CN117050951A

CN117050951A - Construction method of X-linked retinitis pigmentosa model dog

Info

Publication number: CN117050951A
Application number: CN202311073642.XA
Authority: CN
Inventors: 倪庆纯; 黄军就; 胡敏华; 王家慧; 赵志宏; 汪荣根; 周治东; 龙海斌
Original assignee: Guangzhou General Pharmaceutical Research Institute Co ltd
Current assignee: Guangzhou General Pharmaceutical Research Institute Co ltd
Priority date: 2023-08-24
Filing date: 2023-08-24
Publication date: 2023-11-14
Anticipated expiration: 2043-08-24
Also published as: CN117050951B

Abstract

The application relates to a construction method of an X-linked retinitis pigmentosa model dog. Compared with the prior art, the application has the following beneficial effects: the application is based on a gene editing means, specific mutation is carried out on RPGR genes, the fertilized eggs of dogs with the mutation can develop into X-linked retinitis pigmentosa dogs, and the detection of an Electroretinogram (ERG) shows that the function of a cone system of a model dog is damaged, and the model dog provides an effective means for screening effective drugs for X-linked retinitis pigmentosa.

Description

Construction method of X-linked retinitis pigmentosa model dog

Technical Field

The application relates to the field of gene editing, in particular to a method for constructing an X-linked retinitis pigmentosa model dog.

Background

Dogs are an experimental animal commonly used in basic medical research and teaching research, and play an important role in physiological, pharmacological, pathophysiological and other experimental researches. It has now been found that dogs have more than 800 genetic diseases, of which more than half of the diseases, such as muscular dystrophy, progressive retinal atrophy, hemophilia, etc., are manifested in similar clinical characteristics in human patients, and dogs are therefore suitable for use in the study of human genetic diseases. Meanwhile, the dogs are mild in character and easy to teach, and can be well matched with experiments. Compared with rodents such as mice, dogs have longer service lives, can provide a wider time window for drug screening and safety evaluation, and many advantages determine that dogs are ideal model animals for constructing genetic disease models.

In recent years, the rapid development of gene editing technology, CRISPR/Cas9, has demonstrated its great advantages. Through CRISPR, we can construct disease model, improve crop and treat genetic disease more quickly and efficiently. CRISPR/Cas9 can efficiently cut a DNA double strand of a target site, and insertion and deletion mutation is introduced into a DNA double strand break after the repair of an NHEJ pathway, so that efficient knockout of genes is realized. On the other hand, the genetic editing modification generated on the genome by CRISPR can be stably inherited, the constructed animal model can simulate the cause of the genetic disease from the DNA level, the phenotype is stable and continuous, and the offspring disease model can be obtained through natural propagation, so that the method has obvious advantages.

Retinitis pigmentosa (Retinitis pigmentosa, RP) is a type of inherited retinal disease characterized by degeneration of rods and cones, and is a significant cause of vision impairment and blindness. On a global scale, the incidence of retinitis pigmentosa is 1/4000, and 150 or more patients worldwide are afflicted with retinitis pigmentosa. Patients may exhibit impaired vision in the early stages of the disease due to progressive degenerative death of rod cells and secondary death of cone cells, which makes it difficult to accommodate sudden light loss. As the disease progresses, the patient may develop symptoms of night blindness, night vision loss, and peripheral vision loss. The patient's vision is further reduced at the end of the disease, presenting tunnel vision, severely impaired vision, and visual fundus examination with retinal peripheral pigmentation, pale vision and thinning of blood vessels. Finally, the patient is completely blind, and the work and life of the patient are greatly influenced.

It is known that mutations generated in more than 50 genes all cause retinitis pigmentosa, wherein retinitis pigmentosa due to gene mutation on the X chromosome is called X-linked retinitis pigmentosa (X-Linked Retinitis Pigmentosa, XLRP). X-linked retinitis pigmentosa is one of the most severe forms of retinitis pigmentosa, which progresses rapidly, and human patients manifest symptoms of night blindness around 10 years of age and severe vision loss around 40 years of age. RPGR is the most predominant pathogenic gene of XLRP, and 70% -80% of XLRP cases are caused by RPGR gene mutations. It is thought that the RPGR protein is involved in the transport of proteins within photoreceptor cells and may also be involved in the formation of photoreceptor cell structures.

At present, no effective medicine for treating X-linked retinitis pigmentosa exists clinically, so that a safer and more effective XLRP treatment scheme is developed, which not only brings about the hope of curing XLRP patients, but also has a broad market prospect. Therefore, the construction of an animal model for screening X-linked retinitis pigmentosa therapeutic drugs is of great importance, but no relevant report is currently available.

Disclosure of Invention

The embodiment of the application aims at providing an sgRNA combination of a targeted RPGR gene, and an RPGR gene editing system based on the combination can be used for successfully constructing an X-linked retinitis pigmentosa model dog.

In a first aspect of the application, there is provided a gene editing cell whose genome comprises a mutated RPGR gene having a fragment shown as SEQ ID NO. 13 or having fragments shown as SEQ ID NO. 14 and SEQ ID NO. 15.

In some embodiments of the application, the gene editing cell comprises:

(1) A sgRNA combination targeting the RPGR gene; or,

(2) An RPGR gene editing system comprising the sgRNA combination of the targeted RPGR gene;

wherein the sgRNA combination targeting the RPGR gene comprises:

the sgRNA as shown in SEQ ID NO. 22,

sgRNA as shown in SEQ ID NO. 23, and,

the sgRNA is shown as SEQ ID NO. 24.

In some embodiments of the application, the gene editing cell comprises a fertilized egg.

In a second aspect of the application, there is provided a RPGR gene-targeted sgRNA combination comprising:

sgRNA as shown in SEQ ID NO. 22;

sgRNA as shown in SEQ ID NO. 23; the method comprises the steps of,

the sgRNA is shown as SEQ ID NO. 24.

In a third aspect of the present application, there is provided an RPGR gene editing system comprising:

the sgRNA combination targeting the RPGR gene described in the second aspect.

In some embodiments of the application, the RPGR gene editing system further comprises a Cas9 protein and/or an mRNA of a Cas9 protein.

In a fourth aspect of the application, a recombinant vector is provided that transcribes the sgRNA combination, or also transcribes mRNA of the Cas9 protein.

In some embodiments of the application, the recombinant vector further comprises a screening element.

In some embodiments of the application, the screening element comprises a resistance gene BSD.

In a fifth aspect of the present application, there is provided a method of constructing a gene editing cell described in the first aspect, the method comprising the steps of:

and carrying out gene editing on the RPGR genes in the target cells, so that the genomes of the target cells comprise the mutated RPGR genes, and constructing the gene editing cells.

In some embodiments of the application, the construction method comprises the steps of:

and introducing the RPGR gene editing system into target cells to ensure that the genome of the target cells contains the mutated RPGR gene, so as to prepare the gene editing cells.

In some embodiments of the application, the means of introduction comprises microinjection.

In a sixth aspect of the present application, there is provided a method of constructing an X-linked retinitis pigmentosa model dog, the method comprising the steps of:

constructing the gene editing cell in the first aspect by taking a canine fertilized egg as a target cell; the method comprises the steps of,

transplanting the gene editing cells to a canine parent receptor for development, and screening an X-linked retinitis pigmentosa model canine from the birth pups.

In some embodiments of the application, the step of screening comprises: and carrying out PCR amplification on the genomic DNA of the birth pups, detecting the obtained amplification products, and screening out X-linked retinitis pigmentosa model dogs according to the obtained detection.

In some embodiments of the application, the primer pair used for PCR amplification is shown as SEQ ID NO. 16 to SEQ ID NO. 21.

In some embodiments of the application, the dogs comprise Beagle dogs.

In a seventh aspect of the application there is provided a tissue or organ comprising the gene editing cell of the first aspect or a tissue or organ of a model canine constructed in the sixth aspect.

In an eighth aspect of the application, there is provided the use of a gene editing cell as described in the first aspect, a sgRNA combination targeting an RPGR gene as described in the second aspect, an RPGR gene editing system as described in the third aspect, a recombinant vector as described in the fourth aspect, a tissue or organ as described in the seventh aspect for screening for a medicament for the treatment of X-linked retinitis pigmentosa.

In a ninth aspect of the present application, there is provided a gene editing cell as described in the first aspect, a canine constructed by the construction method as described in the sixth aspect, a tissue or organ detection primer pair as described in the seventh aspect, or a kit comprising the detection primer pair, as shown in SEQ ID NO. 16 to SEQ ID NO. 21.

Compared with the prior art, the application has the following beneficial effects:

the application is based on a gene editing means to carry out specific mutation on the RPGR gene, and fertilized eggs with the mutation can develop into X-linked retinitis pigmentosa dogs, thereby providing an effective means for screening effective drugs for X-linked retinitis pigmentosa.

Drawings

In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.

FIG. 1 shows the comparison of the agarose gel electrophoresis results of the RPGR gene fragments of the gene-edited dogs numbered 21P179 and 21P176 with those of the wild type Beagle dogs (# 1-21P179, #2-21P 176);

FIG. 2 is a schematic diagram of the RPGR gene alignment of the gene-edited dogs numbered 21P179, 21P176 with wild type Beagle dogs;

FIG. 3 is a comparison of the results of partial sequencing of the RPGR gene from the gene-edited dogs numbered 21P179, 21P176 and the wild type Beagle dogs;

FIG. 4 is visual electrophysiological statistics of gene-edited dogs and wild type Beagle dogs, numbered 21P 176;

fig. 5 is a photograph of two model dogs at their young age.

Detailed Description

The present application will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the application.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

The terms "plurality", "plural", "multiple", and the like in the present application refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present application can be implemented, the technical problem of the present application is solved, and the technical effect expected by the present application is achieved.

Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the application.

In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.

In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the present application, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1 to 10, and where t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present application,% (w/w) and wt% each represent weight percent,% (v/v) represents volume percent, and% (w/v) represents mass volume percent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.

First aspect of the application

The present application provides a gene editing cell whose genome comprises a mutated RPGR gene having a fragment shown as SEQ ID NO. 13 or having fragments shown as SEQ ID NO. 14 and SEQ ID NO. 15.

Optionally, the gene editing cell comprises:

(1) A sgRNA combination targeting the RPGR gene; or,

wherein the sgRNA combination targeting the RPGR gene comprises:

the sgRNA as shown in SEQ ID NO. 22,

sgRNA as shown in SEQ ID NO. 23, and,

the sgRNA is shown as SEQ ID NO. 24. The genome of the gene editing cell contains the mutated RPGR gene after being edited by the RPGR gene editing system.

The kind of the gene editing cell is not particularly limited in the present application, and may be, but not limited to, fertilized eggs such as fertilized eggs of dogs, and further fertilized eggs of Beagle dogs.

Second aspect of the application

The application provides a sgRNA combination of a targeting RPGR gene, which comprises the following components:

sgRNA as shown in SEQ ID NO. 22;

sgRNA as shown in SEQ ID NO. 23; the method comprises the steps of,

the sgRNA is shown as SEQ ID NO. 24.

Third aspect of the application

The application provides an RPGR gene editing system, which comprises:

the sgRNA combination targeting the RPGR gene described in the second aspect.

Optionally, the RPGR gene editing system further comprises a Cas9 protein and/or an mRNA of a Cas9 protein.

Fourth aspect of the application

The present application provides a recombinant vector that transcribes the sgRNA combination, or also transcribes mRNA of Cas9 protein.

Optionally, the recombinant vector further comprises a screening element. Further alternatively, the screening element comprises a resistance gene BSD.

Fifth aspect of the application

The present application provides a method of constructing a gene editing cell described in the first aspect, the method comprising the steps of:

The technology used for gene editing is not particularly limited in the present application, and for example, CRISPR/Cas9, TALEN or ZFN may be selected.

Optionally, the construction method comprises the following steps: and introducing the RPGR gene editing system into target cells to ensure that the genome of the target cells contains the mutated RPGR gene, so as to prepare the gene editing cells.

The method of introduction is not particularly limited in the present application, and may be selected from, but not limited to, microinjection.

Sixth aspect of the application

The application provides a construction method of an X-linked retinitis pigmentosa model dog, which comprises the following steps:

Alternatively, the canine fertilized egg may be a fertilized egg of a cell stage.

Optionally, the step of screening comprises: and carrying out PCR amplification on the genomic DNA of the birth pups, detecting the obtained amplification products, and screening out X-linked retinitis pigmentosa model dogs according to the obtained detection.

The method of detection in the present application is not particularly limited, and for example, a sequencing method or an agarose gel electrophoresis method may be used.

The application does not limit the detected sample, and the detected sample can be the blood of the baby birth or the tissue of the ear margin. The genomic DNA may be extracted from the sample using a commercial kit.

Further alternatively, the PCR amplification may be performed using a primer pair as shown in SEQ ID NO. 16 to SEQ ID NO. 21.

The application is not particularly limited to canine breeds and Beagle dogs can be selected but not limited thereto.

Seventh aspect of the application

The present application provides a tissue or organ comprising the gene editing cell of the first aspect or a tissue or organ of a model canine constructed in the sixth aspect.

Eighth aspect of the application

The application provides the use of a gene editing cell as described in the first aspect, a sgRNA combination targeting an RPGR gene as described in the second aspect, an RPGR gene editing system as described in the third aspect, a recombinant vector as described in the fourth aspect, a tissue or organ as described in the seventh aspect for screening a medicament for the treatment of X-linked retinitis pigmentosa.

Ninth aspect of the application

The application provides a gene editing cell as described in the first aspect, a canine constructed by the construction method as described in the sixth aspect, a detection primer pair for a tissue or organ as described in the seventh aspect, or a kit comprising the detection primer pair, wherein the primer pair is as shown in SEQ ID NO. 16-SEQ ID NO. 21.

Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present application, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Examples: preparation of RPGR knockout dogs

The preparation method of the RPGR knockout dog comprises the following steps:

(1) Multiple targeting sites and targeting sequences are designed according to the RPGR gene sequence of the dogs for screening.

The targeting site sequence was determined for the partial sequence of exon 4 (SEQ ID NO: 4), the partial sequence of exon 5 (SEQ ID NO: 5), and the partial sequence of exon 6 (SEQ ID NO: 6) of the canine RPGR gene.

3 targeting sites are designed according to the RPGR gene sequence, and the sequence of the targeting sites is as follows:

SEQ ID NO:4：5’-GCAGCTGGAGGAAATAATGA-3’；

SEQ ID NO:5：5’-TGTGGGGTGATAATTCTGAA-3’；

SEQ ID NO:6：5’-TTGTGGTGGAGGGCACACAG-3’。

(2) Constructing a plasmid according to the targeting sequence designed in the step (1), wherein the plasmid can simultaneously express sgRNA and SpCas9 protein in cells.

The sgRNA sequence synthesized according to the target site sequence and the complementary sequence thereof are as follows:

F1(SEQ ID NO:7)：CACCgcagctggaggaaataatga；

R1(SEQ ID NO:8)：AAACtcattatttcctccagctgc；

F2(SEQ ID NO:9)：CACCGtgtggggtgataattctgaa；

R2(SEQ ID NO:10)：AAACttcagaattatcaccccacaC；

F3(SEQ ID NO:11)：CACCGttgtggtggagggcacacag；

R3(SEQ ID NO:12)：AAACctgtgtgccctccaccacaaC。

(3) Firstly, transiently transfecting a gene editing plasmid and a virus packaging plasmid into HEK293T cells, packaging viruses, collecting virus liquid to infect Beagle canine fibroblasts, screening and treating the Beagle canine fibroblasts by using Blasticidin (BSD) medicaments with proper working concentration, and removing cells which do not express SpCas9 protein and sgRNA. The surviving cells are collected, the genome is extracted and subjected to PCR experiments, and then the editing condition of each target point is analyzed through Sanger sequencing results to determine the sgRNA for modeling.

(4) In vitro transcription of sgrnas, followed by mixing of sgrnas with SpCas9 protein forms RNP.

The sgRNA sequences transcribed in step (4) in this example are:

sgRNA2(SEQ ID NO:22):

GGCAGCTGGAGGAAATAATGAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttaaaa。

sgRNA3(SEQ ID NO:23):

GTGTGGGGTGATAATTCTGAAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttaaaa。

sgRNA6(SEQ ID NO:24):

GTTGTGGTGGAGGGCACACAGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttaaaa。

(5) RNP was injected into canine fertilized eggs.

(6) Fertilized eggs are transplanted into the oviduct on one side of the female dogs with the double oviducts subjected to embryo flushing, wherein the female dogs have less bleeding.

Embryo transfer was performed 5 times in total, with 5 recipients. 4 puppies were born, of which 2 positive puppies (see table 1 and fig. 5).

TABLE 1 embryo transfer Table

Experimental batch	Number of fertilized eggs transplanted	Production of puppy count	Gene-edited canine number	Gene editing dog ear number
					1	2	0	0
2	4	0	0
					3	8	2	2	21P176/21P179
4	5	1	0
					5	5	1	0

Sequencing results from the TA clone experiments showed that the puppy RPGR gene, numbered 21P179, had a large DNA fragment deletion, a 4621bp fragment was deleted from the target of sgRNA2, and a 2bp deletion was also present at the target of sgRNA6 (FIGS. 2, 3). Deletion of these DNA sequences causes both frame shift mutation and premature stop codon in the RPGR gene, failing to produce the correct RPGR protein. The male dog had the following sequence (SEQ ID NO: 13):

SEQ ID NO:13

AAGGAGGCAAAGTATATGCAGCTGGAGGAAATAAATCCATGTCCTAATGGGGGCTTAATGTCTAGTCATTTTGCTTTACTATAAAAGGAAGTAGAAGAAGTAAGATGGTTGCTATGCAAAACAGCTGAAGTCATTTTCTAGTTCATTAGTAGCAATGGTCATGAATTCATAAAGATTTTGGATTAGCATAATCAAAGGGAAAAGAAAATCTTAGCTGTATTTCCTTACCATACAATAAAAATAAGAAAGGATAATTGATCAAGTTGTCGTTTTCTAAGTTTTTTATTGAATAAGAATGTGGACCCCCAAAACTAGTATTTTGTCCATAATTATGTCGTCCTTGATCATTGTTCTTTTCCTTCAACAGCAGAGGGACAACTGTACACATTTGGAGAACCGGAGTGTGGAAAGTTAGGTCTTCCCAATCAGCTGCTGGTCAACCACAGAATGCCCCAGCCGGTACCTGGAATTCCCGGGAAGGTGGTCCAAGTAGCTTGTGGTGGAGGGCACAGTGGTTCTTACAG。

sequencing results from the TA clone experiments showed that the parent dog RPGR gene numbered 21P176 also had a large DNA fragment deletion, mainly at the target of the sgRNA2 on exon 4 to the target of the sgRNA6 on exon 6 (FIGS. 2, 3). Deletion of large DNA fragments results in frame shift mutations and premature stop codons in the RPGR gene, which do not result in the correct RPGR protein. The female dog has the following characteristic sequence (SEQ ID NO: 14):

SEQ ID NO:14：

AAGGAGGCAAAGTATATGCAGCTGGAGGAAATAAGGACATTTCCTTGTCCTTATTGGGCACAGCTGTCCTTATTCCACAGCTTGTCCACTGTCCCAGTGGTTCTTACAGG。

at the same time, we also detected the presence of another form of large DNA fragment deletion mutation in 21P176 (fig. 2). The mutation signature sequence is as follows (SEQ ID NO: 15):

SEQ ID NO:15：AAGGAGGCAAAGTATATGCAGCTGGAGGAAATAAAATAAAATAAGTGGGTGAAGGGCAAATTGGTTTAGAAAATGTAACCAATGTGTGTGTCCCTCAACAAGTGACTGTTGGAAAACCTATCTCCTGGATCTCTTGTGGATATTACCATTCAGCTTTTGTAACAA。

mutations in both model canine RPGR genes analyzed resulted in the occurrence of premature stop codons, failing to produce the correct RPGR protein.

The embodiment provides primer pairs for detecting sequences shown as SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15, the primer pairs are used for carrying out PCR reaction by taking the genome of the cub to be detected as a template, then sequencing is carried out on the PCR product, and the sequences obtained by analyzing the sequencing can be used for detecting the sequences; or after PCR reaction, agarose gel electrophoresis is carried out on the PCR product, after electrophoresis is carried out for a period of time, the gel imaging shows that the band sizes corresponding to the sequences of SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and the like are obviously smaller than the band sizes of the wild Beagle canine genome PCR product, so that the sequences can be detected as well (figure 1). The primer is designed aiming at the sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 as follows:

SEQ ID NO:1：

AATACATTCCACACGTTCTGTGCATTTCAATTGCTACTTAAGCTCTAGACCGATTTAGAAAATCTCACAAAAATGTATGACCTCGGTTGTTTTGCTTTATTTTTGACAATGAATCGAAGTTGTTCACCAAATGGTGGGATAATGTCAGCTTTCAGCTTTCTTCAGTGTTAATAGAGGAGATATCTGGTAACTGGCCTGTCTCATAAAGATAGTGACTATAGTATATTGATGTTTCCTTTTCCATGTGCAGAAGGAGGCAAAGTATATGCAGCTGGAGGAAATAATGAAGGACAGCTGGGACTTGGTGACACTGAAGAGAGAAGCACTTTTCATCTAATTAGTTTTTTTACTTCCCAGCGTAAGATTAAGCAGCTTTCTGCTGGATCTAATACTTCAGCTGCACTAACTGGTGAGATTTTGTTCCACTTCAGCCTGGGATCCGTCCCTTTCATTTAGACCAGGATATACCAGCCACGGCAACTGAGGACAGCATTGCTGTTTAGTTTTCTTTCATTAACTATGTGTTTTCAGAAGCTATAAAAATCAAACTTTAAATCATGACATATATTACAGTTTTGTGCTTTTTTAAGAATTAGATGTAAACACACACTTAGCTGCTATGCTGTCCATCTACCCCTGGAGATAGTAAATATTCTCTTCATCATGTGTTCTGAACATTCTATTTGTAGCTGTAATAAAGTTGCTATCAGAAACAGAAATCTTTTGGATATATCTCTGCGAGGCAC。

SEQ ID NO:2：

ACTAAGTGAGGCTGTGTTGGAAATATAATAATTTGATAGCCTTGCTACCTCTTAAAACCTTTTTTTCCCCTCCTTGTGTTTTCTTTATAGAGGATGGAGAGCTTTTCATGTGGGGTGATAATTCTGAAGGGCAAATTGGTTTAGAAAATGTAACCAATGTGTGTGTCCCTCAACAAGTGACTGTTGGAAAACCTATCTCCTGGATCTCTTGTGGATATTACCATTCAGCTTTTGTAACAAGTAAGACAGAATGTTTTGGATTCATCATGTTACCTCCGACTTCTAAGTGTTTACTCAATAATGAAATGATCTAACATTCTACAGTTCTGTGGAAATATTGCTGTGGCAGTCTATGGAATGAAATAATTTTGGTTCATTTTGGGTCACAGTGTGTCTC。

SEQ ID NO:3：

TAAGAATGTGGACCCCCAAAACTAGTATTTTGTCCATAATTATGTCGTCCTTGATCATTGTTCTTTTCCTTCAACAGCAGAGGGACAACTGTACACATTTGGAGAACCGGAGTGTGGAAAGTTAGGTCTTCCCAATCAGCTGCTGGTCAACCACAGAATGCCCCAGCCGGTACCTGGAATTCCCGGGAAGGTGGTCCAAGTAGCTTGTGGTGGAGGGCACACAGTGGTTCTTACAGGTACGTGTGGAGCTGCTGCTCCATCCCCTGCTTTCTGTGATGGCTAATA。

the primer pairs have the following sequences:

RPGRFP3(SEQ ID NO:16):AATACATTCCACACGTTCTGTGC。

RPGRRP3(SEQ ID NO:17):GTGCCTCGCAGAGATATATCCAA。

RPGRFP4(SEQ ID NO:18):ACTAAGTGAGGCTGTGTTGGAAA。

RPGRRP4(SEQ ID NO:19):GAGACACACTGTGACCCAAAATG。

RPGRFP5(SEQ ID NO:20):TAAGAATGTGGACCCCCAAAACT。

RPGRRP5(SEQ ID NO:21):TATTAGCCATCACAGAAAGCAGG。

electroretinogram (ERG) measurements were performed on both model dogs, where only the b-wave statistics of 21P176, which is a bright adaptation 3.0, showed a significant decrease in b-wave amplitude of the bright adaptation 3.0, suggesting impaired cone system function. The ERG results further confirmed that the gene editing XLRP model dogs were obtained. See fig. 4.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. A gene editing cell, wherein the genome of the gene editing cell comprises a mutated RPGR gene having a fragment shown in SEQ ID No. 13, or having a fragment shown in SEQ ID No. 14 and SEQ ID No. 15;

optionally, the gene editing cell comprises:

(1) A sgRNA combination targeting the RPGR gene; or,

wherein the sgRNA combination targeting the RPGR gene comprises:

the sgRNA as shown in SEQ ID NO. 22,

sgRNA as shown in SEQ ID NO. 23, and,

sgRNA as shown in SEQ ID NO. 24;

alternatively, the gene editing cell comprises a fertilized egg.

2. A RPGR gene-targeted sgRNA combination, comprising:

sgRNA as shown in SEQ ID NO. 22;

sgRNA as shown in SEQ ID NO. 23; the method comprises the steps of,

the sgRNA is shown as SEQ ID NO. 24.

3. An RPGR gene editing system, comprising:

the sgRNA combination of claim 2 that targets the RPGR gene;

4. A recombinant vector, wherein the recombinant vector transcribes the sgRNA combination, or further transcribes mRNA of Cas9 protein;

optionally, the recombinant vector further comprises a screening element;

further alternatively, the screening element comprises a resistance gene BSD.

5. The method of constructing a gene editing cell according to claim 1, comprising the steps of:

performing gene editing on the RPGR genes in the target cells to ensure that the genomes of the target cells contain mutated RPGR genes, and constructing gene editing cells;

optionally, the construction method comprises the following steps:

introducing the RPGR gene editing system into target cells to make the genome of the target cells contain mutated RPGR genes, so as to prepare gene editing cells;

further alternatively, the means of introduction comprises microinjection.

6. The construction method of the X-linked retinitis pigmentosa model dog is characterized by comprising the following steps of:

constructing the gene editing cell of claim 1 by taking a canine fertilized egg as a target cell; the method comprises the steps of,

7. The method of constructing an X-linked retinitis pigmentosa model canine of claim 6, wherein the step of screening comprises: carrying out PCR amplification on the genomic DNA of the birth cubs, detecting the obtained amplification products and screening out X-linked retinitis pigmentosa model dogs according to the obtained detection;

alternatively, the primer pair used for PCR amplification is shown as SEQ ID NO. 16 to SEQ ID NO. 21;

optionally, the dog comprises a Beagle dog.

8. A tissue or organ comprising the gene-editing cell of claim 1 or the model canine tissue or organ constructed according to claim 6 or 7.

9. Use of the gene editing cell of claim 1, the sgRNA combination of claim 2 that targets the RPGR gene, the RPGR gene editing system of claim 3, the recombinant vector of claim 4, the tissue or organ of claim 9 for screening a drug for treating X-linked retinitis pigmentosa.

10. The gene editing cell of claim 1, the canine constructed by the construction method of any one of claims 6 to 7, the detection primer pair of the tissue or organ of claim 8, or a kit comprising the detection primer pair, the detection primer pair being as shown in SEQ ID No. 16 to SEQ ID No. 21.