referred to as "CRISPR-associated protein", generally refers to a class of enzymes complementary to CRISPR sequences, which are capable of using CRISPR sequences as a guide (guide) to recognize and cleave a specific DNA strand. Non-limiting examples of Cas proteins include: casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csinl and Csxl 2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Ccll, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, and/or their modified forms. In some embodiments, the Cas protein is a Cas9 protein.

In the present application, the term "Cas 9 protein" or "Cas 9 nuclease", also known as Csn1 or Csx12, generally refers to a class of proteins in type II CRISPR/Cas systems that are involved in both crRNA biosynthesis and in the destruction of invading DNA. Cas9 proteins typically include a RuvC nuclease domain and an HNH nuclease domain, each cleaving two different strands of a double-stranded DNA molecule. Already in different bacterial species such as Streptococcus thermophilus (S.thermophiles) Listeria innocua bacterium (L.) (Listeria innocua) (gasitunas, Barrangou et al.2012; jinek, Chylinski et al.2012) and Streptococcus pyogenes ((S. pyogenes)S.Pyogenes) Cas9 protein is described in (Deltcheva, Chylinski et al.2011). For example, Streptococcus pyogenes: (Streptococcus pyogenes) Cas9 protein, the amino acid sequence of which is found in SwissProt database accession No. Q99ZW 2; neisseria meningitidis (Neisseria meningitides) Cas9 protein, the amino acid sequence of which is shown in UniProt database number A1IQ 68; streptococcus thermophilus (Streptococcus thermophilus) Cas9 protein, the amino acid sequence of which is shown in UniProt database number Q03LF 7; staphylococcus aureus (D) (Staphylococcus aureus) Cas9 protein, the amino acid sequence of which is shown in UniProt database accession number J7RUA 5.

CRISPR/Cas systems can include many short repeated sequences, called "repeats". When expressed, the repeat sequences may form secondary structures (e.g., hairpins) and/or comprise unstructured single-stranded sequences. Repetitive sequences usually occur in clusters and often differ from species to species due to evolution (diverge). These repeated sequences are regularly spaced from a unique intermediate sequence called a "spacer", thereby forming a repeat-spacer-repeat (repeat-spacer-repeat) locus structure. The spacer is identical or highly homologous to known foreign invader sequences. The spacer-repeat unit encodes crisprna (crrna), which is processed into the mature form of the spacer repeat unit. crRNA contains a "seed" or spacer sequence (a form naturally occurring in prokaryotes, the spacer sequence targeting a foreign invader nucleic acid) that targets the target nucleic acid. The spacer sequence is located at the 5 'or 3' end of the crRNA.

The CRISPR/Cas system can also include a polynucleotide sequence encoding a CRISPR-associated protein (Cas protein). The Cas gene encodes a nuclease that is involved in the biosynthesis and interference phases of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and/or tertiary structures.

In nature, trans-activation CRISPR RNA (tracrRNA) is required for crRNA biosynthesis in type II CRISPR systems. tracrRNA can be modified by endogenous RNaseIII and then hybridized to crRNA repeats in pre-crRNA. Endogenous RNaseIII may be recruited to cleave pre-crRNA. The cleaved crRNA can be subjected to exonuclease trimming to produce a mature crRNA form (e.g., 5' end trimming). the tracrRNA can remain hybridized to the crRNA, and the tracrRNA and crRNA are associated with a site-directed polypeptide (e.g., Cas 9). The crRNA in the crRNA-tracrRNA-Cas9 complex can direct the complex to a target nucleic acid that can hybridize to the crRNA. Hybridization of the crRNA to the target nucleic acid can activate Cas9 for target nucleic acid cleavage. The target nucleic acid in a type II CRISPR system is called a Protospacer Adjacent Motif (PAM). Indeed, PAM is essential to facilitate binding of site-directed polypeptides (e.g., Cas 9) to target nucleic acids. Type II systems (also known as Nmeni or CASS 4) can be further subdivided into type II-A (CASS 4) and type II-B (CASS 4 a). CRISPR/Cas9 systems useful for RNA programmable gene editing can be found in Jinek et al, Science, 337 (6096): 8l6-82l (2012), international patent application publication No. WO2013/176772 provides numerous examples and applications of CRISPR/Cas endonuclease systems useful for site-specific gene editing.

gRNA

The methods of the present application include providing a genome-targeted nucleic acid that can direct an associated active polypeptide (e.g., Cas protein) to a specific target sequence within a target nucleic acid (e.g.,RHOan allele). The nucleic acid that targets the genome can be RNA. The genome-targeted RNA may be referred to herein as a "guide RNA" or "gRNA". In certain instances, a gRNA described herein can be complementary to a target nucleic acid.In other cases, the gRNA can be identical to the target nucleic acid (when said to be the same, the "U" in the RNA corresponds to thymine "T" in the DNA due to the difference in bases encoding the RNA and the DNA). In other cases, the nucleic acid sequence (e.g., DNA) encoding the gRNA can be identical or complementary to the target nucleic acid. In the present application, the terms "target nucleic acid," "target nucleic acid," and "target region" are used interchangeably and generally refer to a nucleic acid sequence that can be recognized by a gRNA, either as a double-stranded nucleic acid or as a single-stranded nucleic acid. The gRNA may be transcribed or replicated from sequences encoding the gRNA, e.g., the gRNA may be transcribed from DNA sequences encoding the gRNA. In the present application, the term "sequence encoding a gRNA" generally refers to a DNA sequence from which the gRNA can be obtained by transcription. In the present application, the "sequence encoding a gRNA" may have the same nucleotide sequence as a target sequence of the gRNA.

In certain instances, a gRNA can comprise at least a spacer sequence and CRISPR repeat that hybridizes to a target nucleic acid sequence of interest. In type II systems, the gRNA also contains a second RNA called the tracrRNA sequence. In type II CRISPR systems, the CRISPR repeat and tracrRNA sequences hybridize to each other to form a duplex. The gRNA can bind to the Cas protein, forming a guide RNA-Cas protein complex. The genome-targeted nucleic acid can render the complex target-specific due to its association with the Cas protein. Thus, the genome-targeted nucleic acid can direct the activity of the Cas protein. In some cases, the nucleic acid targeted to the genome may be a double-stranded guide RNA. In certain instances, the gRNA may be a single-stranded guide rna (sgrna). The double-stranded guide RNA or single-stranded guide RNA may be modified.

In some cases, a double-stranded guide RNA can comprise two RNA strands. The first strand may comprise an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat. The second strand may comprise a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat), a 3' tracrRNA sequence and optionally a tracrRNA extension sequence.

In certain instances, the sgRNA can comprise in the 5' to 3' direction an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat, a single molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and optionally a tracrRNA extension sequence. The optional tracrRNA extension may comprise elements for additional functions (e.g., stability) that contribute to the guide RNA. A single guide linker can link the smallest CRISPR repeat and the smallest tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may comprise one or more hairpins.

For example, the sgRNA can comprise a variable length spacer sequence having 17-30 nucleotides at the 5' end of the sgRNA sequence. In other cases, the sgRNA can include a variable length spacer sequence having 17-24 nucleotides at the 5' end of the sgRNA sequence. For example, the sgRNA can comprise a sequence of 21 nucleotides. For example, the sgRNA can comprise a sequence of 20 nucleotides. For example, the sgRNA can comprise a sequence of 19 nucleotides. For example, the sgRNA can comprise a sequence of 18 nucleotides. For example, the sgRNA can comprise a sequence of 17 nucleotides. For example, the sgRNA can comprise a sequence of 22 nucleotides. For example, the sgRNA can comprise a sequence of 23 nucleotides. For example, the sgRNA can comprise a sequence of 24 nucleotides. The sgRNA can be unmodified or modified.

The grnas described herein can bind to sequences in a target nucleic acid of interest. A nucleic acid (or portion thereof) that targets a genome can interact with a target nucleic acid in a sequence-specific manner by hybridization (i.e., base pairing). The nucleotide sequence of the sgRNA can vary depending on the sequence of the target nucleic acid of interest.

In the CRISPR/Cas system of the present application, the gRNA sequence can be designed to hybridize to the target nucleic acid in the vicinity of a PAM sequence recognizable by the Cas protein used in the system. The gRNA may be completely matched or mismatched to the target sequence. Cas proteins typically have a specific PAM sequence that can be recognized in the target DNA.

For example, the Cas9 protein may be from streptococcus pyogenes (ii)S. pyogenes) The Cas9 protein is recognized in the target nucleic acidPAM comprising in particular the sequence 5 '-NRG-3', wherein R comprises A or G, wherein N may be any nucleotide. As another example, the Cas9 protein may be from staphylococcus aureus (s.) (Staphylococcus aureus) The Cas9 protein (SaCas 9) recognizes a PAM comprising the sequence 5 '-nngrr (t) -3' in a target nucleic acid, wherein R comprises a or G, wherein N can be any nucleotide. In some more specific cases, the PAM sequence recognized by SaCas9 can comprise 5 '-NNGRR-3', where R comprises a or G, where N can be any nucleotide. For example, a PAM as described herein may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs 39-43.

The grnas described herein can specifically bind to the mutation site.

In certain instances, the gRNA can be associated with a peptide comprising the mutation siteRHOAt least a portion of the nucleic acids in the alleles specifically bind. For example, the sequence encoding the gRNA may comprise the nucleotide sequence set forth in any one of SEQ ID numbers 1, 2, and 4.

In certain instances, the gRNA may be conjugated with a c.c50t mutationRHOAt least part of the nucleic acid sequence of exon 1 of the allele is specifically complementary. E.g. said and comprising c.c50t mutationsRHOA gRNA that is specifically complementary to at least a portion of the nucleic acid sequence of exon 1 of an allele can comprise the nucleotide sequence set forth in any one of SEQ ID numbers 44-45. For example, the sequence encoding the gRNA may comprise the nucleotide sequence set forth in any one of SEQ ID numbers 1-2.

In certain instances, the gRNA may be conjugated to a c.c 403t-containing mutationRHOAt least part of the nucleic acid sequence of exon 2 of the allele is specifically complementary. For example, the peptide comprises the c.c403t mutationRHOA gRNA that is specifically complementary to at least a portion of the nucleic acid sequence of exon 2 of an allele can comprise the nucleotide sequence set forth in SEQ ID number 47. For example, the sequence encoding the gRNA may comprise the nucleotide sequence set forth in SEQ ID number 4.

In some cases, the percent complementarity between the gRNA and the target nucleic acid can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some cases, the percent complementarity between the gRNA and the target nucleic acid can be up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%.

Grnas for CRISPR systems described herein can be synthesized by chemical methods, e.g., high performance liquid chromatography. For example, two or more RNA molecules are linked together. Longer length RNAs (e.g., Cas 9-encoding RNAs) can be obtained by enzymatic reactions. Various types of RNA modifications can be introduced in the art during or after chemical and/or enzymatic synthesis of RNA, for example, to enhance stability, reduce innate immune response, and/or enhance other properties.

Carrier

The present application provides vectors. The polynucleotide that directs RNA (RNA or DNA) and/or the polynucleotide that encodes an endonuclease (RNA or DNA) can be delivered by viral or non-viral delivery vectors known in the art. Alternatively, the endonuclease polypeptide may be delivered by viral or non-viral delivery vectors known in the art, such as electroporation or lipid nanoparticles. In other aspects, the DNA endonuclease can be delivered as one or more polypeptides alone or pre-complexed with one or more guide RNAs, or one or more crrnas and tracrrnas. Some exemplary non-viral delivery vectors can be found in Peer and Lieberman, Gene Therapy, 18: 1127-1133(2011).

The vectors described herein can comprise the nucleic acid molecules of the present application (e.g., sequences encoding grnas and/or grnas). Polynucleotides can be delivered by non-viral delivery vectors, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes. As previously described, the site-directed polypeptide and the genome-targeted nucleic acid can each be administered to a cell or patient, respectively. In another aspect, the site-directed polypeptide may be pre-complexed with one or more guide RNAs or one or more crrnas and tracrrnas. The pre-composite may then be administered to a cell or patient. This pre-composite is called ribonucleoprotein particles (RNP). RNA is capable of forming specific interactions with polynucleotides (e.g., RNA or DNA). Although this property is exploited in many biological processes, it is also accompanied by the risk of promiscuous interactions occurring in nucleic acid-rich cellular environments. One approach to this problem is to form ribonucleoprotein particles (RNPs) in which RNA is pre-complexed with nucleases. RNPs can protect RNA from degradation. The nuclease in the RNP may be modified or unmodified. Likewise, the gRNA, crRNA, tracrRNA, or sgRNA may be modified or unmodified. There are many modifications known and that can be used in the art. For example, deletion, insertion, translocation, inactivation and/or activation of nucleotides. Such modifications may include the introduction of one or more mutations (including single or multiple base pair changes), increasing the number of hairpins, crosslinking, breaking particular stretches of nucleotides, and other modifications. Modifications may include inclusion of at least one non-naturally occurring nucleotide, or one modified nucleotide, or an analog thereof. The nucleotides may be modified at the ribose, phosphate and/or base moieties.

The vector may also be a polynucleotide vector, for example, a plasmid, cosmid or transposon. Suitable carriers for use have been described extensively and are well known in the art. One skilled in the art will appreciate that vectors comprising the nucleic acid molecules described herein may also comprise other sequences and elements useful for replication of the vector in prokaryotic and/or eukaryotic cells. For example, the vectors described herein may include a prokaryotic replicon, i.e., a nucleotide sequence that has the ability to direct the host's own replication and maintenance in a prokaryotic host cell (e.g., a bacterial host cell). Such replicons are well known in the art. In certain instances, the vector may comprise a shuttle element that renders the vector suitable for replication and integration in prokaryotes and eukaryotes. In addition, the vector may also include a gene capable of expressing a detectable marker (e.g., a drug resistance gene). The vector may also have a reporter gene, e.g., a gene that encodes a fluorescent or other detectable protein.

In certain instances, the vector can include a viral vector, e.g., AAV, lentivirus, retrovirus, adenovirus, herpesvirus, and hepatitis virus. Methods for producing viral vectors comprising a nucleic acid molecule (e.g., an isolated nucleic acid molecule described herein) as part of the vector genome are well known in the art and can be performed by those skilled in the art without undue experimentation. In other cases, the vector can be a recombinant AAV virion that has packaged therein a nucleic acid molecule as described herein. Methods of producing recombinant AAV may comprise introducing a nucleic acid molecule described herein into a packaging cell line, producing AAV infection, helper functions for AAV cap and rep genes, and recovering the recombinant AAV from the supernatant of the packaging cell line. Various types of cells can be used as packaging cell lines. For example, packaging cell lines that can be used include, but are not limited to, HEK 293 cells, HeLa cells, and Vero cells.

In certain instances, the vector can be an adeno-associated vector (AAV). In the present application, the term "adeno-associated vector" generally refers to a vector derived from naturally occurring and available adeno-associated viruses as well as artificial AAV. The AAV may include different serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, as well as any AAV variant or mixture. The AAV genome is typically flanked by Inverted Terminal Repeats (ITRs), and the term "ITRs" or "inverted terminal repeats" refers to segments of nucleic acid sequences present in AAV and/or recombinant AAV that form the T-shaped palindrome required to complete the AAV lytic and latent life cycle. Techniques for producing AAV vectors are standard in the art, and include providing to a cell the polynucleotide to be delivered, the rep and cap genes, and the AAV genome to be packaged that contributes to viral function. Production of AAV vectors typically requires the presence of the following components within a single cell (referred to herein as a packaging cell): rAAV genome, AAV rep and cap genes separated from (e.g., absent from) the rAAV genomeAnd helper virus. The AAV rep and cap genes may be from any AAV serotype, and may also be from an AAV serotype different from the AAV genomic ITRs, including but not limited to the AAV serotypes described herein. The AAV vectors in the present application may comprise targetingRHOgRNA at the site of mutation in the gene. For example, the sequence encoding the gRNA may comprise the nucleotide sequence set forth in any one of SEQ ID numbers 1, 2, and 4.

In certain instances, the gRNA-encoding sequence can be located in the same vector as the Cas9 protein-encoding nucleic acid. In other cases, the gRNA-encoding sequence can be located in a different vector than the Cas9 protein-encoding nucleic acid.

The AAV vectors of the present application can be from a variety of species. For example, the AAV may be an avian AAV, a bovine AAV or a goat AAV. In certain embodiments, the vector is AAV 8.

The methods of the present application may include generating packaging cells, i.e., generating cell lines that can be used to stably express all essential components of AAV. For example, the AAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from the AAV genome, and a plasmid (or plasmids) carrying a selectable marker such as a neomycin resistance gene are integrated into the genome of the cell. AAV genomes have been introduced into bacterial plasmids by methods such as GC tailing (Samulski et al, 1982, Proc. Natl. Acad. S6.ETSA, 79: 2077. 2081). The packaging cell line can then be infected with a helper virus (e.g., adenovirus). In addition to plasmids, adenovirus or baculovirus can be used to introduce AAV genome and/or rep and cap genes into packaging cells.

In the present application, the term "subject" generally refers to any subject for whom diagnosis, treatment, or therapy is desired. For example, in the present application, a subject in need thereof mayRHOThe gene comprises a mutation site selected from the group consisting of: c.c50t and c.c 403t. In certain instances, the subject may comprise a mammal. In certain instances, the subject may comprise a human. In certain instances, the subject may comprise an east asian human.

In another aspect, the present application providesCompositions for treating retinitis pigmentosa in a subject, which may include removalRHOAn active ingredient of a mutation site of a gene and a pharmaceutically acceptable carrier, wherein the mutation site is selected from the group consisting of: c.c50t and c.c 403t. The compositions may comprise a physiologically tolerable carrier and a cellular composition, and optionally at least one biologically active agent dissolved or dispersed in the therapeutic composition as an active ingredient. Generally, the vectors described herein can be administered in the form of a suspension with a pharmaceutically acceptable carrier. One skilled in the art will recognize that pharmaceutically acceptable carriers that can be used can include buffers, compounds, cryopreservatives, preservatives or other agents, and do not interfere with the delivery of the carrier to be used. The composition may also comprise a cell preparation, e.g., an osmotic buffer, which allows for maintenance of the integrity of the cell membrane, and optionally, a nutrient solution, which maintains cell viability or enhances implantation upon administration. Such formulations and suspensions are known to those skilled in the art, or may be adapted for use with the vectors and/or cells of the present application using routine experimentation.

In the present application, the term "administering" may be by a method or route to introduce the cells and/or vector into the subject, or to some desired site of the subject. The cells and/or vectors can express a nucleic acid molecule of the present application (e.g., a sequence encoding a gRNA and/or a gRNA) at a desired site (e.g., a site of injury or repair) to produce a desired effect. The cells (or their differentiated progeny) and/or vectors may be administered by any suitable route that can deliver the cells (or their differentiated progeny) and/or vectors to a desired site in a subject, with at least a portion of the implanted cells (or cellular components) and/or vectors remaining viable. After administration to a subject, the survival of the cells may be as short as several hours, e.g., twenty-four hours, several days, up to several years, or even consistent with the life of the patient. In certain instances, the administering comprises injecting. For example, the carrier may be administered by a systemic route of administration, such as intraperitoneal or intravenous route. For example, the administering may comprise a sub-retinal injection.

gRNA and vector pairs as described hereinRHOThe allelic mutation site may be cleaved with an efficiency of greater than about 50%, e.g., greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 98%, as measured in an in vitro enzymatic cleavage reaction. For example, using a fluorescence detection kit, the grnas and vectors described herein are capable of cleaving a target nucleic acid with a certain cleavage efficiency and specificity. In addition, the gRNA and the vector of the application do not affect the survival of host cells and have certain safety.

Reagent kit

The present application provides a kit that can include a nucleic acid molecule capable of encoding the sgRNA. The kits described herein can further comprise a nucleic acid molecule encoding the Cas protein. In certain embodiments, the nucleic acid molecule capable of encoding a sgRNA and the nucleic acid molecule encoding a Cas protein may be located in the same vector. For example, the sgRNA can comprise a nucleotide sequence set forth in any one of SEQ ID NOs 1, 2, and 4. For example, the Cas protein may be a Cas9 protein. As another example, the Cas protein may be a Cas9 protein from staphylococcus aureus (i.e., Sacas 9).

In the present application, the vector in the kit can be prepared by the following method: the sgRNA-encoding nucleic acid molecule and the Cas protein-encoding nucleic acid molecule are inserted into a blank vector (e.g., a plasmid vector), which can be inserted into the blank vector before, simultaneously with, or after the sgRNA-encoding nucleic acid molecule is inserted into the Cas protein-encoding nucleic acid molecule.

The kits described herein may comprise any one or more of the nucleic acid molecules, compositions, vectors, and cells mentioned above. They may be provided individually or in combination and may be provided in any suitable container, such as a vial, bottle or tube.

In certain instances, the kit includes one or more carriers, which can be an agent that facilitates use of one or more of the nucleic acid molecules, compositions, vectors, and cells in the kit. The carrier may be provided in any suitable container. The reagents may be provided in a form which is useful in a particular assay or in a form which requires the addition of one or more other components prior to use (e.g. in concentrated or lyophilized form). For example, the kit may comprise one or more reaction or storage buffers. The buffer may be any buffer including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some cases, the buffer may be basic. In some cases, the buffer may have a pH of from about 7 to about 10. In certain instances, the kit may include one or more oligonucleotides that may correspond to a guide sequence for insertion into a vector so as to operably link the guide sequence and regulatory elements. In certain instances, the kit can include a homologous recombination template polynucleotide.

In certain instances, the kit may also include a donor molecule of interest, such as a selection or screening marker. For example, the donor molecule of interest may be wild-typeRHOA gene or a fluorescent marker (e.g., GFP).

In certain instances, the kit comprises a detection reagent that can be used to detect one or more selected from the group consisting ofRHOGene mutation: c.c50t and c.c 403t. The detection reagent may be a substance, including a small molecule, a nucleic acid molecule, a polypeptide and/or a protein, capable of specifically recognizing the mutation site of the gene. For example, the detection reagent may be capable of specifically recognizingRHOProbes for the target region of a gene mutation, and/or capable of specific amplificationRHOPrimers to the target region of the gene mutation.

In certain instances, the pharmaceutically acceptable carrier is suitable for subretinal administration.

The kits described herein can further include other materials required from the commercial and user standpoint, including other buffers, diluents, filters, pillows, syringes, and inserts with instructions for performing any of the methods described herein. Suitable packaging materials may also be included and may be any packaging material consistent with the art, such as vials, ampoules, canisters, flexible packaging, and the like. These articles of manufacture may be further sterilized and/or sealed. The kits of the present application may further comprise instructions for use, a dosing regimen, one or more fine needles, one or more syringes, and a solvent.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

Example 1 Collection of information on genetic diagnosis of patients

In this study, a large number of RP patients who had been diagnosed in the third Hospital of Beijing university in recent years were collected for clear genetic diagnosisRHOThe RP family of the gene mutation discoversRHOTwo mutation hotspots of the gene, see table 2 below.

TABLE 2RHOHot spot of mutation of (2)

Example 2 SgRNA design and pX601-SgRNA plasmid construction

For the 2 mutation sites, a total of 5 sgrnas were designed for the SaCas9 system using the Benchling website, and the nucleotide sequences are shown in table 3 below.

TABLE 3 SgRNA sequences

The targeting vectors used in this application are: pX601-AAV-CMV: NLS-SaCas9-NLS-3xHA-bGHpA; U6: BsaI-sgRNA, map as shown in FIG. 1 (for vector information see https:// www.addgene.org/61591 /).

To is directed atRHOA schematic of sgRNA for the p.Thr17Met design is shown in FIG. 2, forRHOWeave of p.arg135trp designA sequence diagram of the code gRNA is shown in FIG. 3.

The specific steps of plasmid construction are as follows:

(1) SgRNA annealing

The T4 PNK and 10X T4 Ligation Buffer were thawed on ice for use. The following reaction system was prepared:

TABLE 4 reaction System

The prepared reaction system is placed on a PCR instrument, and the following reaction program is operated:

TABLE 5 reaction procedure

(2) Vector cleavage

The binding sites of the DNA sequences encoding the grnas were released using BSaI digestion, and the following digestion reaction was formulated in 1.5ml PCR tubes:

TABLE 6 reaction System

Enzyme digestion is carried out for 1-2 h/(K overnight enzyme digestion), recovery and purification are carried out, the concentration is measured, and the concentration is diluted to 50 ng/mu l.

(3) Connection of

The following ligation system (200 μ l PCR tube) was formulated using the recovery vector of the previous step and the annealed DNA sequence encoding the gRNA:

TABLE 7 connection system

And (3) placing the ligation reaction system in the last step into 37 ℃ for ligation for about 1-2h, and completing construction of a DNA sequence vector containing the coding gRNA.

(4) Plasmid transformation

1) Taking out competent cells, and thawing on ice

2) Taking 1 mu l and connecting the 1 mu l into a 50 mu l competence, and keeping the temperature for 2min on ice at 42 degrees for 90s for half an hour on ice

3) Shaking 500 mul of non-resistance culture medium for 1h,

4) 100 mu l taking plank

5) Picking bacteria (500 mu l of culture medium) on the second day, shaking for 3-4 hours, taking 200 mu l and sending to sequencing

(5) Plasmid extraction (according to Omega endotoxin removal plasmid big extract kit)

1) Correctly sequenced px601-SacAS9-RHO-SgRNA was shaken overnight (50-200 mL), shaken at 37 ℃ for 12-16 h to amplify the plasmid, and extracted the next day (shaking time is less than 16 h).

2) Taking 50-200mL of bacterial liquid, centrifuging at 4000Xg for 10min at room temperature, and collecting thalli.

3) The medium was discarded. 10mL of Solution I/RNaseA mixture was added to the pellet and the cells were resuspended completely by pipetting or vortexing.

4) Add 10mL Solution II, cover, gently invert the tube 8-10 times to get a clear lysate. If necessary, the lysate may be left to stand at room temperature for 2-3 min.

5) 5mL of pre-cooled N3 Buffer was added, the cap was closed, the tube was gently inverted up and down 10 times until a white floc was formed, and the tube was allowed to stand at room temperature for 2 min.

6) Preparing a syringe filter, pulling out the piston in the syringe, vertically placing the syringe on a suitable test tube rack, placing a collecting tube at the outlet of the lower end of the injector, and enabling the opening of the syringe to be upward. The lysate was immediately poured into the syringe of the filter. The cell lysate was left in the syringe for 5 min. The white floc floats on the surface of the lysate. The cell lysate may have flowed out of the filtration syringe port. The cell lysate was collected in a new 50mL tube. Carefully insert the syringe plunger gently into the barrel, and push the plunger slowly to allow the lysate to flow into the collection tube.

7) 0.1 volume of ETR Solution (blue) was added to the eluted filtered lysate, the tube was inverted 10 times, and then allowed to stand in an ice bath for 10 min.

8) The above lysate was bathed at 42 ℃ for 5 min. The lysate will again appear cloudy. At this point, centrifugation at 4,000Xg for 5min at 25 ℃ will result in a blue layer formation at the bottom of the tube by the ETR Solution.

9) Transferring the supernatant to another new 50mL tube, adding 0.5 volume of absolute ethanol at room temperature, slightly inverting the tube 6-7 times, and standing at room temperature for 1-2 min.

10) Sleeving a HiBind DNA Maxi combination column into 50mL collecting test tubes, and adding 20 mL of filter liquid into the HiBind DNA Maxi combination column. Centrifuge at 4,000Xg for 3min at room temperature. The filtrate was discarded.

11) Sleeving the HiBind DNA Maxi combination columns into the same collecting pipe, repeating the step 10 until all the residual filtrate is combined into the HiBind DNA Maxi combination columns, and centrifuging according to the same conditions.

12) Sleeving the HiBind DNA Maxi combined column into the same collecting pipe, adding 10mL of HBC Buffer to the HiBind DNA Maxi combined column, centrifuging for 3min at room temperature at 4,000Xg, and discarding the filtrate.

13) Sleeving the HiBind DNA Maxi combined column into the same collecting pipe, adding 15mL of DNA Wash Buffer (diluted by absolute ethyl alcohol) into the HiBind DNA Maxi combined column, centrifuging for 3min at room temperature at 4,000Xg, and discarding the filtrate. Note: the concentrated DNA Wash Buffer had to be diluted with ethanol as per the instructions before use. If the DNA washing buffer is placed in a refrigerator before use, it is taken out and left at room temperature.

14) Sleeving the HiBind DNA Maxi combined column into the same collecting pipe, adding 10mL of DNA Wash Buffer (diluted by absolute ethyl alcohol) into the HiBind DNA Maxi combined column, centrifuging for 3min at room temperature at 4,000Xg, and discarding the filtrate.

15) The highest speed (not more than 6000 Xg) was air-thrown to dry the matrix of HiBind DNA Maxi binding columns for 10 min.

16) (optional) further air-drying the HiBind DNA Maxi binding columns (optional) one of the following methods is selected to further dry the HiBind DNA Maxi binding columns and then elute DNA (if necessary):

a) putting the HiBind DNA Maxi combination columns in a vacuum container for 15min to dry ethanol: the column was moved to a vacuum chamber at room temperature, and the apparatus was connected to all vacuum chambers. The vacuum chamber is sealed and evacuated for 15 min. And removing the HiBind DNA Maxi combination column for next operation. b) Drying the column in a vacuum oven or drying at 65 deg.C for 10-15 min. And removing the HiBind DNA Maxi binding columns and carrying out the next step of operation.

17) The HiBind DNA Maxi combination column is placed on a clean 50mL centrifuge tube, 1-3mL of Endo-Free elasticity Buffer is directly added onto the HiBind DNA Maxi combination column substrate (the added amount depends on the concentration of the expected final product), and the mixture is kept standing for 5min at room temperature.

18) The DNA was eluted by centrifugation at 4,000Xg for 5 min.

19) The column was discarded and the DNA product was stored at-20 ℃.

The constructed pX601-SgRNA plasmid vector was named as follows:

TABLE 8 plasmid vectors

Example 3 detection of SaCas9-sgRNA target efficiency detection kitRHOEfficiency of in vitro editing of each sgRNA

The specific experimental steps are as follows:

(1) in vitro transcription of sgrnas:

1) primer design for SgRNA in vitro transcription

TABLE 9 primer sequences for SgRNA in vitro transcription

2) Construction of sgRNA in vitro transcription template

The PCR reaction system is as follows:

TABLE 10 reaction System

3) The steps of running gel by using 2.0% DNA gel and recovering gRNA in-vitro transcription template gel by using an OMEGA gel recovery kit are as follows:

a) adding equal volume of membrane binding solution into PCR reaction products, adding 1 mu L of membrane binding solution into every 1mg required for gel cutting recovery, heating at 50-60 ℃ for 7min until all gels are completely dissolved, uniformly mixing by vortex, and passing through a column for recovery;

b) lowering the liquid into a recovery column, centrifuging for 1min at 10000 x g, and removing filtrate;

c) adding 700 mu L of Washing Buffer, >13000 x g, centrifuging for 1min, and removing filtrate;

d) repeating step c);

e) centrifuging for 10min by using an empty tube >13000 x g;

f) transferring the column to a new Ep tube of 1.5mL, marking, and adding 20-30 mu L of Elution Buffer or ddH₂O, standing at room temperature for 2 min;

g) centrifuging for 1min at 13000 x g, discarding the adsorption column, storing DNA at 2-8 deg.C, measuring concentration and recording, and storing at-20 deg.C for a long time.

4) In vitro transcription of sgRNA (20 µ L system):

TABLE 11 transcription System

Mixing, placing in a constant-temperature incubator at 37 ℃ for reaction; and after the reaction is finished, adding 2 muL Dnase I, and carrying out gel running after reacting for 30min at 37 ℃.

5) Recovery of gRNA was performed as described in the OMEGA gel recovery kit.

（2）RHO Preparation of sgRNA cleavage template

1) Extraction ofRHOGenomic DNAs of iPSCs (p.Thr17Met, p.Arg135Trp) derived from induced pluripotent stem cells of adrP patients and normal iPSCs not carrying mutation sites were prepared using the genomic DNAs as templatesRHO sgRNA cleaves template dsDNA.

The genome DNA extraction steps are as follows:

a) 400x g centrifuging for 5min to collect cells, and discarding the supernatant. Adding 220 mul PBS, 10mL RNase Solution and 20 mul PK working Solution into the sample, and re-suspending the cells. Standing at room temperature for more than 15 min;

b) adding 250 mul Buffer GB into the cell resuspension, uniformly mixing by vortex, carrying out water bath at 65 ℃ for 15-30 min, and purifying by passing through a column;

c) adding 250 microliters of absolute ethyl alcohol into the digestive juice, and uniformly mixing for 15-20 seconds in a vortex manner;

d) the gDNA Columns were placed in a Collection Tubes 2 ml. Transferring the mixed liquid (including precipitate) obtained in the last step to an adsorption column. Centrifuge for 1min at 12,000 x g. If the column blockage occurs, centrifuging for 3-5 min at 14,000 x g. If the mixed liquid exceeds 750 mu L, the column is required to be passed by times.

e) The filtrate was discarded and the adsorption column was placed in the collection tube. Adding 500 mul Washing Buffer A into the adsorption column. Centrifuge for 1min at 12,000 x g.

f) The filtrate was discarded and the adsorption column was placed in the collection tube. Adding 650 mu l Washing Buffer B into the adsorption column. Centrifuge for 1min at 12,000 x g.

g) And (4) repeating the step.

h) The filtrate was discarded and the adsorption column was placed in the collection tube. Centrifuge in an empty tube at 12,000 Xg for 2 min.

i) The column was placed in a new 1.5ml centrifuge tube. Adding 30-100 mul of Elution Buffer preheated to 70 ℃ to the center of the membrane of the adsorption column, and standing at room temperature for 3 min. Centrifuge at 12,000 Xg for 1 min.

Note that: for tissues with abundant DNA, 30-100 μ l of Elution Buffer can be added for repeated Elution.

j) The adsorption column is discarded, the DNA is stored at 2-8 ℃, the concentration is measured and recorded, and the DNA is stored at-20 ℃ for a long time.

Each cleavage site in this study required two sets of dsDNA, one containing the mutation site, withRHO17-M-dsDNA and RHO135-M-dsDNA, wherein the PCR process takes iPSCs gDNA of a patient as a template; another group is dsDNA containing no mutation sites, havingRHO17-C-dsDNA andRHO135-C-dsDNA, and the PCR process takes normal human iPSCs gDNA as a template.

The primers used are as follows in table 12:

TABLE 12 cleavage template primer sequences

2) The PCR reaction system is as follows:

TABLE 13 reaction System

3) The PCR product was recovered using a 1.5% DNA gel run and an OMEGA gel recovery kit, as described above.

(3) SaCas9-SgRNA in vitro enzyme digestion reaction:

the reaction system is as follows:

TABLE 14 reaction System

Fully and uniformly mixing, reacting for 30min at 37 ℃, adding 3 microliter DNA loading buffer solution, mixing, boiling for 5min at 65 ℃, and running 2% agarose gel to analyze the enzyme digestion result.

The results of the detection of the in vitro efficiency of the RHO17-SgRNA are shown in FIG. 4, the RHO17-SgRNA1 and the SgRNA2 cut the in vitro templates of the experimental group, the cutting efficiency of the SgRNA1 reaches more than 50%, and the cutting efficiency of the SgRNA2 approaches 100%; and the in vitro template of the control group is not cut, so that the cutting efficiency and specificity of the two SgRNAs are good, and the two SgRNAs can be applied to subsequent cell experiments and in vivo experiments; and the RHO17-SgRNA3 cuts the templates of the experimental group and the control group, has poor specificity and cannot be used. In fig. 4, M: experimental groups; c: control group (wild type dsDNA).

The detection result of the in vitro efficiency of the RHO135-SgRNA is shown in figure 5, the RHO135-SgRNA1 cuts the in vitro template of the experimental group, the cutting efficiency is nearly 50%, and the in vitro template of the control group is not cut, so that the cutting efficiency and specificity of the sgRNA are proved, and the method can be applied to subsequent cell experiments and in vivo experiments; and the RHO135-SgRNA2 has cleavage on the templates of the experimental group and the control group, has poor specificity and cannot be used. In fig. 5, M: experimental groups; c: control group (wild type dsDNA).

Example 4 in vitro detection of gRNA editing efficiency using gRNA active fluorescence detection kit

(1) Principle of experiment

The fluorescent reporter gene mKate (a red fluorescent protein) in the fluorescent reporter plasmid in the kit is terminated by a stop codon in advance, the truncated mKate has no activity, in order to detect the gRNA detection activity, a target site identified by Cas9/gRNA can be inserted into the stop codon, under the action of Cas9 and the gRNA, double-stranded DNA at the target site is cut to form DSB, cells form active fluorescent protein through homologous recombination effect, and whether the activity of the fluorescent protein is increased or not is detected through a fluorescent microscope or a flow cytometer to judge the activity and knockout efficiency of the gRNA.

(2) Design of primers required for vector construction

The primer sequences are shown in Table 15.

TABLE 15 primer sequences

(3) Experimental procedure for vector construction

1) Annealing of primers

The synthesized primer sequences were used to prepare a system as shown in the following table, and after annealing, a double strand of DNA having a cohesive end was formed.

TABLE 16 reaction System

The prepared reaction system is placed on a PCR instrument, and the following reaction program is operated:

TABLE 17 reaction procedure

2) Carrier ligation reaction

TABLE 18 reaction System

Placing the prepared reaction system on a PCR instrument, and carrying out the following procedures: 30 min-1 h at 16 ℃.

3) And (3) transformation: adding 5 muL of the ligation product into 50 muL DH5 alpha competent cells which are just thawed, flicking and uniformly mixing, after ice bath for 30min, thermally shocking for 45 s at 42 ℃, immediately standing on ice for 2min, adding 950 muL and LB liquid culture medium preheated at 37 ℃, performing shake culture for 45 min at 37 ℃, and taking 100 muL to coat on an ampicillin-resistant plate.

4) And (3) positive clone identification: pairing a forward sgRNA primer and a sequencing primer TS-SP001, performing colony PCR, selecting 2-3 positive colonies with a product size of 632bp, shaking the bacteria, extracting plasmid DNA, sequencing, and performing sequence comparison after a reverse complementary sequencing result, wherein the sequencing primer is TS-SP 001: CTGATAGGCAGCCTGCACCTG (SEQ ID NO: 36). The sequencing sequence was as follows: vector sequence I (SEQ ID NO: 37) -sgRNA (SEQ ID NO: 1-5) -PAM (NNGRR (T), SEQ ID NO: 39) -vector sequence II (SEQ ID NO: 38).

5) The bacterial liquid with the correct sequencing is selected, shaken overnight, and the plasmid is extracted (the steps are the same as above).

(4) Plasmid transfection and analysis of results

1) The 293T cells were thawed and placed in 6-well plates.

2) And co-transfecting the constructed plasmid containing the target sequence and the plasmid vector containing gRNA and Cas9 to obtain a target cell, and setting a negative control group.

3) Fluorescence signal detection by fluorescence microscope: 48 h after transfection, flow cytometry was used to observe: if the experimental group detects a stronger fluorescence signal, the gRNA activity is higher. If the fluorescence signal is reduced or not detected in the experimental group, the gRNA is weak or inactive.

4) Flow cytometry for detecting gRNA activity

And co-transfecting the constructed plasmid containing the target sequence and the plasmid vector containing gRNA and Cas9 to obtain a target cell, and setting a negative control group. Statistical analysis can be performed by repeating each set of results three times (two-tailed t-test, p < 0.05).

The flow sorting steps are as follows:

a. the medium in the six-well plate was aspirated and washed 2 times with DPBS;

b. adding 500 mul of 0.05% pancreatin, and incubating and digesting for 4min at 37 ℃;

c. adding DMEM with the volume of 3-5 times to neutralize pancreatin, centrifuging at 800r/min for 2 min;

d. sucking off the supernatant, adding PBS to resuspend the cells, centrifuging at 800r/min for 2min, and repeating once;

e. the supernatant was aspirated off, and 200 μ l of PBS containing 2% FBS was added to resuspend the cells;

f. adding the liquid obtained in the previous step into a filter pipe, and enabling the liquid to completely pass through a filter screen;

g. and (6) loading on a machine.

The results are shown in FIGS. 6-10.

FIG. 6 shows the in vitro detection of the editing efficiency of RHO17-SgRNA1 using a gRNA active fluorescence detection kit. Wherein, A is an experimental group, the co-transfected plasmids are RHO17-mkate-mut-sgRNA1 and pX601-R17-sg1, and the pX601-R17-sg1 cuts the target site of the RHO17-mkate-mut-sgRNA1 plasmid; panel B shows experimental control groups RHO17-mkate-mut-sgRNA1 and pX601 empty plasmids without cutting; panel C control, co-transfected plasmids RHO17-mkate-wt-sgRNA1 and pX601-R17-sg1, pX601-R17-sg1 did not cleave the target site of the RHO17-mkate-wt-sgRNA1 plasmid. The results prove that the sgRNA can be applied to subsequent cell experiments and in vivo experiments, and the cutting efficiency and specificity of the sgRNA are the same as those of the in vitro cutting experiment.

FIG. 7 shows the in vitro detection of the editing efficiency of RHO17-SgRNA2 using a gRNA active fluorescence detection kit. Wherein, A is an experimental group, the co-transfected plasmids are RHO17-mkate-mut-sgRNA2 and pX601-R17-sg2, and the pX601-R17-sg2 cuts the target site of the RHO17-mkate-mut-sgRNA2 plasmid; panel B shows experimental control groups RHO17-mkate-mut-sgRNA2 and pX601 empty plasmids without cutting; panel C control, co-transfected plasmids RHO17-mkate-wt-sgRNA2 and pX601-R17-sg2, pX601-R17-sg2 did not cleave the target site of the RHO17-mkate-wt-sgRNA2 plasmid. The results prove that the SgRNA can be applied to subsequent cell experiments and in vivo experiments, and the cutting efficiency and specificity of the SgRNA are the same as those of the in vitro cutting experiment.

FIG. 8 shows the in vitro detection of the editing efficiency of RHO17-SgRNA3 using a gRNA active fluorescence detection kit. Wherein, A is an experimental group, the co-transfected plasmids are RHO17-mkate-mut-sgRNA3 and pX601-R17-sg3, and the pX601-R17-sg3 cuts the target site of the RHO17-mkate-mut-sgRNA3 plasmid; panel B shows experimental control groups RHO17-mkate-mut-sgRNA3 and pX601 empty plasmids without cutting; panel C control group, co-transfected plasmids RHO17-mkate-wt-sgRNA3 and pX601-R17-sg3, pX601-R17-sg3 cut the target site of the RHO17-mkate-wt-sgRNA3 plasmid. The results prove that the SgRNA has poor cutting specificity, has the same result as the in vitro cutting experiment, and cannot be applied to subsequent cell experiments and in vivo experiments.

FIG. 9 shows the in vitro detection of the editing efficiency of RHO135-SgRNA1 using a gRNA active fluorescence detection kit. Wherein, A is an experimental group, the co-transfected plasmids are RHO135-mkate-mut-sgRNA1 and pX601-R135-sg1, and the pX601-R135-sg1 cuts the target site of the RHO135-mkate-mut-sgRNA2 plasmid; panel B shows the empty plasmids of experimental control group RHO135-mkate-mut-sgRNA1 and pX601 without cutting; panel C control, co-transfected plasmids RHO135-mkate-wt-sgRNA1 and pX601-R135-sg1, pX601-R135-sg1 did not cleave at the target site of the RHO135-mkate-wt-sgRNA1 plasmid. The results prove that the SgRNA can be applied to subsequent cell experiments and in vivo experiments, and the cutting efficiency and specificity of the SgRNA are the same as those of the in vitro cutting experiment.

FIG. 10 shows the in vitro detection of the editing efficiency of RHO135-SgRNA2 using a gRNA active fluorescence detection kit. Wherein, A is an experimental group, the co-transfected plasmids are RHO135-mkate-mut-sgRNA2 and pX601-R135-sg2, and the pX601-R135-sg2 cuts the target site of the RHO135-mkate-mut-sgRNA2 plasmid; panel B shows the empty plasmids of experimental control group RHO135-mkate-mut-sgRNA2 and pX601 without cutting; panel C control, co-transfected plasmids were RHO135-mkate-wt-sgRNA2 and pX601-R135-sg2, with pX601-R135-sg2 cleaving the target site of the RHO135-mkate-wt-sgRNA2 plasmid. The results prove that the SgRNA has poor cutting specificity, has the same result as the in vitro cutting experiment, and cannot be applied to subsequent cell experiments and in vivo experiments.

Fig. 11 shows flow sorting statistics for in vitro sgRNA editing efficiency detection using a gRNA active fluorescence detection kit. As can be seen from FIG. A, there was a statistical difference in the cleavage efficiency between the experimental group (RHO 17-m 1) and the control group (RHO 17-W1); as can be seen from FIG. B, there was a statistical difference in the cleavage efficiency between the experimental group (RHO 17-m 2) and the control group (RHO 17-W2); as can be seen from FIG. C, the cleavage efficiencies of the experimental group (RHO 135-m 1) and the control group (RHO 135-W1) were statistically different, and were useful for subsequent cell and in vivo experiments. Three replicates of each experimental and control group were set up for flow sorting.

Example 5 in vitro validation of gRNA safety and specificity Using 293T cells

(1) 293T cell culture

The medium used for 293T cells was high-sugar DMEM, 5% CO, supplemented with 10% fetal bovine serum and 100U/ml dual cyan/streptomycin antibody₂The culture was carried out at 37 ℃.

1) Recovery of cryopreserved cells

a) The temperature of the thermostatic waterbath is adjusted to 37 ℃, the frozen cells are taken out from the liquid nitrogen, the cover is clamped by tweezers, and the cells are quickly shaken in the water.

b) The frozen stock solution was transferred to a 15ml graduated centrifuge tube, 10ml of cell culture solution was added slowly and the solution was mixed gently by shaking. The cap was screwed down, fired at 1000rpm/min and centrifuged for 3 minutes.

c) And (4) after the fire is over, adding a proper amount of culture solution, slightly blowing and beating the cell sediment at the bottom, transferring the cells into a culture bottle, and putting the culture bottle into an incubator for culture.

2) Cell passage

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%, passaging of the cells was started.

b) Old culture medium in the cell culture flask was washed out and washed 3 times with PBS. Adding 500 mu l of trypsin containing EDTA into a culture bottle, placing the culture bottle into an incubator for incubation for about 1 minute, immediately adding 1ml of culture solution into the culture bottle to stop digestion when cell gaps become large and cells become round, gently blowing and beating the cells by using a straw, transferring the liquid in the culture bottle into a centrifuge tube after the cells all float from the bottom of the bottle, and centrifuging at 1000rpm/min for 2 minutes.

c) The supernatant was discarded and the pelleted cells were resuspended by adding 2ml of medium to the centrifuge tube. The cell suspension was split into 4 new culture flasks, each with 4ml of culture medium, gently shaken to mix the cells evenly and spread over the flasks, and placed into a cell incubator for culture.

d) 293T cells were seeded into 6-well plates 1-2d prior to transfection and plasmid transfection was performed when cells grew to 80-90% confluence.

(2) Transfection of 293T cells

1) We numbered the pX601-SaCas9 plasmid constructed in example 2, see table below:

TABLE 19 plasmid vectors

2) The plasmid transfection procedure and procedure were as follows:

numbering 1.5mL of EP tubes according to the sequence, adding 250 mu L of DMEM culture medium (serum-free) into each tube, sequentially adding 1.5 mu g of pX601-RHO-SgRNA plasmid and 1 mu g of pLenti-GFP plasmid according to the table, fully and uniformly mixing by vortex, adding 7.5 mu L of PEI transfection reagent into each tube, uniformly mixing by finger flicks (not uniformly mixing by vortex), and carrying out transfection after placing for 20min at room temperature.

Taking out the 6-well plate cultured with 293T, observing the confluence degree of cells under a microscope, changing the liquid of the cells, removing waste liquid, adding 1.75mL of new complete culture medium, numbering the empty cells according to the above, adding the prepared transfection system into each well according to the number, and culturing overnight.

The next day, GFP expression was observed under a fluorescence microscope to evaluate the transfection efficiency, and the plasmid after transfection was cultured in the presence of good transfection efficiency.

Two days after transfection, a proper amount of puromycin is added into each well (including a negative control group) (note that the antibiotic concentration can be gradually increased from 0.1 mug/mL to 0.5 mug/mL), cells positive for transfection are screened, the survival condition of the cells is observed every day, the liquid is changed every 2 days, and the puromycin with a corresponding amount is added during liquid change.

And (3) stopping antibiotic screening when the cells in the negative control wells completely die and the cells in the experimental group and the control group are alive (indicating successful transfection), using a normal culture medium, subculturing to a 6cm culture dish after the cells in the 6-well plate grow to 80-90% confluency, collecting the cells after the cells grow to 80-90% confluency, and preparing to extract genome DNA, wherein the whole process is about 7-10 d.

3) Extraction of 293T cell genome DNA

The steps are the same as the previous steps.

(3) gRNA efficiency validation

1) T7E1 enzyme digestion experiment

And carrying out PCR according to the system, running glue, and recovering a PCR product, wherein the recovery steps are the same as the previous steps.

Carrying out T7E1 enzyme digestion reaction on the PCR recovery or gel cutting recovery product obtained above

a) T7E1 enzyme digestion annealing system (19.5 muL):

TABLE 20 reaction System

b) T7E1 enzyme cutting annealing program:

95℃ 2 min

the temperature of 95 ℃ to 85 ℃ is-2 ℃/s

The temperature of 85 ℃ to 25 ℃ is-0.1 ℃/s

16℃ ∞。

a) T7E1 enzyme digestion reaction system

TABLE 21 reaction System

37℃ 20 min

d) The enzyme digestion product runs glue

Preparing glue: 2.5% gel, double dye

Glue running procedure: 140V, 20min to 30min

e) And checking the glue running result. As shown in FIG. 12, the plasmid did not cleave the target genome after transfection of 293T cells with pX601-R17-Sg1 as shown in A in FIG. 12, demonstrating good specificity of RHO17-SgRNA 1; as shown in B in FIG. 12, after 293T cells were transfected with pX601-R17-Sg2, the plasmid did not cleave the target genome, demonstrating good specificity for RHO17-SgRNA 2; as shown by C in FIG. 12, the plasmid did not cleave the target genome after transfection of 293T cells with pX601-R135-Sg1, demonstrating good specificity for RHO135-SgRNA 1. In fig. 12, M: pX601-R17-Sg1, pX601-R17-Sg2 or pX601-R135-Sg1, C: pX 601.

Example 6 verification of gRNA editing efficiency in patient-derived iPSCs

(1) Extraction and culture of patient renal epithelial cells

Patient A carries in this applicationRHO c.50C>T mutation, patient B harborsRHO c.403C>T, Normal human C does not have disease nor carries any gene mutation sites.

The method is carried out by using a renal epithelial cell separation and culture kit provided by Beijing Saibei company, and comprises the following experimental steps:

1) urineasy was isolated complete medium, additives, Gelatin, wash brought intercellular. The additive was thawed in a refrigerator, and the remainder was placed outside.

2) Irradiating with an ultraviolet lamp, a 12-hole plate, a 50ml centrifugal tube, a 15ml centrifugal tube, an electric pipettor, a pipette, a 5ml gun and a gun head, and a 1ml gun and a gun head; and opening the water bath kettle at 37 degrees.

3) Taking urine: gloved, disinfected, preferably midstream urine, and sealed with PARAFILM.

4) Gelatin is 750 muL/hole, the bottom (3 holes) of the capsule is covered for no less than half an hour and placed at 37 ℃.

5) Sterilizing the outer surface of the urine bottle by 75% alcohol, subpackaging into a 50mL conical-bottom centrifuge tube, sealing, and then 400Xg for 10 min.

6) Urineasy was removed and the complete medium + additive was isolated and prepared (5 mL basal medium per 0.5mL additive).

7) The supernatant was slowly pipetted to 1ml along the upper liquid level with a minimum speed pipette.

8) Resuspending into a 15ml centrifuge tube, adding 10ml of washing solution, mixing uniformly, and 200Xg for 10 min.

9) The 12-well plate was removed, Gelatin was aspirated, washed once with washing solution (500 μ L), and 750 μ L UrinEasy was added to each well to separate complete medium, which was placed at 37 degrees.

10) Remove 15mL centrifuge tube, leave 0.2mL cell pellet.

11) Urineeasy isolation complete medium resuspend cell pellet: one hole for male and two holes for female, as D0.

12) And (4) observation:

d1: observing whether pollution exists or not;

d2: supplement isolation medium-women: 500 mul/hole; male: 250 mu l/hole;

d4: if no adherence exists: half the volume of the medium was changed every two days, and 1mL of complete medium was slowly added along the wall.

13) Until adherence occurs: after the cells adhere to the wall (3-7 days or 9-10 days), the Urineasy expands the complete culture medium

Culturing for two days, 500 mu L, and changing the liquid in a full amount. And (4) after the wall is attached, carrying out passage at about 9-12D (not more than 14D) and 80-90% confluence degree, and sequentially carrying out passage to a 6-well plate, and freezing and storing after 6cm culture dish and 10cm culture dish for later use.

(2) Induction of iPSCs

Patient-derived (p.arg135trp and p.thr17met) renal epithelial cells were induced into iPSCs, the procedure was as follows:

1) the somatic cell confluency reaches 70-90%, then the digestion passage can be carried out, and the cells are inoculated in a 96-well plate; the inoculation density is controlled at 5000-15000/well, 3 density gradients can be set according to the cell condition, and each gradient is provided with 3 multiple wells. The day of cell inoculation was recorded as day-1.

2) Day 0: observing the confluence degree and state of cells under a mirror, selecting multiple wells with different gradients for digestion counting, and selecting wells with the cell amount of 10000-. Please configure reprogramming media a as follows:

TABLE 22 culture Medium formulation

3) Centrifuging the reprogramming additive II, adding 97 mu L of reprogramming culture medium A into a tube of the reprogramming additive II, uniformly mixing to prepare a reprogramming culture medium B, adding 100 mu L of reprogramming culture medium B into one 96 selected holes meeting the conditions, and putting the culture plate back to the incubator.

4) Day 1-2: the cells were observed under a mirror and photographed to record morphological changes of the cells. If the cell morphology changes obviously, the reprogramming culture medium B can be removed, and the cell is switched to the reprogramming culture medium A for continuous culture; if the morphological change is not obvious, the liquid can not be changed.

5) Day 3: if the cell morphology is obviously deformed in the first two days and the cell growth speed is high, the pancreatin digestion passage can be carried out. Transferring the cells to 2-6 holes of a six-hole plate according to the cell state and the cell quantity, and adding a reprogramming culture medium C to form single cell adherence as much as possible. Please configure reprogramming media C as follows:

TABLE 23 culture Medium formulation

6) Day 4: observing the adherence condition of the cells, and if most of the cells adhere well, replacing a fresh somatic cell culture medium for continuous culture.

7) Day 5: when a small cluster of clones (a colony mass of 4 or more cells) is formed, the somatic cell culture medium is changed to a Reproneasy human cell reprogramming medium. If no small cluster clone is formed temporarily, observation can be continued for one to two days, and the ReProeasy human cell reprogramming culture medium is replaced.

8) Day 6-8: under the observation of a microscope, if a small cluster of clones becomes large, one clone block has more than 10 cells, and a Reroyasy human cell reprogramming culture medium can be directly changed into a PSCeasy human pluripotent stem cell culture medium (or a PGM1 human pluripotent stem cell culture medium). If more dead cells are observed before changing the solution, the solution can be changed after being washed by PBS (phosphate buffer solution) after being balanced at room temperature.

9) Day 9-20: the cells were observed under the mirror and photographed to record the morphological changes of the cells. The room temperature equilibrated fresh pscaasy human pluripotent stem cell medium was changed daily.

10) Day 21: under-lens observation, if a single cell clone fills the entire 10-fold field of view, the clone can be cut with a 1mL syringe needle (or other instrument such as a glass needle) and picked into a 48-well plate previously coated with Matrigel (if the clone is in good condition, the cells are thick and grow faster, and can be picked directly into a 24-well plate).

11) After cloning and picking out, inoculating the cells by using a PSCeasy human pluripotent stem cell recovery culture medium, and continuously culturing the cells to a required generation number by using the PSCeasy human pluripotent stem cell culture medium after the cells are attached to the wall.

(3) iPSCs electricity transfer

1) The plasmid pX601 is modified to have puromycin resistance and is used for screening after electrotransformation, the modified plasmids are named as pX601-R17-puro-sg1, pX601-R17-puro-sg2 and pX601-R135-puro-sg1, and the blank plasmid is the pX601-GFP-puro plasmid.

2) The iPSCs are electrically converted as follows:

a) and (4) recovering the frozen iPSCs to a 6-well plate, and performing electrotransformation when the cells grow to 60-80% confluence.

b) Discarding the culture medium, adding 1ml PBS to clean the cells, discarding the waste liquid, and repeating twice.

c) Adding 1mL of EDTA digestive solution with the concentration of 0.05% to iPSCs, and standing and digesting for 3min at 37 ℃;

d) taking out the 6-hole plate, confirming the cell digestion condition under a light microscope, adding the complete culture medium to neutralize the digestive juice, slightly knocking the wall of the culture dish, slightly blowing the cells by using a gun head, collecting the cell suspension to a 15mL centrifuge tube, counting, and centrifuging at room temperature at 200Xg for 5 min.

e) Discarding the supernatant as much as possible, and resuspending the cells by using a prepared 100 muL electrotransfer solution (82 muL electrotransfer base solution +18 muL electrotransfer additive +5 mug plasmid), wherein the number of all electrotransfer cells is 1x10⁶And (4) respectively. And (3) lightly blowing, uniformly mixing, sucking to the bottom of the electrode cup by using a suction pipe to ensure that the cup bottom is completely covered and no bubble is generated, and placing the electrode cup in a Lonza electric rotating instrument for electric rotation, wherein the electric rotating procedure is CA-137.

f) After the electroporation was completed, iPSCs were re-inoculated into 6-well plates for culture.

g) Cell survival was observed 24h after transfection and selection with puromycin was initiated the next day after transfection.

h) Later cell cultures, screens and see example 5.

(4) Verification of gRNA editing efficiency

1) gDNA for extracting iPSCs

The method is the same as the previous method.

2) PCR reaction

The primers used are as in Table 24 below, the procedure is as before.

TABLE 24 primer sequences

3) T7E1 enzyme digestion experiment

The steps are the same as the previous steps.

The results of the patient A experiment are shown in FIG. 13, and the Sanger sequencing results in the left panel show patient A carrierRHO c.50C>Heterozygous mutations of T, not carried by normal persons; right picture displayRHO17-SgRNA1 andRHO17-SgRNA2 gene editing effects in patient a and normal human C iPSCs, a-GFP indicating that patient a iPSCs were transfected with pX601-GFP-puro blank control plasmid only; a-17sgRNA1 or A-17sgRNA2 indicates that iPSCs of patient A are transfected with pX601-R17-puro-sg1 or pX601-R17-puro-sg2 plasmids; c-17sgRNA1 or C-17sgRNA2 showed that normal human iPSCs were transfected with pX601-R17-puro-sg1 or pX601-R17-puro-sg2 plasmid, and the results of T7E1 showed that,RHO17-SgRNA1 andRHO17-SgRNA2 for iPSCs of patient A onlyRHOThe gene mutation site has an editing effect, but has no editing effect on the corresponding gene position of a normal person. Consistent with the results of the in vitro experiments above.

The results of the patient B experiment are shown in FIG. 14, and the Sanger sequencing results in the left panel show patient B carryoverRHO c.403C>Heterozygous mutations of T, not carried by normal persons; right picture displayRHO135-SgRNA1 has gene editing effect in patient B and normal human C iPSCs, and B-GFP shows that the iPSCs of patient B are only transfected with pX601-GFP-puro blank control plasmids; b-135sgRNA1 shows that patient B's iPSCs were transfected with pX601-R135-puro-sg1 plasmid; c-135sgRNA1 shows that normal human iPSCs were transfected with pX601-R135-puro-sg1 plasmid, and the results of T7E1 show that,RHO135-SgRNA1 for patient B only iPSCsRHOThe gene mutation site has an editing effect, but has no editing effect on the corresponding gene position of a normal person. Consistent with the results of the in vitro experiments above.

Example 7 in vitro validation of Gene editing efficiency of gRNA Using 3D retinal tissue

(1) Patient A, B and normal human C-derived iPSCs were induced into 3D retinal tissue.

The specific steps are shown in table 25 below:

TABLE 25 Experimental procedures

(2) Construction and coating of AAV8 viruses

The RHO17-SgRNA1 and RHO17-SgRNA2 edit the same site, and the cutting effect of the latter is stronger than that of the former, and the RHO17-SgRNA2 is adopted for the 3D retina experiment and the mouse experiment in view of the results of the in vitro experiment and the stem cell experiment.

The plasmid pX601 is transformed to carry GFP fluorescent protein for later screening of GFP + cells, the transformed plasmids are named as pX601-R17-GFP-sg2 and pX601-R135-GFP-sg1, and the blank control plasmid is the pX601-GFP plasmid.

1) And (5) plasmid amplification. The constructed AAV vector, packaging plasmid and helper plasmid need to be subjected to massive endotoxin removal extraction, and a Qiagen large-extraction kit is used for massive extraction of the plasmid, and the steps are the same as the steps.

2) AAV8-293T cells. The cell density was observed on the day of transfection and 80-90% full vector, packaging and helper plasmids were transfected.

3) AAV8 virus: viral particles are present in both the packaging cells and the culture supernatant. Both the cells and the culture supernatant can be collected to obtain the best yield.

4) Purification of AAV, Long-term storage at-80 ℃.

(3) 3D retinal tissue in vitro validation of gRNA gene editing efficiency

1) Infection with AAV8 induced successful 3D retinal tissue. Observing the expression condition of GFP under a fluorescence microscope 3 days after infection, collecting GFP + 3D retinal tissue, digesting the 3D retinal tissue by using a papain system to prepare single cell suspension, screening by using a flow cytometry instrument, extracting gDNA (deoxyribonucleic acid), and the method is the same as the method.

2) PCR reaction

The primers used are as in Table 26 below, the procedure is as before.

TABLE 26 primer sequences

3) T7E1 enzyme digestion experiment

The method is the same as the previous method.

The results are shown in FIG. 15. FIG. 15A showsRHOGene editing effects of SgRNA2 on 3D retinal tissue of patient a and normal human C, a-GFP indicating that the 3D retina of patient a is infected with pX601-GFP blank control virus only; a-17sgRNA2 indicates that patient A's 3D retina was infected with pX601-R17-GFP-sg2 virus; c-17sgRNA2 shows that normal human 3D retina was transfected with pX601-R17-GFP-sg2 virus, and the results of T7E1 show,RHO17 of SgRNA2 on 3D retina of patient A onlyRHOThe gene mutation site has an editing effect, but has no editing effect on the corresponding gene position of a normal person; FIG. 15B showsRHO135-SgRNA1 gene editing effects in 3D retinal tissue of patient B and normal human C, B-GFP indicating that the 3D retina of patient B was infected with only pX601-GFP blank control plasmid; b-135sgRNA1 indicates that patient B's iPSCs were infected with pX601-R135-GFP-sg1 virus; c-135sgRNA1 shows that the 3D retina of a normal human is infected with pX601-R135-GFP-sg1 virus, and the result of T7E1 shows that,RHO135-SgRNA1 on 3D retina of patient B onlyRHOThe gene mutation site has an editing effect, but has no editing effect on the corresponding gene position of a normal person. Consistent with the results of the in vitro experiments above.

Example 8 verification of Gene editing efficiency in humanized mouse model

(1) Construction of humanized mouse

The work of constructing the humanized mice was performed by Baioser diagram company, Beijing.

1) Humanized mouse production protocol

Against the human mutation site, constructedRHOHumanized mouse model (biotech of baiosacharis, beijing). The humanized mouse model has two kinds, one is portableRHOGene mutation site (p.Arg135Trp orThr17met), one is a mouse model in which only the humanized fragment is knocked in without mutation sites.

2) The method comprises the following steps:

a) development ofRHOGene humanization carries mutant (p.Thr17Met or p.Arg135Trp or) point-mutant mice:

designing and constructing gRNA for recognizing a target sequence; the test was carried out according to scheme 1, which was determined by both parties; namely the Targeting strategy-1-EGE-System;

constructing a CRISPR/Cas9 vector for target gene cutting;

③ detecting the activity of sgRNA/Cas 9;

designing and constructing a gene knock-in targeting vector; performed according to the content of the double-agreed scheme 1, a substitution of the coding region at the genomic level (4.9 kb for 4.7 kb) was carried out, with the introduction of mutation sites, i.e.c.50C > T, p.Thr17Met or c.403C > T, p.Arg135Trp;

transcribing sgRNA/Cas9 mRNA in vitro;

sixthly, injecting sgRNA/Cas9 mRNA and a targeting vector into the fertilized eggs of the mice;

⑦RHOdetecting and propagating the gene knocked-in F0 generation mouse;

⑧RHOacquisition and genotype identification of gene knock-in F1 generation heterozygote mice

b) Development ofRHOGene humanization knock-in mice:

designing and constructing sgRNA for recognizing a target sequence; the method is carried out according to scheme 1 which is commonly negotiated by two parties, namely Targeting strategy-1-EGE-System in the experimental scheme;

constructing a CRISPR/Cas9 vector causing target gene cutting;

③ detecting the activity of sgRNA/Cas 9;

designing and constructing a gene knock-in targeting vector; the replacement of the coding region at the genomic level (4.9 kb instead of 4.7 kb) was performed as per the contents of scheme 1;

transcribing sgRNA/Cas9 mRNA in vitro;

sixthly, injecting sgRNA/Cas9 mRNA and a targeting vector into the fertilized eggs of the mice;

⑦RHOdetecting and propagating the gene knocked-in F0 generation mouse;

⑧RHOobtaining and genotyping of heterozygote mice of the F1 generation by gene knock-in.

(2) Feeding and breeding

1) After obtaining two kinds of humanized mice, mice heterozygous for the F1 generation were allowed to undergo internal crossing, and a sufficient number of mice homozygous for the F2 generation or the F3 generation were obtained as soon as possible and used as AAV virus injection to evaluate the in vivo editing efficiency of AAV8-pX601-RHO-SgRNA obtained as described above.

(3) Genotyping of humanized mice

Primer pair WT-F/WT-R was designed in the wild gene sequence, and this pair of primers did not amplify the product of the mutant allele but only the wild type allele when subjected to PCR, whereas primer/Mut-R was designed to be humanized in miceRHOIn the gene sequence, when the primer pair WT-F/Mut-R is used for PCR, the product of the wild type allele cannot be amplified, and only the product of the mutant type allele can be amplified.

The primer sequences are shown in Table 27 below:

TABLE 27 primer sequences

The pair of WT-F/WT-R primers is mainly used for identifying the existence of wild type allele, and the PCR result of the pair of WT-F/Mut-R primers is combined to judge the specific genotype of the animal: homozygous/heterozygous/wild type.

The criteria for genotyping are given in table 28 below:

TABLE 28 genotype judgment standards

Description of the drawings: y: detecting PCR products with expected lengths by gel electrophoresis; n: the gel electrophoresis did not detect the expected length of the PCR product; H/H: a homozygous genotype; h/+: a heterozygote genotype; +/+: and (4) a wild type.

The PCR reaction system and procedure are described in "example 3", and the PCR products were sequenced to detect humanized miceRHOWhether the gene includes a mutation site that is desired to be knocked in.

(4) Mouse subretinal space AAV8 virus injection

The AAV virus injected into mice is the virus used for 3D retinal tissue infection.

1) Mydriasis with 1% atropine 30 minutes prior to injection; mydriasis is again performed before anesthesia.

2) After 80 mg/kg ketamine +8 mg/kg xylazine is injected intraperitoneally for anesthesia, the mouse is placed in front of an animal experiment platform of an ophthalmic surgical microscope, and a drop of 0.5% proparacaine local anesthetic is dropped on the mouse eye. The method comprises the following steps of (1) dividing by 100: 1, adding sodium fluorescein stock solution into AAV virus, and centrifuging at low speed.

3) An insulin needle is used for pre-pricking a small hole in the pars plana of the ciliary body of a mouse, a needle head of a micro-syringe is used for penetrating through the small hole and then entering the vitreous chamber of the eye of the mouse, at the moment, a proper amount of 2% hydroxymethyl cellulose is dripped on the eye of the mouse, so that the eyeground of the mouse can be clearly seen under a microscope, the needle head is continuously inserted into the subretina of the periphery of the opposite side by avoiding the vitreous body, AAV virus with fluorescein sodium is slowly pushed in, the injection amount of each eye is 1 mu l, and the fluorescein sodium is used as an indicator for judging whether to be injected into the subretina.

4) After operation, mice are observed whether the mice have abnormality or not, and neomycin eye ointment is given to prevent infection.

(5) Evaluation of the editing efficiency of AAV8-pX601-RHO-SgRNA in humanized mice

1) 3 months after the operation, a mouse living body imaging system is used for observing the expression of GFP in mouse eyeballs, the GFP + the mouse eyeballs are taken, a papain system is used for digesting 3D tissues to prepare single cell suspension, GFP + positive retinal cells are screened by a flow cytometry instrument, and gDNA is extracted.

2) PCR reaction

The primers used are shown in Table 29 below, in the same manner as above.

TABLE 29 primer sequences

3) T7E1 enzyme digestion experiment

The method is the same as the previous method.

FIG. 16A showsRHOAs a result of PCR running for genotype identification of the humanized mice, the mouse of number 20 was a humanized mouse carrying a heterozygous mutation, the mice of numbers 21 and 22 were humanized mice carrying WT, and the mouse of number 23 was a humanized mouse carrying a homozygous mutation; FIG. 16B shows a portable beltRHO c.50C>Humanized mouse with homozygous mutation of T, FIG. 16C shows a mouse harboring RHO c.403C>Humanized mice homozygous for the mutation T.

FIG. 17A showsRHOGene editing efficiency of 17-SgRNA2 on humanized mice, M-17sgRNA2, carrying c.50C>The humanized mouse with homozygous mutation of T is injected with AAV-R17-sg2-SaCas9 virus, while C-17sgRNA2 shows that the humanized mouse carrying WT is injected with AAV-R17-sg2-SaCas9 virus, and the T7E1 result showsRHO17SgRNA2 had an editing effect only on the humanized mouse carrying the mutation, but not on the corresponding site of the humanized mouse carrying WT; FIG. 17B showsRHOGene editing efficiency of 135-SgRNA1 on humanized mice, M-135sgRNA1, carrying c.403C>The humanized mouse with homozygous mutation of T is injected with AAV-R135-sg1-SaCas9 virus, while C-135sgRNA1 shows that the humanized mouse carrying WT is injected with AAV-R135-sg1-SaCas9 virus, and the T7E1 result showsRHO135SgRNA1 had only an editing effect on humanized mice carrying mutations, but not on the corresponding sites in humanized mice carrying WT. The results of the in vivo experiments are consistent with those of the in vitro experiments described above.

Sequence listing

<110> third Hospital of Beijing university (third clinical medical college of Beijing university); beijing Zhongyin science and technology Co., Ltd

<120> nucleic acid molecule and kit for binding mutant RHO gene

<130> 0138-PA-011

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO135 SgRNA2-T7 forward primer

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO 17-reverse primer 1

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<223> RHO 135-Forward primer 1

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO 135-reverse primer 1

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO17-mKate-mut-sgRNA1 reverse primer

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO17-mKate-wt-sgRNA1 forward primer

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO17-mKate-mut-sgRNA2 reverse primer

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO17-mKate-wt-sgRNA2 forward primer

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO17-mKate-wt-sgRNA2 reverse primer

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO17-mKate-mut-sgRNA3 forward primer

<400> 24

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<210> 25

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO17-mKate-mut-sgRNA3 reverse primer

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO17-mKate-wt-sgRNA3 forward primer

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO17-mKate-wt-sgRNA3 reverse primer

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<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO135-mKate-mut-sgRNA1 forward primer

<400> 28

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<210> 29

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO135-mKate-mut-sgRNA1 reverse primer

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<211> 30

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO135-mKate-wt-sgRNA1 forward primer

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<210> 31

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO135-mKate-wt-sgRNA1 reverse primer

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<210> 32

<211> 30

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<213> Artificial Sequence (Artificial Sequence)

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<223> RHO135-mKate-mut-sgRNA2 forward primer

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> RHO135-mKate-mut-sgRNA2 reverse primer

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<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO135-mKate-wt-sgRNA2 forward primer

<400> 34

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<210> 35

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> RHO135-mKate-wt-sgRNA2 reverse primer

<400> 35

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<210> 36

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<213> Artificial Sequence (Artificial Sequence)

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<223> TS-SP001

<400> 36

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> vector sequence I

<400> 37

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aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca 240

ctcggctggg aggcctccac cgagatgctg taccccgctg acggcggcct ggaaggcaga 300

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<210> 38

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> vector sequence II

<400> 38

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cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac 120

gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc 180

tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa 240

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gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa ttgacccggg 540

tacccagctt tcttgtacaa agtggtacgc gtgaattcac tcctcaggtg caggctgcct 600

atcag 605

<210> 39

<211> 5

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PAM1

<400> 39

gagaa 5

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<223> PAM2

<400> 40

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<223> PAM3

<400> 41

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<210> 42

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<220>

<223> PAM4

<400> 42

cagga 5

<210> 43

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<220>

<223> PAM5

<400> 43

aagga 5

<210> 44

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> RHO17-SgRNA1

<400> 44

cguaccacac ccaucgcauu g 21

<210> 45

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> RHO17-SgRNA2

<400> 45

ugcguaccac acccaucgca u 21

<210> 46

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> RHO17-SgRNA3

<400> 46

cugcguacca cacccaucgc a 21

<210> 47

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> RHO135-SgRNA1

<400> 47

caccacguac cacucgaugg c 21

<210> 48

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> RHO135-SgRNA2

<400> 48

cacucgaugg ccaggaccac c 21

<210> 49

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 17-Forward primer 2

<400> 49

ttatgaacac ccccaatctc cc 22

<210> 50

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 17-reverse primer 2

<400> 50

gagggctttg gataacattg ac 22

<210> 51

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 17-Forward primer 3

<400> 51

ctagtgtcac cttggcccct c 21

<210> 52

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 17-reverse primer 3

<400> 52

tgctgcaaac atggcccgag a 21

<210> 53

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 135-Forward primer 2

<400> 53

tggctcctag gagaggcccc 20

<210> 54

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 135-reverse primer 2

<400> 54

ctgtttcttc ttctgcccta 20

<210> 55

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> WT-F

<400> 55

ggcagcagtg ggattagcgt tagta 25

<210> 56

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> WT-R

<400> 56

tgtgtagagg gtggtggtga atcct 25

<210> 57

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Mut-R

<400> 57

acgatcagca gaaacatgta ggcgg 25

<210> 58

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 17-Forward primer 4

<400> 58

gagtgtgggg actggatgac 20

<210> 59

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 17-reverse primer 4

<400> 59

gggactctcc cagacccctc 20

<210> 60

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 135-Forward primer 3

<400> 60

tgtccgggtt atttcatttc cc 22

<210> 61

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RHO 135-reverse primer 3

<400> 61

gagatgggac cagcccttgt 20

Claims (9)

1. A kit comprising 1) a vector, 2) a pharmaceutically acceptable carrier, wherein the vector comprises a nucleic acid molecule capable of encoding a sgRNA having a nucleotide sequence set forth in any one of SEQ ID NOs 1, 2, and 4.

2. The kit of claim 1, wherein the vector comprises a viral vector.

3. The kit of claim 1, wherein the vector comprises AAV.

4. The kit of claim 1, comprising a nucleic acid molecule encoding a Cas protein.

5. The kit of claim 4, wherein the nucleic acid molecule capable of encoding a sgRNA and the nucleic acid molecule encoding a Cas protein are in the same vector.

6. The kit of claim 1, comprising a detection reagent that can be used to detect one or more selected from the group consisting ofRHOGene mutation: c.c50t and c.c 403t.

7. The kit of claim 1, wherein the pharmaceutically acceptable carrier is suitable for subretinal administration.

8. The kit of claim 1, comprising one or more fine needles, one or more syringes, and/or a solvent.

9. A nucleic acid molecule for encoding sgRNA, the nucleotide sequence of which is shown in any one of SEQ ID NO 1, 2 and 4.

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