CN111850044A

CN111850044A - Method for constructing rhesus monkey model for retinitis pigmentosa based on in-vivo gene knockout

Info

Publication number: CN111850044A
Application number: CN202010685115.4A
Authority: CN
Inventors: 薛天; 李守振; 章梅
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2020-07-16
Filing date: 2020-07-16
Publication date: 2020-10-30

Abstract

The invention utilizes a CRISPR gene editing system and an AAV transduction system to directly knock out an RHO gene in adult macaque retina photoreceptor cells so as to simulate retinal degeneration caused by hereditary RHO mutation and construct a macaque Retinitis Pigmentosa (RP) animal model in 3-6 months. The model can promote the research and treatment of human RP diseases.

Description

Method for constructing rhesus monkey model for retinitis pigmentosa based on in-vivo gene knockout

Technical Field

The present invention relates to the field of molecular biology. In particular, the invention relates to the realization of animal disease modeling by gene knockout through a gene editing technology.

Background

Since the research and development of CRISPR/Cas9 gene editing technology (PMID: 24157548) by Zhang Feng professor at the Massachusetts institute of technology in 2013, the gene editing technology is widely applied to the gene modification of mammals, including the animal modeling of genetic diseases. Retinitis Pigmentosa (RP) is one of the most extensive genetic blinding diseases, the pathogenic genes of the Retinitis Pigmentosa (RP) are various, single-gene mutation pathogenic genes also account for a large proportion, and mutation of genes such as RHO, USH2A, RP1, PRPF31 and the like can cause dominant or recessive RP (PMID: 23701314). The animals currently used for RP studies are focused on rodent disease models, such as the P23H knock-in mouse model constructed by Krzysztof Palczewski (PMID: 21224384). However, the mouse has a long distance from the human evolution, and has obvious differences in retinal photoreceptor cell distribution and viability, and cannot well simulate human RP diseases. This naturally allows one to construct an RP animal model using non-human primates. Nevertheless, due to the extremely long breeding cycle and the extremely small number of the produced seeds, the construction of transgenic macaque RP models still has great limitation, and the extensive use of such macaque genetic RP models is more impractical, thus seriously hindering the development of RP disease research and treatment. Therefore, the creation of a macaque animal model which can well simulate the human hereditary retinitis pigmentosa becomes a problem to be solved urgently under the conditions of short time and low experimental cost. By using CRISPR gene editing technology, in-vivo gene knockout is realized in adult macaque photoreceptor cells, and a large number of available RP model animals are hopefully and rapidly manufactured for the pathological mechanism and treatment development research of RP diseases in the future.

Disclosure of Invention

The present inventors have solved the problems existing in the art through long-term studies. Taking the most extensive RP pathogenic gene RHO as an example, the RHO gene is directly knocked out in adult rhesus monkey retinal photoreceptor cells by using a CRISPR gene editing system and an AAV transduction system so as to simulate retinal degeneration caused by hereditary RHO mutation, and a rhesus monkey retinal pigment degeneration (RP) animal model is constructed in 3-6 months. The model can promote the research and treatment of human RP diseases. By adopting the scheme of the invention, the knockout of other RP related pathogenic genes (such as USH2A, RP1 and PRPF31) is also applicable to the construction of the macaque Retinitis Pigmentosa (RP) animal model. Specifically, the present invention solves the technical problems in the art by the following technical solutions.

1. A method for knocking out, preferably in vivo, a retinitis pigmentosa disease-causing gene in a mammalian photoreceptor cell, comprising constructing one or more, e.g., 3, sgrnas coding sequence targeting said disease-causing gene, preferably the coding sequence set forth in SEQ id nos 3-5 and the coding sequence for CRISPR enzyme, in an empty viral expression vector, transfecting a host cell with the constructed viral expression vector and expressing and packaging as an active virus, and infecting said photoreceptor cell with said active virus, thereby effecting knock-out of said retinitis pigmentosa disease-causing gene in said photoreceptor cell, wherein each of said sgrnas coding sequence is complementary to said disease-causing gene sequence; the retinitis pigmentosa disease-causing genes are, for example, RHO, USH2A, RP1, PRPF31 genes, preferably RHO genes; preferably the mammal is a primate, more preferably of the family actinidiaceae, still more preferably of the genus macaca, most preferably of the species macaca mulatta; the viral expression vector is, for example, an expression vector of adeno-associated virus, adenovirus or lentivirus; the CRISPR enzyme is, for example, Cas9 or SaCas 9.

2. The method of item 1, wherein the empty viral expression vector is an adeno-associated viral vector, and the adeno-associated viral expression vector and helper vector are transfected into the host cell and expressed and packaged as an adeno-associated virus, preferably an adeno-associated virus derived from AAV6, more preferably ShH 10.

3. The method of any one of items 1-2, wherein the host cell is a 293T cell.

4. A viral expression vector obtained by constructing one or more, e.g., 3, coding sequences of sgrnas targeting retinitis pigmentosa disease-causing genes, e.g., RHO, USH2A, RP1, PRPF31 genes, and coding sequences of CRISPR enzymes, in an empty viral expression vector, wherein each of the coding sequences of the sgrnas is complementary to the sequence of the disease-causing gene; the viral expression vector is, for example, an expression vector of adeno-associated virus, adenovirus or lentivirus; the CRISPR enzyme is, for example, Cas9 or SaCas 9.

5. A virus capable of knocking out a retinitis pigmentosa disease-causing gene, e.g., RHO, USH2A, RP1, PRPF31 gene, in a mammalian photoreceptor cell, wherein said virus is obtained by expressing and packaging the viral expression vector of item 4 in a host cell.

6. The virus of item 5, wherein the empty viral expression vector is an adeno-associated viral vector, and the adeno-associated viral expression vector and helper vector are transfected into the host cell and expressed and packaged as an adeno-associated virus, preferably the adeno-associated virus is derived from AAV6, more preferably ShH 10.

7. The virus of any one of items 5 to 6 wherein the host cell is a 293T cell.

8. A composition comprising a virus according to any one of items 5 to 7 and a pharmaceutically acceptable carrier.

9. A kit comprising a virus according to any one of items 5 to 7 or a composition according to item 8.

10. Use of the virus of any one of items 5-7 or the composition of item 8 or the kit of item 9 to infect a mammalian photoreceptor cell to knock out, e.g., in vivo, a retinitis pigmentosa disease-causing gene in the mammalian photoreceptor cell, wherein the retinitis pigmentosa disease-causing gene is, e.g., the RHO, USH2A, RP1, PRPF31 genes; preferably the mammal is a primate, more preferably of the family actinidiaceae, even more preferably of the genus macaca, most preferably of the species macaca mulatta.

11. Use of a virus according to any one of items 5 to 7 or a composition according to item 8 or a kit according to item 9 in the manufacture of a mammalian model of retinitis pigmentosa, wherein the mammal is preferably a non-human primate, more preferably of the family actinidiaceae, still more preferably of the genus actinidia, most preferably of the species macaca mulatta.

Drawings

FIG. 1 schematic of sgRNAs targeting the RHO gene.

FIG. 2 is a schematic representation of an AAV vector used herein.

Fig. 3 is a schematic view of the subretinal injection of rhesus macaque.

FIG. 4 is an in vivo gene editing resulting in Indels.

FIG. 5 Kiwi retina immunofluorescence staining microscopy.

Figure 6 rhesus monkey retina in vivo Optical Coherence Tomography (OCT) imaging.

FIG. 7 Kiwi retina photoreceptor cell transmission electron microscopy imaging.

Figure 8 rhesus monkey retina in vitro electroretinogram recordings.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

The present invention is directed to solving the problems of the prior art. Taking the most extensive RP pathogenic gene RHO as an example, the RHO gene is directly knocked out in adult rhesus monkey retinal photoreceptor cells by using a CRISPR gene editing system and an AAV transduction system so as to simulate retinal degeneration caused by hereditary RHO mutation, and a rhesus monkey retinal pigment degeneration (RP) animal model is constructed in 3-6 months. The model can promote the research and treatment of human RP diseases.

The specific technical content is as follows:

first, we designed three sgrnas targeting the RHO gene, as shown in fig. 1, to efficiently knock out the rhe gene of cynomolgus monkeys in vivo.

The invention adopts SaCas9, so that SaCas9 and sgRNA can be constructed in the same expression vector, and the design is not the first creation of the invention and is a direct application of Zhang Feng pX602 plasmid. (1) The three sgRNAs were constructed after the pX602 vector U6 promoter, respectively, as described in fig. 2. Three AAV vectors were transfected into 293T cells with ShH10 and pHelper helper plasmids, and three high-titer AAV viruses (5E12/ml) were packaged. The AAV is a core product, can efficiently infect photoreceptor cells through subretinal space injection, and knock out RHO genes.

(2) The virus is directly used in adult macaque eyes by means of subretinal cavity injection, and three injections are carried out, wherein each injection is 30-50 ul. The injection pattern is shown in figure 3. The virus bleb formed by injection can be fully recovered within 3 weeks.

The RHO gene is knocked out in vivo in adult macaque retina photoreceptor cells through an AAV virus transduction CRISPR system, photoreceptor cell degeneration can be caused in 3-6 months, human Retinitis Pigmentosa (RP) phenotype is simulated, and a macaque model based on in vivo gene knockout of retinitis pigmentosa is created.

In summary, the technical invention comprises two aspects. First, in vivo gene knockout was achieved in adult cynomolgus photoreceptor cells by a viral transduction system, which is exemplified by the RHO gene, but the technique is not limited to the RHO gene. Secondly, the RHO gene is knocked out in vivo in adult macaque photoreceptor cells, a macaque RP animal model capable of well simulating human RP diseases is created, and the technology can be similarly promoted to realize modeling of RP diseases of macaques with other pathogenic genes.

The use of sgrnas described herein may be single or multiple. The use of Cas9 is not limited to SaCas9, and all CRISPR enzymes with nucleic acid cleavage activity can be used. CRISPR transduction systems are not limited to AAV viruses, lentiviruses, adenoviruses, etc. may also be employed. AAV is the first recommended virus. Subretinal injection is not the only injection that can be used, and such as aqueous humor injection, intravenous injection, and modification can also be used.

The mammal described herein may be any mammal, preferably a primate, more preferably of the family actinidiaceae, still more preferably of the genus macaca, most preferably of the species macaca mulatta.

The host cell described herein is any host cell suitable for expression of the expression vectors herein, such as a mammalian host cell, e.g., a 293T cell.

In some embodiments, the invention provides a composition comprising a virus and a pharmaceutically acceptable carrier.

In some embodiments, the invention provides kits comprising a virus or composition described herein.

In some embodiments, the invention provides the use of the virus, composition or kit described herein to infect a mammalian photoreceptor cell to knock out, e.g., in vivo knock out, a retinitis pigmentosa disease-causing gene in the mammalian photoreceptor cell, wherein the retinitis pigmentosa disease-causing gene is, e.g., the RHO, USH2A, RP1, PRPF31 genes; preferably the mammal is a primate, more preferably of the family actinidiaceae, even more preferably of the genus macaca, most preferably of the species macaca mulatta.

The kits described herein comprise a container and a label or package insert on or with the container. In some embodiments, suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various materials such as glass or plastic. A container holds a composition comprising an adeno-associated virus as described herein. At least one agent in the composition is a carrier herein. The kit of the invention may further comprise a package insert indicating that the composition may be used to infect a mammalian photoreceptor cell to knock out, e.g., knock out in vivo, a causative gene of retinitis pigmentosa in the mammalian photoreceptor cell or to prepare a mammalian model of retinitis pigmentosa. The kit may also include other containers containing buffers such as water for injection, phosphate buffered saline, glucose solution, and may also include other materials such as other buffers, diluents, filters, needles, and syringes.

Advantages and advantageous technical effects of the invention

Compared with rodent models in the background art, the macaque adopted by the invention has an evolutionary relationship which is more close to human, has a macular structure, and can better simulate the characteristics of human RP diseases.

Compared with transgenic macaque animal models in the background art, the in-vivo gene knockout technology realized by the invention has no selectivity to macaques, any common macaque can be used, the cost is low, and the expensive cost for propagating transgenic macaques for a long time is avoided. Because the macaque has long birth seed period and low birth seed rate, the invention can rapidly (3-6 months, 3-5 years of breeding period is needed for transgenic macaques) obtain a large amount of available RP model animals.

In addition, the gene which can be operated by the invention is not limited to the RHO gene, and other genes can be knocked out in vivo in the retina photoreceptor cell to simulate the influence of other human gene abnormalities on RP diseases. Compared with other animal models, the invention only needs to replace sgRNAs in the viral vector, and can be conveniently and quickly applied to other gene knockout animal models.

Examples

Materials and reagents

The main reagents and materials used in this application:

The DNA sequence of the plasmid I is shown as SEQ ID NO. 1.

The DNA sequence of the plasmid II is shown as SEQ ID NO. 2.

sgRNA1, whose coding DNA sequence is GCGGGCGTGGTGCGCAGCCCCT (SEQ ID NO: 3).

sgRNA2, whose coding DNA sequence is GTCGGCCACGGCTAGGTTGAG (SEQ ID NO: 4).

sgRNA3, whose coding DNA sequence is GATACTTCGTCTTCGGGCCCA (SEQ ID NO: 5).

ShH10 the whole DNA sequence of which is shown in SEQ ID NO. 6.

The whole DNA sequence of the pHelper plasmid is shown in SEQ ID NO 7.

The experimental macaques were from the Kunming animal institute (theory: SMKX-2017023).

In vitro ERG recording instrument: electrophysiological platform (scientifica), digitada 1440A (axon CNS), MuticClamp 700B (axon CNS), cell culture medium DMEM (Gibco), fetal bovine serum FBS (Gibco), ultrasonic cell disruptor (Xinzhi, SCIENTZ-2D), iodixanol (sigma) for virus purification, ultracentrifuge L100-XP (Beckman), ultracentrifuge tube (Beckman, 344058), ultrafilter tube for virus concentration (Amicon Ultra-15), optical coherence tomography (Heidelberg), microscope (sixty) for ophthalmic surgery, transmission electron microscope (Hitachi HT7700), laser confocal microscope (Leica SP8), and cryosection instrument (Leica).

Example 1 Actinidia retinal photoreceptor cell in vivo Gene transduction

In this example, taking the RHO gene as an example, we designed three sgRNAs targeting the RHO gene of macaque, as shown in fig. 1, the specific sequences of sgRNAs 1-3 are SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, respectively. Subsequently, three sgRNAs were constructed after the U6 promoter in the plasmid-AAV vector of fig. 2, respectively, so that subsequently packaged AAV can express both Cas9 gene editing enzyme and sgRNAs. This vector was designed to be engineered from the Zhang FengpX 602 plasmid. The method specifically comprises the following steps: a. theThe TBG promoter in ddgene plasmid #61593(pX602) is digested by Xba1/Age1 and replaced by hSyn promoter, the modified plasmid is shown as plasmid I in figure 2, and the whole vector sequence is SEQ ID NO. 1. Taking sgRNA1 as an example, two short DNA sequences CACCGCGGGCGTGGTGCGCAGCCCCT (SEQ ID NO:8) and AAACAGGGGCTGCGCACCACGCCCGC (SEQ ID NO:9) are annealed and then ligated into a plasmid I digested with Bsa1 to construct a SaCas9/sgRNA1 plasmid, and in the same way, a SaCas9/sgRNA2 and a SaCas9/sgRNA3 can be constructed. The plasmid II pAAV-GFP is shown in figure 2, the specific sequence is SEQ ID NO:2, and the plasmid is an AAV vector for expressing Green Fluorescent Protein (GFP), and can enable cells infected by AAV virus to emit green fluorescence so as to achieve the purpose of visualization. Plasmid one or plasmid two, respectively with ShH10(SEQ ID NO:6) and pHelper (SEQ ID NO:7) constitute a three-plasmid viral packaging system transfected into HEK293T cells to generate the corresponding AAV, and the whole viral packaging and purification method can be found in reference PMID: 17406430. AAV-containing nuclei were sonicated to release virions, followed by purification of the virus by means of iodixanol density gradient ultracentrifugation (SW32 rotor, 32000 rpm, 4h, 18 ℃), the virus at 40% density was aspirated, and then concentrated using Amicon Ultra-15 filter (Millipore) to finally obtain titers of 5X 10 ¹²ShH10 AAV virus with individual viral genome/ml for subsequent infection of cynomolgus retinal photoreceptor cells. The virus titer determination mode is fluorescent quantitative PCR, and the sequence of the used primer is GFP:

GACAACCACTACCTGAGCAC (SEQ ID NO:10, forward primer) and

CAGGACCATGTGATCGCG (SEQ ID NO:11, reverse primer); SaCas 9:

AACTGACCAATCTGAACTCCG (SEQ ID NO:12, forward primer) and

TCTGGTTGTCGTTGGTGTG (SEQ ID NO:13, reverse primer). Some companies in the market can produce high-titer and high-quality AAV, and can also meet the requirements.

In example 1, we packaged AAV viruses of ShH10 subtypes, namely AAV-GFP and SaCas9/sgRNAs, and mixed four viruses, namely AAV-GFP, SaCas9/sgRNA1, SaCas9/sgRNA2 and SaCas9/sgRNA3, and injected into adult rhesus as shown in FIG. 3 by means of subretinal injectionThe sub-retinal space of the monkey is specifically infected with photoreceptor cells, and the RHO gene is knocked out. The subretinal space injection shown in fig. 3 is embodied in the following manner: adult macaques are anesthetized by intramuscular injection of ketamine (10mg/kg) and sodium pentobarbital (40mg/kg) and propofol (10-12 mg-kg) is instilled intravenously during surgery^-1·h^-1) Continuous anesthesia was performed. We passed two holes through the corneoscleral edge for insertion of a modified hamilton needle (38G, 40G) and illumination fiber, while performing a vitrectomy of the cynomolgus monkey eye to lower intraocular pressure prior to injection of AAV virus. For a single injection site, AAV virus injection volume was 30-50ul, and within three weeks, the injection blebs could be completely absorbed.

It should be noted that AAV-GFP is only a visualization tool and is not a virus that is essential for the present method, the sgRNA can be used singly or in multiple, the sub-retinal injection format is not limited, and the injection volume can be more or less.

In some embodiments, we can design an AAV transduction system targeting a certain gene, that is, sgrnas targeting a certain gene are constructed in a vector shown in fig. 2, and produce AAV viruses with high titer and capable of efficiently infecting cynomolgus monkey retinal photoreceptor cells, and by means of in vivo injection, in vivo knockout of retinal photoreceptor cell genes is achieved, the number of sgrnas is not limited, and the targeted gene is not limited to RHO.

Example 2 rhesus monkey retinal photoreceptor cell RHO knock-out assay

In example 2, the same as in example 1, AAV viruses were packaged and the subretinal injection of macaque was completed, and at the same time, AAV-GFP and SaCas9 were injected into the left eye of macaque, i.e. this group of viruses did not contain sgRNA, and therefore targeted RHO could not be achieved and knocked out, which was called a control virus group; and AAV-GFP and SaCas9/sgRNAs are injected into the right eye, the virus group can effectively target RHO and realize knockout, and the virus group is called an experimental virus group.

Macaques were sacrificially sampled three months after injection and surgically isolated, we obtained macaque retinal samples, the whole retinal genome was extracted with a genome extraction kit (Qiagen), and the RHO first exon was specifically amplified by PCR (NEB, Q5) using primers CATTCTTGGGTGGGAGCAGA (SEQ ID NO:14) and CAAGGTAGCGTTCAGAGCCA (SEQ ID NO:15), and the PCR products were subjected to deep sequencing (BGI). As shown in FIG. 4, in the experimental virus group, the sgRNA3 is targeted to generate a large number of indels (insertions and deletions), namely the theoretical cleavage site of SacAS9/sgRNA3, and a plurality of base additions or deletions occur between 18 th base and 19 th base, which can cause open reading frame shift (ORFshift) of the RHO gene, thereby causing the RHO protein to lose function. In contrast, no production of Indels was found in the control virus group. In conclusion, we effectively knock out rhesus monkey retinal photoreceptor cell RHO gene by in vivo injection of SaCas9/sgRNAAAV virus.

EXAMPLE 3 photoreceptor degeneration status (4 months of injection)

As in example 2, we performed sacrificial sampling of 4 month virally injected rhesus macaques and cryo-sections (14 microns) of retinas of virally injected rhesus macaques, immunohistochemical staining with antibody RHO (sigma), and microscopic observation with confocal laser microscopy. Immunohistochemistry is a common biological assay method, and will not be described again here.

As shown in FIG. 5, GFP is AAV-GFP expressed green fluorescent protein, which is seen to be distributed in photoreceptor cells in the outer nuclear layer, i.e., our method realizes efficient in vivo gene transduction of cynomolgus monkey retinal photoreceptor cells. Meanwhile, in the RHO immunohistochemical display, the RHO immunofluorescence signals are obviously reduced, and the structures of the photosensitive extracellular segment and the photosensitive intracellular segment almost disappear, namely, after the photosensitive cell RHO gene is effectively knocked out, the expression level of the coded protein RHO is obviously reduced, and the structure of the photosensitive cell is obviously degenerated.

The above phenotype mimics the human RP situation, demonstrating the effectiveness of the rhesus monkey RP model created by this technique.

Example 4 OCT in vivo imaging exhibits photoreceptor degeneration (6 months of injection)

In example 4, we performed in vivo optical coherence tomography (OCT, spectra, Heidelberg Engineering, Heidelberg, germany) imaging of 6 month virus-injected rhesus macaques. Adult macaques were anesthetized by intramuscular injection of ketamine (10mg/kg) and sodium pentobarbital (40 mg/kg) and mydriasis was performed with atropine and live fundus imaging was performed on an OCT instrument.

As shown in fig. 6, the control virus group (left side) was injected with a control virus that did not carry sgRNAs, and rhesus retinas were substantially normal and not affected by injection, virus toxicity, and the like. However, in the experimental virome (right side), the structure of the outer nuclear layer where the photoreceptor cells are located is obviously degenerated, and the structures such as the inner segment of the photoreceptor cell segment and the like almost disappear, namely, the photoreceptor cells of the macaque are obviously degenerated after the RHO gene is knocked out, the human RP condition is simulated, and the effectiveness of the macaque RP model created by the technology is demonstrated.

Example 5 degradation of subcellular structures of photoreceptor cells

In example 5, we performed sacrificial sampling of 6 month injected rhesus macaques and transmission electron microscopy imaging of retinal tissue. The detached retinal tissue was immediately fixed with 2.5% glutaraldehyde and 2% paraformaldehyde at room temperature for 2 hours, after which about 1mm was cut³The retina with Retinal Pigment Epithelium (RPE) was fixed with 2.5% glutaraldehyde and 2% paraformaldehyde at 4 ℃ overnight. After washing three times with the arsenious acid buffer, 1.5% (w/v) potassium ferrocyanide and 1% (w/v) osmium tetroxide were added for postimmobilization. Uranyl acetate 2% (w/v) was used for the first dyeing, followed by rinsing in ethanol and acetone and dehydration. The sample was then infiltrated with a gradient Epon and the test block was hardened at 60 ℃ for 48 hours. Sections (70nm) were cut with a microtome (Leica) and collected on a formvar/carbon coated copper mesh. The grid was rinsed and dropped into a 2% uranyl acetate aqueous solution, followed by a second dyeing with lead citrate. The samples were photographed using a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 80 kV.

As shown in fig. 7, the control virus group (upper) retina showed well-arranged rods with normal subcellular morphology, i.e. long and dense mitochondria in the inner segment (B), intact membrane disc structure in the outer segment (C) and regular nuclei with high nuclear to cytoplasmic ratio (D). However, in the experimental virome (lower) retina, we observed that mitochondria in the inner segment were vacuolated, even in a disrupted state (F), some disrupted and shortened outer segments were interspersed in the Outer Nuclear Layer (ONL), and the membrane discs were randomly arranged (G). Meanwhile, photoreceptor cells in the retinas of experimental virogroups showed strong apoptosis (H). In conclusion, RHO mutant rhesus macaques show typical RP-deficient subcellular structures and apoptosis, well mimicking human RP disease states.

Example 6 RP model macaque is severely impaired in visual function

In example 6, ex vivo Electroretinograms (ERG) recordings were made of rhesus monkey retinas injected for 4 months with virus. Fig. 8A shows a schematic diagram of an apparatus for recording an electroretinogram from an isolated retina, which is briefly described as a system for recording a voltage signal between an electrode located above and below the retina and a ground line for a conventional electrophysiological platform (scientific), wherein the signal is amplified by MutiClamp700B (Axon CNS), and then read and visualized by digitdata 1440a (Axon CNS). A flash (20 ms, 535nm wavelength) with an intensity of 3.8-1515.6 photons-. mu.m-2 was used to stimulate the photoreceptor cells. The ERG signal is amplified, low pass filtered at 20Hz, and then digitized for further analysis.

As shown in fig. 8, there was no significant difference in the ex vivo ERG recorded light responses between control virus group retinas (C) and retinas without viral infection (B); however, in the experimental virome retina (D), photoreceptor cell photoreaction was severely damaged. In E, the retina of the control virus group and the retina photoreceptor cell of the experimental virus group are statistically shown to have significant difference (P <0.01, uniperedstent t-test).

This result demonstrates that rhesus macaques with the RHO gene knockout in vivo RP model have severely impaired visual function. This phenotype mimics the human RP disease state, demonstrating the effectiveness of this macaque RP model.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for knocking out, preferably in vivo, a retinitis pigmentosa disease-causing gene in a mammalian photoreceptor cell, comprising constructing one or more, e.g., 3, sgrnas coding sequence targeting said disease-causing gene, preferably the coding sequences set forth in SEQ ID NOs 3-5 and CRISPR enzyme coding sequence, in an empty viral expression vector, transfecting a host cell with the constructed viral expression vector and expressing and packaging as an active virus, and infecting said photoreceptor cell with said active virus, thereby effecting knock-out of said retinitis pigmentosa disease-causing gene in said photoreceptor cell, wherein each of said sgrnas coding sequence is complementary to said disease-causing gene sequence; the retinitis pigmentosa disease-causing genes are, for example, RHO, USH2A, RP1, PRPF31 genes, preferably RHO genes; preferably the mammal is a primate, more preferably of the family actinidiaceae, still more preferably of the genus macaca, most preferably of the species macaca mulatta; the viral expression vector is, for example, an expression vector of adeno-associated virus, adenovirus or lentivirus; the CRISPR enzyme is, for example, Cas9 or SaCas 9.

2. The method of claim 1, wherein the empty viral expression vector is an adeno-associated viral vector, and the adeno-associated viral expression vector and helper vector are transfected into the host cell and expressed and packaged as an adeno-associated virus, preferably an adeno-associated virus derived from AAV6, more preferably ShH 10.

3. The method of any one of claims 1-2, wherein the host cell is a 293T cell.

5. A virus capable of knocking out a retinitis pigmentosa disease-causing gene, e.g., RHO, USH2A, RP1, PRPF31 gene, in a mammalian photoreceptor cell, wherein the virus is obtained by expressing and packaging the viral expression vector of claim 4 in a host cell.

6. The virus of claim 5, wherein the empty viral expression vector is an adeno-associated viral vector, and the adeno-associated viral expression vector and helper vector are transfected into the host cell and expressed and packaged as an adeno-associated virus, preferably an adeno-associated virus derived from AAV6, more preferably ShH 10.

7. The virus of any one of claims 5 to 6 wherein the host cell is a 293T cell.

8. A composition comprising the virus of any one of claims 5-7 and a pharmaceutically acceptable carrier.

9. A kit comprising a virus according to any one of claims 5 to 7 or a composition according to claim 8.

10. Use of the virus of any one of claims 5-7 or the composition of claim 8 or the kit of claim 9 for infecting a mammalian photoreceptor cell to knock out, e.g., in vivo, a retinitis pigmentosa disease-causing gene in the mammalian photoreceptor cell, wherein the retinitis pigmentosa disease-causing gene is, e.g., the RHO, USH2A, RP1, PRPF31 genes; preferably the mammal is a primate, more preferably of the family actinidiaceae, even more preferably of the genus macaca, most preferably of the species macaca mulatta.

11. Use of a virus according to any one of claims 5 to 7 or a composition according to claim 8 or a kit according to claim 9, preferably in a non-human primate, more preferably of the family Actinidiaceae, even more preferably of the genus Actinidia, most preferably of the species Actinidia chinensis, for the preparation of a mammalian model of retinal pigment degeneration.