Establishment method of gene point mutation non-human primate model

Abstract

The invention relates to the technical field of medical animal disease models, in particular to a method for establishing a gene point mutation non-human primate model.According to the method, editing is conducted through a CRISPR / Cas9 gene editing system, and finally a non-human primate model carrying CHD8c.3724Cgt is obtained; according to the cynomolgus monkey with T (p.Arg1242Ter) point mutation, different types of point mutations occur at the 3724th basic group of the CHD8 gene, the mutation of T enables arginine (Arg) at the 1242nd site to be replaced by a termination codon to form truncated protein, so that the dosage of CHD8 protein haploid coded by the gene is insufficient. The animal model for simulating human CHD8 mutation related nervous system diseases is constructed and can be used as an ideal research model for researching autism related phenotypes caused by insufficient haplodose, and a foundation is laid for revealing a neurological dysfunction mechanism and exploring a potential treatment strategy.

Description

Technical Field

[0001] This invention relates to the field of medical animal disease model technology, specifically to a method for establishing a non-human primate model with gene point mutations. Background Technology

[0002] Autism Spectrum Disorder (ASD) is a relatively common and complex mental illness with high heterogeneity. In recent years, candidate gene-based and whole-genome sequencing methods have been used to explore genetic variations associated with autism. Recent large-scale whole-genome sequencing and exome sequencing have identified hundreds of susceptibility genes significantly associated with autism, including SHANK3, PTEN, and CHD8. Among them, CHD8 is one of the susceptibility genes with the highest mutation frequency and penetrance. This gene encodes a member of a chromosomal domain helicase DNA-binding protein family, which plays an important role in transcriptional regulation, epigenetic remodeling, promotion of cell proliferation and RNA synthesis, and apoptosis. The human CHD8 gene shares 98.4% homology with primates; therefore, establishing a CHD8 gene-deficient model in monkeys has significant guiding implications for the study of autism in humans.

[0003] In existing technologies, CRISPR / Cas9-based gene editing technology has been used to construct animal models, but non-human primate models (especially cynomolgus monkeys) targeting single-base mutations in the CHD8 gene have not yet been effectively established. Traditional autism modeling methods, which involve treating pregnant primates with VPA and MIA antibodies, produce offspring exhibiting autistic-like behaviors such as socialization and stereotyped behaviors, but fail to mimic the mechanism of action of a specific pathogenic gene. Publicly available methods for constructing CHD8 monkey models using gene editing involve injecting lentiviral vectors into the brain during embryonic development; however, this method cannot accurately determine the mutation status of tissue DNA sequences. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention utilizes the CRISPR / Cas9 gene editing system to induce a c.3724C>T (p.Arg1242Ter) mutation in exon 18 of the cynomolgus monkey CHD8 gene, within a cynomolgus monkey genetic background. This invention constructs an animal model simulating human CHD8 mutation-related neurological diseases, laying the foundation for elucidating the mechanisms of neurological dysfunction and exploring potential therapeutic strategies.

[0005] This invention utilizes CRISPR / Cas9 technology to precisely edit the CHD8 gene and, combined with non-human primates in the background of cynomolgus monkeys, successfully constructs a cynomolgus monkey model carrying the CHD8R1242 point mutation.

[0006] The first aspect of this invention provides a method for establishing a non-human primate model with gene point mutations, comprising the following steps:

[0007] (1) sgRNA design, screening, and point mutation efficiency detection:

[0008] An online tool was used to design an sgRNA targeting exon 18 of the cynomolgus monkey CHD8 gene, the nucleotide sequence of which is 5'-aggcacgatgtcatcgaatt-3'.

[0009] (2) Superovulation oocyte retrieval and sperm collection:

[0010] Superovulation was performed on female cynomolgus monkeys to collect oocytes; sperm was collected from male cynomolgus monkeys to ensure sperm quality and optimize its use for in vitro fertilization.

[0011] (3) In vitro fertilization and microinjection:

[0012] In vitro fertilization was performed using intracytoplasmic sperm injection (ICSI), followed by the injection of a mixture of 20 μL of sgRNA (final concentration 50 ng / μL) and YE1 mRNA (final concentration 100 ng / μL) into the fertilized egg using a micromanipulator.

[0013] (4) Embryo transfer:

[0014] The injected embryos were transferred into the uterus of female cynomolgus monkeys to carry out surrogacy and produce F0 offspring after pregnancy and delivery. Multiple recipient female cynomolgus monkeys were used for transplantation to improve the success rate of the embryo implantation experiment.

[0015] (5) Genotyping of F0 generation monkeys:

[0016] Genotypes of F0 generation monkeys were detected using PCR and sequencing to screen for individuals carrying the CHD8 gene c.3724C>T(p.Arg1242Ter) mutation.

[0017] Further, in step (1), the sgRNA of exon 18 of the cynomolgus monkey CHD8 gene is used as the cleavage site, and the cleavage efficiency is detected by in vitro cleavage experiment. The in vitro cleavage experiment uses T7EI or SurveyorAssay, and the most efficient sgRNA is selected for subsequent experiments.

[0018] Furthermore, in step (2), the superovulation treatment is performed by injecting the donor female cynomolgus monkey with Gonal-F and Aze.

[0019] Furthermore, in step (2), the female cynomolgus monkeys are 3-5 years old and the male cynomolgus monkeys are 5-11 years old.

[0020] Further, in step (3), Piezo microinjection is used. The pulse intensity range is typically 3-50V (the specific voltage depends on the piezoelectric controller model and needle tip resistance), and the injection time range is 0.1-0.5 seconds (the time to maintain injection pressure). This ensures successful injection and improves embryo survival rate. The sgRNA concentration is 50-150 ng / μL, and the YE1-mRNA concentration is 100-300 ng / μL.

[0021] Furthermore, in step (4), the embryo transfer is performed during the morula stage; the recipient female cynomolgus monkeys are 4-12 year old cynomolgus monkeys of the same age.

[0022] Further, in step (5), a pair of primers F1 / R1 are designed at both ends of the mutation site based on the sgRNA site of the CHD8 gene; PCR primer amplification is performed on the F0 generation monkeys and they are sent for sequencing. The product amplified by the primer in the gene knockout monkeys is 505bp. Sanger sequencing is used, and the monkeys are identified using sequencing primer F1.

[0023] F1:5'-gaatgggtggtcccacctgt-3'

[0024] R1:5'-agtcaaatccctgtgtaaaaacagctttt-3'.

[0025] The second aspect of this invention provides a CHD8 gene point mutation cynomolgus monkey model prepared by the above method.

[0026] The third aspect of this invention provides the application of the CHD8 gene point mutation cynomolgus monkey model prepared by the above method in the study of autism mechanisms or drug development.

[0027] The present invention has the following advantages over the prior art:

[0028] 1. This invention introduces a single-base mutation, c.3724C>T, into exon 18 of the CHD8 gene in cynomolgus monkeys using the CRISPR / Cas9 gene editing system. This mutation results in a premature translation terminator in the CHD8 gene, with the mutated form being p.Arg1242Ter. This leads to the loss or impaired function of the CHD8 protein. This premature translation termination signal has been observed in patients with CHD8-associated overgrowth syndrome (PMID: 31001818), who were diagnosed with autism spectrum disorder. This invention successfully constructs a monkey model carrying the CHD8 c.3724C>T mutation, providing an ideal tool for in-depth research into the role of CHD8 mutations in autism and other neurological disorders, and providing a crucial experimental foundation for gene therapy and drug development.

[0029] 2. This invention ultimately yielded cynomolgus monkeys carrying the CHD8 c.3724C>T (p.Arg1242Ter) point mutation. Different types of point mutations occurred at the 3724th base of the CHD8 gene. The C>T mutation replaced the arginine (Arg) at position 1242 with a stop codon, forming a truncated protein and resulting in insufficient haploid dosage of the CHD8 protein encoded by this gene. Numerous studies have shown that insufficient haploid dosage of CHD8 is a high-risk factor for autism. CHD8 plays a crucial role in neurodevelopment through chromatin remodeling and gene expression regulation. Loss-of-function variants disrupt Wnt signaling, epigenetic networks, and neuronal differentiation, leading to abnormal brain structure and function, and consequently triggering autism-related phenotypes. Our constructed CHD8 mutant monkey model can serve as an ideal research model for studying autism-related phenotypes caused by haploid dosage insufficiency. Attached Figure Description

[0030] Figure 1 The results are from genotyping of monkeys using Sanger sequencing with sequencing primer F1. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments.

[0032] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0033] Example 1:

[0034] A method for establishing a non-human primate model with gene point mutations includes the following steps:

[0035] (1) sgRNA design:

[0036] To design a point mutation sgRNA targeting the 3724th base of CHD, we designed a highly efficient single-base editing sgRNA: 5'-aggcacgatgtcatcgaatt-3'. The sgRNA was directly synthesized by GenScript and the YE1-BE4max mRNA was obtained through in vitro transcription.

[0037] (2) Rapid ovulation and sperm collection:

[0038] Superovulation was induced by injecting follicle-stimulating hormone (FSH) into 3-year-old female cynomolgus monkeys for 10 days using a sterile syringe. 37 hours later, human chorionic gonadotropin (hCG) was injected intraperitoneally. Oocytes were collected within 35 hours via laparoscopic minimally invasive abdominal extraction and placed in an oocyte collection solution. The oocytes were then retrieved for fertilization. For sperm collection, adult male cynomolgus monkeys aged 5-11 years were selected, and sperm was collected using electrical stimulation and placed in the semen collection solution.

[0039] (3) In vitro fertilization and microinjection:

[0040] Intracytoplasmic sperm injection (ICSI) was used to fertilize oocytes to obtain zygotes. Subsequently, YE1-mRNA and gRNA were injected into the zygotes using a microinjector. Different concentrations of YE1-mRNA and gRNA were injected into the zygotes, and the highest concentration that enabled the zygotes to divide normally was recorded to determine the concentrations of Cas9 mRNA and gRNA.

[0041] (4) Embryo transfer:

[0042] The injected embryos were transferred into the oviducts of adult female cynomolgus monkeys. One month after the transfer, an ultrasound could be performed to confirm pregnancy. Five months after the transfer, the pregnant monkeys gave birth, and the umbilical cords of the newborn monkeys were numbered and subjected to PCR testing.

[0043] (5) Genotyping of F0 generation monkeys:

[0044] 1. Extraction of DNA from the umbilical cord of a baby monkey:

[0045] (1) After cutting the umbilical cord from the monkey farm, it was sent to the molecular laboratory with dry ice and the DNA extraction kit (Tiangen, DP304) was used for the operation.

[0046] (2) Cut off about 30 mg of umbilical cord sample with sterilized scissors and put it into 200 μl Buffer GA, add 20 μl Proteinase K and mix well.

[0047] (3) Incubate overnight at 56℃;

[0048] (4) Add 200 μl of buffer GB, invert thoroughly to mix, place at 70°C for 10 min, the solution should be clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0049] (5) Add 200 μl of anhydrous ethanol and shake well for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0050] (6) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0051] (7) Add 500 μl of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0052] (8) Add 600 μl of washing solution PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0053] (9) Repeat step 8.

[0054] (10) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual rinsing liquid in the adsorption material.

[0055] (11) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50 μL of dH2O dropwise to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0056] (12) After measuring the concentration using Nanodrop, store it at -20℃ as a PCR template.

[0057] 2. PCR amplification

[0058] Based on the sgRNA site of the CHD8 gene, a pair of primers F1 / R1 were designed at both ends of the mutation site. We performed PCR primer amplification on newborn F0 monkeys and sent them for sequencing. The product amplified by the primers in the gene knockout monkeys was 505bp. Sanger sequencing was used, and the monkeys were identified using sequencing primer F1.

[0059] F1:5'-gaatgggtggtcccacctgt-3'

[0060] R1:5'-agtcaaatccctgtgtaaaaacagctttt-3'

[0061] The PCR amplification system is as follows:

[0062] 组件 体积 DNA模板 1微升(50 - 200纳克) F1引物(10微摩尔) 1微升 R1引物(10微摩尔) 1微升 GXL DNA聚合酶 25微升 GXL PCR缓冲液 10微升 2.5毫摩尔脱氧核苷三磷酸 4微升 <![CDATA[ddH2O]]> 至多50微升

[0063] The PCR reaction procedure is as follows:

[0064]

[0065]

[0066] Figure 1 The genotyping results of the CHD8 c.3724C>T(p.Arg1242Ter) monkey constructed for the example were obtained from... Figure 1 The sequencing results show that, compared with wild-type monkeys, the C base at the 3724th base of the CHD8 gene in the model monkey has been changed to a T base.

[0067] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for establishing a non-human primate model with gene point mutations, characterized in that, Includes the following steps: (1) sgRNA design and screening: Design an sgRNA targeting exon 18 of the cynomolgus monkey CHD8 gene, wherein the nucleotide sequence of the sgRNA is 5'-aggcacgatgtcatcgaatt-3'; (2) Superovulation oocyte retrieval and sperm collection: Superovulation was performed on female cynomolgus monkeys to collect oocytes; sperm was collected from male cynomolgus monkeys. (3) In vitro fertilization and microinjection: In vitro fertilization was performed using intracytoplasmic sperm injection (ICSI), followed by the injection of a mixture of sgRNA and YE1 mRNA into the fertilized egg using a micromanipulator. (4) Embryo transfer: The injected embryos were transferred into recipient female cynomolgus monkeys, and F0 generation baby monkeys were obtained after pregnancy and delivery. (5) Genotyping of F0 generation monkeys: Genotypes of F0 generation monkeys were detected using PCR and sequencing to screen for individuals carrying the CHD8 gene c.3724C>T(p.Arg1242Ter) mutation.

2. The method for establishing a non-human primate model with gene point mutation according to claim 1, characterized in that, In step (1), the sgRNA of exon 18 of the cynomolgus monkey CHD8 gene is used as the cleavage site, and the cleavage efficiency is detected by in vitro cleavage experiment. The in vitro cleavage experiment uses T7EI or SurveyorAssay.

3. The method for establishing a non-human primate model with gene point mutation according to claim 1, characterized in that, In step (2), the superovulation treatment is performed by injecting the donor female cynomolgus monkey with Gonal-F and Aze.

4. The method for establishing a non-human primate model with gene point mutation according to claim 1, characterized in that, In step (2), the female crab-eating macaques are 3-5 years old and the male crab-eating macaques are 5-11 years old.

5. The method for establishing a non-human primate model with gene point mutation according to claim 1, characterized in that, In step (3), Piezo microinjection is used; the concentration of sgRNA is 50 ng / uL and the concentration of YE1-mRNA is 100 ng / uL.

6. The method for establishing a non-human primate model with gene point mutation according to claim 1, characterized in that, In step (4), the embryo transfer is performed during the morula stage; the recipient female cynomolgus monkeys are 4-12 year old cynomolgus monkeys of the same age.

7. The method for establishing a non-human primate model with gene point mutation according to claim 1, characterized in that, In step (5), a pair of primers F1 / R1 were designed at both ends of the mutation site based on the sgRNA site of the CHD8 gene. The F0 generation monkeys were amplified by PCR primers and sent for sequencing. The product of the primers amplified in the gene knockout monkeys was 505bp. Sanger sequencing was used, and the monkeys were identified by sequencing primer F1. F1:5'-gaatgggtggtcccacctgt-3' R1:5'-agtcaaatccctgtgtaaaaacagctttt-3'.

8. The application of the CHD8 gene point mutation cynomolgus monkey model prepared by any of the methods described in claims 1-7 in the study of autism mechanisms or drug development.

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