CN111979241B

CN111979241B - Method for preparing non-human mammal model of retinitis pigmentosa

Info

Publication number: CN111979241B
Application number: CN202010715185.XA
Authority: CN
Inventors: 万晓玲; 孙晓东
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2021-10-01
Anticipated expiration: 2040-07-23
Also published as: CN111979241A

Abstract

The invention relates to a method for preparing a non-human mammal model of retinitis pigmentosa. The method comprises replacing an endogenous CTSD gene on one of the animal's homologous chromosomes with a h-CTSD gene; the h-CTSD gene is a human CTSD gene with c.262dupC site variation. The application discovers that the h-CTSD gene is related to RP for the first time, and establishes a mouse animal model for knocking in the h-CTSD gene. The invention provides a convenient, reliable and economic means for researching the relation between CTSD mutation and retinitis pigmentosa and the pathogenic mechanism thereof, and provides a reliable theoretical basis for the research. More importantly, the animal does not show the symptoms of nervous diseases such as spasm, neuronal ceroid lipofuscinosis, ataxia and the like except RP, has simpler pathological background and is very suitable for being used as the construction of an RP animal model.

Description

Method for preparing non-human mammal model of retinitis pigmentosa

Technical Field

The invention relates to the technical field of molecular biology and biomedicine, in particular to a method for preparing a non-human mammal model with retinitis pigmentosa.

Background

The CRISPR/Cas9 technology is a new gene editing technology developed in recent years, is developed from adaptive immune defense formed by archaea and bacteria under long-term selective pressure, and can effectively resist invasion of exogenous DNA. The immune system consists of three components of Cas9 protein, CRISPR RNAs (crRNAs) and trans-activating crRNAs (tracrRNA), and during immune defense, the guide crRNA guides the Cas9 protein to target foreign genes and cuts the foreign genes. Accordingly, people modify crRNA-tracrRNA in vitro to obtain a sgRNA which can identify an NGG PAM (proto-acredjacent motif) sequence and a target gene, and can be combined with Cas9 protein to guide the sgRNA to be cut at the position 3-8 bp upstream of the PAM to form DSB (double strand break). DSBs can be repaired in two ways, one is Non-Homologous recombinant End Joining (NHEJ) mode and the other is Homologous recombinant Repair (HDR) mode, which requires introduction of Homologous fragments as Repair templates. The NHEJ repair may result in base insertions or deletions, or may be mutated to stop codons, thereby affecting the expression of the gene of interest.

Retinitis Pigmentosa (RP) is a group of progressive retinal degenerative diseases caused by a variety of pathogenesis, mainly involving photoreceptors and pigment epithelium. Retinitis pigmentosa is the most common hereditary retinal dystrophy, and the incidence rate of the retinitis pigmentosa in the population is about l/3500-1/7500. RP is highly heterogeneous in genetics, and it has been found that at least 87 genes can cause RP (see RetNet website: http:// sph. uth. edu/RetNet /). Mutations in these genes can result in autosomal dominant inheritance, autosomal recessive inheritance, or X-linked inherited RPs, respectively.

Cathepsin D (cathepsin D, CTSD) is an aspartic lysosomal endopeptidase found by Westley, et al 1979, and the CTSD Gene (Gene ID:1509) is located at 11p15.5, along with 9 exons. Its normal function is to hydrolyze proteins in the acidic environment of the lysosome. CTSD (optimum pH 4) can degrade hormones, polypeptide precursors, polypeptides, structural and functional proteins within a pH range of 2.8 to 5.0. CTSD knockout mice died 26 days postnatally (P26) with blindness, cramping, and Neuronal Ceroid Lipofuscinoses (NCL). At present, missense mutation (autosomal recessive mutation) of the CTSD gene has been reported to cause NCL, and CTSD is also involved in the pathogenesis of several other diseases, including breast cancer and alzheimer's disease.

Based on the above characteristics of CTSD, there are many studies on its mutation site in the prior art, and some of the results are summarized as follows:

it can be seen that although partial mutation of CTSD in the prior art can cause related reports of RP, it is often accompanied with various nervous system symptoms, such as problems of NCL, ataxia, cognitive decline, etc., and the pathological background is complex, so that animals with the above mutation are difficult to be used for RP mechanism research or related drug screening research.

Disclosure of Invention

The invention relates to a method for preparing a non-human mammal model of retinitis pigmentosa, which comprises the following steps:

replacing an endogenous CTSD gene on one of the animal's homologous chromosomes with a h-CTSD gene;

the h-CTSD gene is a human CTSD gene with c.262dupC site variation.

Optionally, knocking out the endogenous CTSD gene using the criprpr-Cas 9 system and replacing the knocked-out endogenous CTSD gene with the h-CTSD gene;

wherein the Crispr-Cas9 system comprises sgRNA1, sgRNA2 and Cas9 enzymes, the sequences of target sites corresponding to the sgRNA1 and the sgRNA2 are shown as

SEQ ID NO

1 and 2;

the nucleotide sequence of the h-CTSD gene is shown as SEQ ID NO. 3.

Optionally, the h-CTSD gene is further linked to a reporter gene or a sequence encoding a tag protein.

Optionally, the h-CTSD gene is replaced by a knockout endogenous CTSD gene by homologous recombination.

Optionally, nucleotide sequences are added on both sides of the h-CTSD gene, and are respectively shown as a left homologous arm and a right homologous arm in SEQ ID NO. 4 and 5, so as to realize homologous recombination.

Optionally, the subject of gene knockout and h-CTSD gene replacement treatment is an endogenous CTSD gene of a fertilized egg.

Alternatively, the criprpr-Cas 9 system and the method of transferring the h-CTSD gene into the fertilized egg are microinjection.

Optionally, the method further comprises transplanting the treated fertilized egg into a pseudopregnant magnetic animal and producing F0 generations, and mating the F0 generation animal correctly expressing the h-CTSD gene with a wild type animal to obtain F1 generations.

Optionally, the non-human mammal is a rodent.

Alternatively, the non-human mammal is a mouse (Mus musculus).

According to a further aspect of the invention, the invention also relates to the use of an animal obtained as described above for identifying and/or testing a drug;

the medicament is used for preventing and/or treating the symptoms of retinitis pigmentosa and/or treating complications related to retinitis pigmentosa.

Compared with the prior art, the invention has the beneficial effects that:

the application discovers that the h-CTSD gene is related to RP for the first time, and establishes a mouse animal model for knocking in the h-CTSD gene. The invention provides a convenient, reliable and economic means for researching the relation between CTSD mutation and retinitis pigmentosa and the pathogenic mechanism thereof, and provides a reliable theoretical basis for the research. More importantly, the animal does not show the symptoms of nervous diseases such as spasm, neuronal ceroid lipofuscinosis, ataxia and the like except RP, has simpler pathological background and is very suitable for being used as the construction of an RP animal model.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of CRISPR/Cas9 gene targeting;

FIG. 2 is a schematic diagram of the targeting strategy of the present invention;

FIG. 3 shows a target band (left insertion site) obtained by electrophoresis after PCR amplification in one embodiment of the present invention;

FIG. 4 shows a target band (right insertion site) obtained by electrophoresis after PCR amplification in one embodiment of the present invention;

fig. 5 is a photograph of the fundus of a h-CTSD-KI mouse according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

the h-CTSD gene is a human CTSD gene with c.262dupC site variation.

The invention constructs a CTSD humanized mutation (c.262dupC) KI non-human animal model based on the fact that the CTSD gene in an RP patient has c.262dupC mutation, wherein the gene mutation can cause RP which is not reported by other laboratories (refer to a RetNet website: http:// sph.uth.edu/RetNet /), and the relation between the gene mutation and the RP and the pathogenic mechanism are not clear. The h-CTSD gene homozygote results in death of the animal shortly after birth, but the heterozygote embryo develops normally and the animal can develop into a sexually mature individual after birth; and the animal does not show symptoms of neurological diseases, and does not find that the animal has other obvious disease symptoms except RP, so the animal model provides a convenient, reliable and economic means for researching the relation between CTSD mutation and retinal pigment degeneration and the pathogenic mechanism thereof in vivo.

In some embodiments, the method knocks out the endogenous CTSD gene using the criprpr-Cas 9 system and replaces the knocked-out endogenous CTSD gene with the h-CTSD gene;

SEQ ID NO

1 and 2;

the nucleotide sequence of the h-CTSD gene is shown as SEQ ID NO. 3.

At present, no relevant report is found on a mouse model of CTSD humanized mutant KI. Therefore, the inventor designs a construction method and application of a CTSD humanized mutation KI animal model, the CTSD humanized mutation (c.262dupC) knock-in animal model is constructed based on CRISPR/Cas9 gene knock-in technology, cutting is carried out near the first exon initiation codon ATG of an animal CTSD gene transcript, the CTSD gene is destroyed, and the human mutated CTSD gene is inserted under the condition that a homologous recombination template exists. The CRISPR/Cas9 technology can directly edit embryos, has high yield and better overall embryo quality, and has the advantage of high knock-in rate.

In some embodiments, the h-CTSD gene is further linked to a reporter gene or a sequence encoding a tag protein.

The reporter gene can be selected from metabolic markers, catalytic reporter genes, antibiotic markers, antibiotic resistance genes, herbicide resistance genes, auxotrophic reporter genes, compound detoxification enzyme genes, and carbohydrate metabolism enzyme selection marker genes that are well known to those skilled in the art;

in some preferred embodiments, for ease of observation and detection, the expression product of the reporter gene is a substance that can self-emit light or produce a color change by catalyzing a substrate reaction, or can cause a substrate to emit light or produce a color change by catalyzing a substrate reaction, or produce emitted light or produce a color change upon irradiation with excitation light. Such substances typically include fluorescent protein, luciferase and LacZ. Both the fluorescent protein and the luciferase are luminescent proteins, and the expression of fluorescence can be detected by a camera or the like. Fluorescent proteins work by absorbing light of one color (excitation) and then emitting a different color (emission) of lower energy light. In contrast, luciferase (and other bioluminescent enzymes) emit light by catalyzing a chemical reaction of a substrate (i.e., luciferin). Unlike the two labels above, LacZ does not emit light. The product of the LacZ gene, beta-galactosidase, catalyzes the conversion of X-gal to an opaque blue compound similar to indigo.

Further, the fluorescent protein may be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein. The green fluorescent protein can adopt common GFP, and can also adopt modified GFP genes, such as enhanced GFP gene EGFP and the like; the blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP and the like; the yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP and the like; the orange fluorescent protein can be selected from mKO, mOrange, mBanana and the like; the red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate and the like.

The tag protein is preferably an HA tag.

In some embodiments, the method of h-CTSD gene replacement for a knockout endogenous CTSD gene is homologous recombination.

The length of the homologous recombination arm is preferably about 1kb

In some embodiments, the h-CTSD gene is flanked by left and right homology arms as shown in

SEQ ID NOs

4 and 5, respectively, to achieve homologous recombination.

In some embodiments, the subject of the gene knockout and h-CTSD gene replacement treatment is an endogenous CTSD gene of a fertilized egg.

In some embodiments, the method of transferring the criprpr-Cas 9 system and the h-CTSD gene into the fertilized egg is microinjection.

In some embodiments, the method further comprises transplanting the treated fertilized egg into a pseudopregnant magnetic animal and producing F0 generations, and mating the F0 generation animal correctly expressing the h-CTSD gene with a wild-type animal to obtain F1 generations.

In some embodiments, the non-human mammal is a rodent.

In some embodiments, the non-human mammal is a mouse (Mus musculus).

The mouse strain can be BALB/C, C57BL, C3H/He, Kunming mouse, ICR, NIH, CFW, LACA, nude mouse or Scid mouse, etc., preferably C57BL mouse.

According to a further aspect of the invention, the invention also relates to the use of an animal obtained by a method as described above for identifying and/or testing a drug;

Embodiments of the present invention will be described in detail with reference to examples.

Examples

This example provides the construction and identification of the h-CTSD-KI mouse model.

Materials (I) and (II)

The mice used in this example were: c57BL/6J, the surrogate mother mouse is C57BL/6J, purchased from Shanghai Slek laboratory animals Co., Ltd, and the mice were randomly divided into control groups and experimental groups.

Second, method

Experimental groups experiments were performed as follows:

1. searching for suitable target sequence in target gene intron

Through an online design tool (http:// crispr. mit. edu /) and a design principle of the gRNA, the gRNA is designed by evaluating a target site with higher score near the ATG sequence of the mouse Ctsd gene, and the sequence of the target site is SEQ ID NO:1-SEQ ID NO: 2.

The gene sequence and sgRNA information are as follows:

sgRNA1：GCCGCGACCATGAAGACTCC CGG(SEQ ID NO:1)；

sgRNA2：GGGAGTCTTCATGGTCGCGG CGG(SEQ ID NO:2)；

the gene sequences are shown below:

---GGAAAGTGGGCGGGCGCGTTCCCGAGAGCCGTGCGTAGGCCTGGAGTAGGGCACGCCTGCGCCCGGGCAGTGTTGGAGGCGGGTTCGGGTCGCCCCGCCCCTCGCCCGCGTCTCACGTGACCCGTTGAGGGCCAAACAAGCGGGTCAGCTGACTCCGCGGGACTGCGGCGTCATCCTGCCTATAAGCCGGCGACCTCTGGCTTTAAGCTTTGCTCTCTTCGGGCCGCCGCGACCATGAAGACTCCCGGCGTCTTGCTGCTCATTCTCGGCCTCCTGGCTTCGTCCTCCTTCGC GATTATCAGgtgaggacccgctctgggtccggagatgcggggctcgtcacctggagtgccgcgtgctccggccgtgctggaatgcacctgtgcacccagcgcagccttcctcagggtcccacagactatggggcgagacactcaaaaggcaggggtctcctgggcccctctccactcctatgactcctatggtcgaacaggagggtacagtagggtggccacaagtgggtctgactccaacctcgcctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtagcggggcggccgcttctgaggagaagtgctgccccatggataggattctgttgaacccagctgtggaagttggaaaacctagtaggcccttgcccccctgaacttcctcaggcctttccttttaagggaccctgtttgacagctgtgcactggtgcatagccctagaagtttactcaaagatgatcccccctgt----

the upper underlined part of the above gene sequence represents the coding region; the upper case non-underlined part represents a non-coding region, and the lower case part represents an intron region.

2. Construction of targeting vectors

The targeting vector core sequence is shown below:

gaacctggggaaccagaacagctcatgtgtccatgtgtagagtggacttagaggcatccacctcctca atacaggtagaacatgtccaaaagcgctcccgggatcagaccctggcaaggctcctctgcgttcctgtccttgtat ggacaccacctctcagagaggacattccccttaaatgcctttctgggctcagctctccatgatggagagctggact ctgcctctctcaaacagcacccaattcatttgctgacaccaagcagggcattaaagagtgaagtagttaagaccca agtggcgattgcaaccctggtctcatctcactgtacaccctaccccttgtatcaatagatgcatagtccaggacta ggcctgcaatcaacattgctacattatggaatgtgcatgttaggtgttccagcttaaataaaaagggtgttgctct ggggagatgagtaggtagtggagctttgaagagggtgaagtttgctggtccaagagtccagagaggctccagggaa attctcaaggaggaacagagctgaaagagaaaaggcttaggtttaagctggcatccaggagagggtcagggcccaa tcagacaaagaccacatcctggtagtgggcaccccaattatgcaaaaaagacagattaagggaggggcggagctga gggcaggatccgcccctctggatgcttccgggcctgcctggtcttccttcccccacgtgacctgtatccagtcatt ctccggtcccaggaaagtgggcgggcgcgttcccgagagccgtgcgtaggcctggagtagggcacgcctgcgcccg ggcagtgttggaggcgggttcgggtcgccccgcccctcgcccgcgtctcacgtgacccgttgagggccaaacaagc gggtcagctgactccgcgggactgcggcgtcatcctgcctataagccggcgacctctggctttaagctttgctctc ttcgggccgccgcgacc(SEQ ID NO:4)ATGCAGCCCTCCAGCCTTCTGCCGCTCGCCCTCTGCCTGCTGGCTGCACCCGCCTCCGCGCTCGTCAGGATCCCGCTGCACAAGTTCACGTCCATCCGCCGGACCATGTCGGAGGTTGGGGGCTCTGTGGAGGACCTGATTGCCAAAGGCCCCGTCTCAAAGTACTCCCAGGCGGTGCCAGCCGTGACCGAGGGGCCCATTCCCGAGGTGCTCAAGAACTACATGGACGCCCAGTACTACGGGGAGATTGGCATCGGGACGCCCCCCCCAGTGCTTCACAGTCGTCTTCGACACGGGCTCCTCCAACCTGTGGGTCCCCTCCATCCACTGCAAACTGCTGGACATCGCTTGCTGGATCCACCACAAGTACAACAGCGACAAGTCCAGCACCTACGTGAAGAATGGTACCTCGTT(SEQ ID NO:3)TACCCATACGACGTACCAGATTACGCT(HA Tag)TGAaagactcccggcgtcttgctgctcattctcggcctcctggcttcgtcctccttcgcgattatcaggtgaggacccgctctgggtccggagatgcggggctcgtcacctggagtgccgcgtgctccggccgtgctggaatgcacctgtgcacccagcgcagccttcctcagggtcccacagactatggggcgagacactcaaaaggcaggggtctcctgggcccctctccactcctatgactcctatggtcgaacaggagggtacagtagggtggccacaagtgggtctgactccaacctcgcctctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtagcggggcggccgcttctgaggagaagtgctgccccatggataggattctgttgaacccagctgtggaagttggaaaacctagtaggcccttgcccccctgaacttcctcaggcctttccttttaagggaccctgtttgacagctgtgcactggtgcatagccctagaagtttactcaaagatgatcccccctgtcctgggtggtgaatctctcctcctccctcctgagttgcacaagtcccagagagagaaggtatggagtggcccagttcaaataccttggagctgaggtctaccccactggggaccaggcccttctcaggcccgtggacttggcctttgtgctagatgaggtcttggtggtagcccgtgtgttttcccactctgtgagtccctggtcgtggcaggtggctgaggccgtaggctgcgggagtagagctggctctgccggctgggcaccagggttgggtgggaaggagcccagtctgtaggctgcaagctgtgggtgggtagcctcagtggagccattgtagggagtggcctaggcttgatcctgccctgggcttgcctgccctgccctcaggcttctcttctctgggaagaggtgttgtggactccaggatccctgagactaggcaccagaaacaaagccatgaagactcccggcgt(SEQ ID NO:5)

the upper case part of the gene sequence represents a h-CTSD coding sequence; the upper italic part represents the HA sequence; the lower case underlined section represents the left homology arm, and the lower case non-underlined section represents the right homology arm.

3. Cas9mRNA preparation

Linearized and purified DNA was transcribed in vitro with Cas9 nuclease to mRNA: the sgrnas were purified to the purity of appropriate transgene injections.

4. Fertilized egg injected microscopically

And (3) uniformly mixing the sgRNA, the Cas9mRNA and the targeting vector according to a certain concentration ratio by using a microinjection instrument, and injecting the mixture into a cytoplasmic part of a fertilized egg of an in vitro fertilized mouse to construct and form a specific mouse embryonic cell (fertilized egg). And transplanting the surviving fertilized eggs into the oviduct of a pseudopregnant female mouse after culturing in vitro for 1-2 hours, wherein the born embryo-transplanted mouse is the F0 mouse.

5. Mouse genotype identification

(1) Extracting the genomic DNA of the offspring mice obtained in the step for genotype identification;

(2) mice in which the target gene is knocked in are respectively crossed with wild type mice.

6. Genotyping

(1) Extracting genome DNA:

A. digestion: within about one week of the birth of the mouse, 0.5cm of the toe of the mouse was cut, placed in a 1.5ml EP tube, centrifuged slightly, and 500ul of a lysis solution (formulation: 100mM Tris pH8.0, 5mM EDTA pH8.0, 0.5% SDS, NaCl 1.17g/100ml), 0.5ul of proteinase K (concentration: 20mg/ml, dissolved in pH7.4, 20mM Tris and 1mM CaCl)₂In the preparation, 50% glycerol buffer solution is stored at minus 20 ℃), mixed evenly and digested overnight in water bath at 55 ℃;

B. phenol chloroform extraction:

1) taking out the EP tube, reversing and mixing evenly, and centrifuging at 1,2000rpm for 10 min;

2) sucking 400ul of supernatant into a new EP tube, adding equal volume of phenol/chloroform, turning upside down, mixing for 3min, slightly standing, and centrifuging at 1,2000rpm for 5 min;

3) gently sucking the supernatant into a new 1.5ml EP tube (not sucking the lower phenol or precipitate), adding equal volume of chloroform into the supernatant, turning upside down, mixing for 3min, slightly standing, and centrifuging at 1,2000rpm for 3 min;

4) sucking the supernatant into another new EP tube of 1.5ml, adding sodium acetate solution of 1/10 volume and anhydrous ethanol of 2.5 times volume, shaking, and standing at-20 deg.C for about 30 min;

5) centrifuging at 1,2000rpm for 10min at 4 deg.C, removing supernatant, and placing EP tube on absorbent paper upside down to suck dry ethanol;

6) after the DNA had dried, 30ul of sterile ddH2O was added and dissolved, and the solution was concentrated and stored at-20 ℃.

The control group has no sgRNA design step, the target gene is directly knocked in, and the rest steps are the same as those of the experimental group.

(2) PCR identification

Forward and reverse PCR primers are designed respectively for the upstream and downstream regions of the insertion site.

1) The primer information is shown in Table 1

TABLE 1

2) The PCR reaction system is shown in Table 2.

TABLE 2

Note: if the sequence is complex, PCR can be performed with other enzymes, 5% DMSO, GC enhancer, etc. are added (the TM value is marked with DMSO, and the customer is advised to add DMSO to the PCR system

(3) Sequencing analysis

A. Direct sequencing of PCR products (50ul system);

B. or the PCR product is reclaimed by tapping, is connected with a PMD18T vector, is coated after DH5 alpha is transformed and is directly sent to a plate for sequencing (Taq enzyme is recommended to be used in PCR);

C. and comparing sequencing results, and analyzing whether KI is successful or not.

Three, result in

The key to gene knock-in is target selection, which can be used to insert the target sequence into the correct target by homologous recombination. By analyzing the structure of a mouse ctsd gene, sgrnas are designed at the ATG of the first exon of a ctsd gene transcript, and the sgRNA1-2 with the highest knock-in efficiency is selected.

3.1 fountain information

Generation 3.1.1F 0 information

2019.8.12 unprocessed, 2019.8.24 cut 1-34#

3.1.2 gel electrophoresis as in FIGS. 3 and 4:

4#, 23#, 25# and 31# are positive mice, PCR is a single small band, and sequencing is carried out by using a forward primer. WT and H₂O is wild type and blank control, respectively.

3.1.3 sequencing results analysis (Forward primer sequencing)

Sequencing the PCR product, and comparing the sequencing result with the sequence of the targeting vector to confirm that the h-CTSD sequence is specifically inserted into the ATG of the mouse Ctsd gene. The sequence alignment results are as follows:

4#、23#、25#、31#：KI

3.2, fundic photographs of h-CTSD-KI mice (8 weeks) are shown in FIG. 5, showing that knock-in groups of h-CTSD genes exhibit marked retinitis pigmentosa symptoms.

Most genetically modified mice are constructed to study the effects of gene expression deletions, additions or target gene sequence changes on body functions; biological processes (reporter strains) can also be monitored by designing genetically modified mice to label specific cell populations (reporter staining). CRISPR/Cas9 is a new generation of gene editing technology that has recently been developed. When the h-CTSD gene knock-in mouse model is constructed, various factors are considered, and finally, a more simple and efficient CRISPR/Cas9 technology is selected. The CRISPR/Cas9 technology has high targeting efficiency, convenient design and simple steps, and has profound influence on the technical development in the field of biology.

The invention adopts CRISPR/Cas9 gene knock-in technology to establish a mouse animal model of h-CTSD gene knock-in for the first time. The optimal sgRNA1-2 is selected, and the sgRNA1-2 and the method designed by the invention have the characteristic of high knock-in efficiency.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> first-person hospital in Shanghai City

<120> method for preparing non-human mammal model of retinitis pigmentosa

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> artificial sequence

<400> 1

gccgcgacca tgaagactcc cgg 23

<210> 2

<211> 23

<212> DNA

<213> artificial sequence

<400> 2

gggagtcttc atggtcgcgg cgg 23

<210> 3

<211> 414

<212> DNA

<213> artificial sequence

<400> 3

atgcagccct ccagccttct gccgctcgcc ctctgcctgc tggctgcacc cgcctccgcg 60

ctcgtcagga tcccgctgca caagttcacg tccatccgcc ggaccatgtc ggaggttggg 120

ggctctgtgg aggacctgat tgccaaaggc cccgtctcaa agtactccca ggcggtgcca 180

gccgtgaccg aggggcccat tcccgaggtg ctcaagaact acatggacgc ccagtactac 240

ggggagattg gcatcgggac gcccccccca gtgcttcaca gtcgtcttcg acacgggctc 300

ctccaacctg tgggtcccct ccatccactg caaactgctg gacatcgctt gctggatcca 360

ccacaagtac aacagcgaca agtccagcac ctacgtgaag aatggtacct cgtt 414

<210> 4

<211> 997

<212> DNA

<213> artificial sequence

<400> 4

gaacctgggg aaccagaaca gctcatgtgt ccatgtgtag agtggactta gaggcatcca 60

cctcctcaat acaggtagaa catgtccaaa agcgctcccg ggatcagacc ctggcaaggc 120

tcctctgcgt tcctgtcctt gtatggacac cacctctcag agaggacatt ccccttaaat 180

gcctttctgg gctcagctct ccatgatgga gagctggact ctgcctctct caaacagcac 240

ccaattcatt tgctgacacc aagcagggca ttaaagagtg aagtagttaa gacccaagtg 300

gcgattgcaa ccctggtctc atctcactgt acaccctacc ccttgtatca atagatgcat 360

agtccaggac taggcctgca atcaacattg ctacattatg gaatgtgcat gttaggtgtt 420

ccagcttaaa taaaaagggt gttgctctgg ggagatgagt aggtagtgga gctttgaaga 480

gggtgaagtt tgctggtcca agagtccaga gaggctccag ggaaattctc aaggaggaac 540

agagctgaaa gagaaaaggc ttaggtttaa gctggcatcc aggagagggt cagggcccaa 600

tcagacaaag accacatcct ggtagtgggc accccaatta tgcaaaaaag acagattaag 660

ggaggggcgg agctgagggc aggatccgcc cctctggatg cttccgggcc tgcctggtct 720

tccttccccc acgtgacctg tatccagtca ttctccggtc ccaggaaagt gggcgggcgc 780

gttcccgaga gccgtgcgta ggcctggagt agggcacgcc tgcgcccggg cagtgttgga 840

ggcgggttcg ggtcgccccg cccctcgccc gcgtctcacg tgacccgttg agggccaaac 900

aagcgggtca gctgactccg cgggactgcg gcgtcatcct gcctataagc cggcgacctc 960

tggctttaag ctttgctctc ttcgggccgc cgcgacc 997

<210> 5

<211> 1019

<212> DNA

<213> artificial sequence

<400> 5

aagactcccg gcgtcttgct gctcattctc ggcctcctgg cttcgtcctc cttcgcgatt 60

atcaggtgag gacccgctct gggtccggag atgcggggct cgtcacctgg agtgccgcgt 120

gctccggccg tgctggaatg cacctgtgca cccagcgcag ccttcctcag ggtcccacag 180

actatggggc gagacactca aaaggcaggg gtctcctggg cccctctcca ctcctatgac 240

tcctatggtc gaacaggagg gtacagtagg gtggccacaa gtgggtctga ctccaacctc 300

gcctctgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt agcggggcgg 360

ccgcttctga ggagaagtgc tgccccatgg ataggattct gttgaaccca gctgtggaag 420

ttggaaaacc tagtaggccc ttgcccccct gaacttcctc aggcctttcc ttttaaggga 480

ccctgtttga cagctgtgca ctggtgcata gccctagaag tttactcaaa gatgatcccc 540

cctgtcctgg gtggtgaatc tctcctcctc cctcctgagt tgcacaagtc ccagagagag 600

aaggtatgga gtggcccagt tcaaatacct tggagctgag gtctacccca ctggggacca 660

ggcccttctc aggcccgtgg acttggcctt tgtgctagat gaggtcttgg tggtagcccg 720

tgtgttttcc cactctgtga gtccctggtc gtggcaggtg gctgaggccg taggctgcgg 780

gagtagagct ggctctgccg gctgggcacc agggttgggt gggaaggagc ccagtctgta 840

ggctgcaagc tgtgggtggg tagcctcagt ggagccattg tagggagtgg cctaggcttg 900

atcctgccct gggcttgcct gccctgccct caggcttctc ttctctggga agaggtgttg 960

tggactccag gatccctgag actaggcacc agaaacaaag ccatgaagac tcccggcgt 1019

Claims

1. A method of making a non-human mammalian model of retinitis pigmentosa comprising:

the h-CTSD gene is a human CTSD gene with c.262dupC site variation.

2. The method of claim 1, wherein the endogenous CTSD gene is knocked out using the criprpr-Cas 9 system and the knocked-out endogenous CTSD gene is replaced with the h-CTSD gene;

wherein the Crispr-Cas9 system comprises sgRNA1, sgRNA2 and Cas9 enzymes, the sequences of target sites corresponding to the sgRNA1 and the sgRNA2 are shown as SEQ ID NO 1 and 2;

the nucleotide sequence of the h-CTSD gene is shown as SEQ ID NO. 3.

3. The method of claim 2, wherein the h-CTSD gene is further linked to a reporter gene or a sequence encoding a tag protein.

4. The method according to claim 2, wherein the h-CTSD gene is replaced by a knockout endogenous CTSD gene by homologous recombination.

5. The method according to claim 4, wherein the h-CTSD gene is flanked by left and right homology arms as shown in SEQ ID NO 4 and 5, respectively, to achieve homologous recombination.

6. The method according to any one of claims 2 to 5, wherein the gene knockout and h-CTSD gene replacement are performed on endogenous CTSD genes of fertilized eggs.

7. The method according to claim 6, wherein the Crispr-Cas9 system and the h-CTSD gene transfer method into the fertilized egg is microinjection.

8. The method of claim 6, further comprising transplanting the treated fertilized egg into a pseudopregnant female animal and producing F0 generations, and mating the F0 generation animal correctly expressing the h-CTSD gene with a wild type animal to obtain F1 generations.

9. The method of claim 6, wherein the non-human mammal is a rodent.

10. The method of claim 9, wherein the non-human mammal is a mouse (Mus musculus).

11. Use of an animal obtained by the method of any one of claims 1 to 10 for the identification and/or testing of a medicament;