CN107043787B - A kind of construction method and application that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9 - Google Patents

Abstract

A method and application of CRISPR/Cas9-based mouse model for site-directed mutation of MARF1, designing efficient sgRNA sequences that recognize specific genomic PAM regions; and designing Donor DNA on the basis of sgRNA sequences; mixing Donor DNA, sgRNA and Cas9 mRNA After pronuclear injection of fertilized eggs, embryo transfer, strain gene identification, founder mice were obtained, cloned and sequenced, and it was confirmed that a genetic mouse mutation model was finally obtained. The invention will have important reference value for improving artificial assisted reproductive technology, preventing female infertility and female reproductive related diseases, and developing new and effective female contraceptive drugs, and has great significance for promoting the reproductive health of women in my country and even the world.

Description

A method for constructing MARF1 site-directed mutation mouse model based on CRISPR/Cas9 law and application

technical field

The present invention relates to the field of gene coding technology, in particular to a method and application of using CRISPR/Cas9 gene coding technology to construct a MARF1 site-directed mutation mouse model.

Background technique

Introduction to MRAF1 Protein

Su et al. recently discovered a Riken gene (4921513D23Rik) encoding a novel protein whose function was unknown in the past. Mice carrying a single point mutation of this gene and a gene trap mutation all showed the same female infertility and male normal phenotype. Further studies have shown that the direct cause of female infertility is that female oocytes cannot resume meiosis and expel immature oocytes. Accordingly, we named the gene Marf1 (meiosis arrest female 1). Clinically, MARF1 protein is also of great significance. On the basis of our experiments, Chen Zijiang et al. sequenced the key exons of genes in patients with egg maturation disorders to explore whether human egg maturation disorders are related to MARF1 gene variation.

MARF1 is an RNA-binding protein. In addition to having the RRM (RNA recognition motif) that this type of protein generally has, it has an RNase-like domain at the N-terminus and a domain at the C-terminus. A newly discovered domain named OST (oskar, tudor) or LOTUS (limkain, oskar and tudor domain containing proteins 5 and 7). The OST/LOTUS domain is also present in the Drosophila protein Oskar and the mammalian proteins TDRD5 and 7 (tudor domaincontaining proteins 5 and 7), and is speculated to bind double-stranded RNAs, especially those formed by hybridization with piRNAs RNA, and then carry them to specific sites in germ cells such as nuage or germ granule. Oskar and TDRD5,7 play an important role in Drosophila germ cell specification and mouse male germ cell meiosis and retrotransposon silencing, respectively (Su.et.al.PNAS.2012). See Figure 1 for details.

Site-directed mutation of MARF1 protein

In the protein structure database NCBI- Structure ( https://www.ncbi.nlm.nih.gov/Structure/cdd/ ), the conserved protein domain analogy was compared with the same conserved XNI, 5'-EXONUCLEASE, TAQ in NYN DNA POLYMERASE and RIBONUCLEASE H were found to have four conserved Asp nuclease catalytic residues. NYN in the MARF1 protein corresponds to D178, D215, D246, and D272 in turn. In the study of protein structure, alanine is the chiral amino acid with the shortest side chain among amino acids. By mutating a certain amino acid residue into alanine, it is possible to locate the amino acid residue that has a key influence on the function of the protein by examining whether the function of the mutant protein is lost or changed due to the mutation. Substitution to alanine removes the active group on the side chain and replaces it with a methyl group that is small in size and has no other functional groups, and has less impact on the protein structure. Therefore, the present invention selects the mutation site No. 272 to mutate aspartic acid (D) into alanine (A). After aspartic acid (D) No. 272 of MARF1 is mutated to alanine (A), it can be used to specifically study the effect of the inactivation of the NYN domain of MARF1 on the entire MARF1 protein, and further study the loss of NYN enzyme in MARF1 protein After activity, effects on regulation of post-transcriptional mRNA homeostasis in oocytes. See Figure 2 for the specific sequence comparison.

Introduction to CRISPR/Cas9 System

CRISPR (clustered, regularly interspaced, short palindromic repeats) is an immune mechanism from bacteria to degrade invading viral DNA or other foreign DNA. In bacteria and archaea, CRISPR systems are divided into three types, among which type I and type III require multiple CRISPR-associated proteins (Cas proteins) to function together, while type II systems only need one Cas protein, which is the basis for the CRISPR system. It provides convenient conditions for wide application. Currently, the CRISPR/Cas9 system from Streptococcus pyogenes is most widely used. Cas9 protein (contains two nuclease domains, which can cut two single strands of DNA respectively. Cas9 first combines with crRNA and tracrRNA to form a complex, and then binds and invades DNA through the PAM sequence to form an RNA-DNA complex structure, and then target The DNA double strand is cut to break the DNA double strand. Due to the simple structure of the PAM sequence (5'-NGG-3'), a large number of targets can be found in almost all genes, so it is widely used. CRISPR/Cas9 The technology has been successfully applied to plants, bacteria, yeast, fish and mammalian cells, and is currently the most efficient genome editing technology. The crRNA and tracrRNA are modified by genetic engineering and connected together to obtain sgRNA (single guide RNA) The fused RNA has similar activity to wild-type RNA, but because the structure is simplified, it is more convenient for researchers to use. See Figure 3 for details.

Contents of the invention

Technical problem to be solved: the present invention provides a method and application of a mouse model for MARF1 site-directed mutation obtained based on CRISPR/Cas9, and the research on the gene MARF1 protein predominantly expressed in oocytes is carried out at the animal level. In particular, it can be explored whether the NYN domain will be inactivated when a point mutation occurs, so as to understand how MARF1 regulates three key links in the growth and development of oocytes—"acquisition of meiosis recovery ability", "mRNA post-transcriptional regulation” and “retrotransposon silencing” to carry out more in-depth research. The present invention is expected to reveal the mechanism of action of the NYN domain of MARF1 for the first time, and further reveal the key mechanism for regulating the growth, development and maturation of oocytes. This is of great reference value for improving artificial assisted reproductive technology, preventing female infertility and female reproductive-related diseases, and developing new and effective female contraceptive drugs, and is of great significance for promoting the reproductive health of women in my country and the world.

Technical solution: A method for constructing a mouse model of MARF1 site-directed mutation based on CRISPR/Cas9, comprising the following steps: Step 1: Design an efficient sgRNA sequence that recognizes a specific genomic PAM region, and the sgRNA sequence is as shown in SEQID NO.1 and design Donor DNA on the basis of sgRNA sequence, which is shown in SEQ ID NO.2 of the Donor DNA sequence; Step 2: inject the Donor DNA, sgRNA and Cas9 mRNA mixture into the fertilized egg pronucleus, embryo transfer, and strain gene identification , to obtain the founder mouse, cloned and sequenced, and confirmed the finally obtained genetic mouse mutation model of the MARF1-D272A point mutation.

The application of the CRISPR/Cas9-based MARF1 site-directed mutation mouse model obtained by the above construction method in the screening of contraceptive drugs.

The application of the CRISPR/Cas9-based MARF1 site-directed mutation mouse model obtained by the above construction method in screening drugs for the prevention and treatment of female infertility or female reproductive diseases.

A kit for obtaining a MARF1 site-directed mutation mouse model, containing the sgRNA shown in SEQ ID NO.1.

The above kit also contains the Donor DNA sequence shown in SEQ ID NO.2.

The above kit also contains Cas9 mRNA.

The specific construction process of the transgenic mouse model is as follows:

1. Construction of CRISPR-Cas system components

On the UCSC website, download the genomic DNA sequence of MARF1. Enter the http://crispr.mit.edu/ website, enter the CDS sequence to find the available sgRNA, and select 250bp around the position of the MARF1-D272A point mutation. The px330-sgRNA plasmid was constructed, and the sgRNA in vitro transcription template was prepared, and the sgRNA was obtained by in vitro transcription. In vitro transcription of Cas9 mRNA was performed.

2. MARF1-sgRNA efficiency and Cas9 mRNA quality detection

After the superovulation, the mouse is closed with the male mouse, and the thrombus is checked to obtain more fertilized eggs. Prepare sgRNA and Cas9 mRNA mix. Detection of sgRNA cleavage sites was determined by PCR, cloning, and sequencing.

3. Design of Donor DNA, pronuclear injection and embryo transfer of MARF1 point mutant mice

Design Donor DNA according to sgRNA cleavage site and synthesize ssDNA. Preparation of donor DNA, sgRNA and Cas9mRNA mixture, pronuclear injection, embryo transfer.

4. Genotype identification of MARF1 point mutation mice

The newborn pups were numbered and part of the toes were clipped to extract genomic DNA. PCR, cloning, and sequencing were used to determine whether the precise recombination of the MARF1-D272A point mutation occurred. Finally, a mouse model with a point mutation of MARF1-D272A in the mouse genome was established, and the first generation of mice was born as founder mice; each founder will be regarded as an independent strain for breeding, and each founder strain will be toe clipped for the first time, The PCR identification of the secondary trimming, and the respective cloning and sequencing verification. The aspartic amino acid at position 272 of the NYN domain of MARF1 protein is mutated to alanine, and the mutated amino acid sequence is shown in SEQ ID NO.1.

Beneficial effects: (1) The method for constructing a MARF1 site-directed mutation mouse model based on CRISPR/Cas9 provided by the present invention is convenient for studying the mechanism of action of multifunctional MARF1 in mouse reproductive development, especially oocyte development, in practical applications, And the model can be passed down stably. (2) Human eggs are rare and restricted by medical ethics and cannot be easily used as experimental objects. "Genetically modified mice" are important models for studying human eggs. The present invention provides a stable genetic method for clinical female infertility research, prenatal diagnosis, third generation "test-tube baby" PGD (preimplantation genetic diagnosis), that is, pre-implantation genetic diagnosis, advancement of assisted reproductive technology and development of contraceptive drugs. effective research platform.

Description of drawings

Figure 1. Sequence comparison of conserved sites in the MARF1-NYN domain.

Figure 2. Sequence comparison of conserved sites in the MARF1-NYN domain.

Figure 3. Brief diagram of the CRISPR-Cas9 system.

Figure 4. Genome splicing efficiency diagram of MARF1-272-sgRNA-F1 and MARF1-272-sgRNA-F2.

Figure 5. MARF1-272 site-directed mutation Donor DNA sequence, MARF1-272-sgRNA-F1, MARF1-272-sgRNA-F2 position in the genome and the corresponding PAM region, and a schematic diagram of the corresponding synonymous mutation.

Figure 6. Identification of MARF1-D272A site-directed mutant mice-PCR and StyI restriction map.

Figure 7. Identification-sequencing map of MARF1-D272A site-directed mutant mice.

Figure 8. Identification of MARF1-D272A site-directed mutation mice after cloning-PCR and StyI restriction map.

Figure 9. Post-cloning identification-sequencing map of MARF1-D272A site-directed mutation mice.

Figure 10. Comparison of oocytes from female mice with point mutations in MARF1 and normal cells.

Figure 11. Comparison of detection indicators between MARF1 point mutant mouse oocytes and normal cells. The left data column of each data set in the figure is normal cells, and the right data column is point mutant cells.

detailed description

The experimental materials needed in this experiment are as follows:

Q5 High-fidelity 2XMaster Mix(NEB,M0492S)

2×Taq PCR Master Mix with loading dye(TIANGEN,KT201)

mMESSAGE mMACHINE T7 ULTRA kit (Ambion, AM1345)

MEGAshortscript TM T7ULTRA kit (Ambion, AM1354)

plasma miniprep plus purification kit (genemark, Dp01-plus/Dp01-plus-300)

Example 1

A construction method and application of a mouse model for MARF1 site-directed mutation based on CRISPR/Cas9. The present invention will be further described below in conjunction with specific examples.

1.1 Construction of CRISPR-Cas system components

1.1.1 Design of sgRNA sequence for gene knock-in

(1) Enter the UCSC website, download the genomic DNA sequence of MARF1, and edit the sequence with SnapGene software.

(2) Enter the NCBI website, and mark exons, introns, promoters, and coding sequence (coding sequence, CDS) features on the sequence according to the gene information.

(3) Enter the http://crispr.mit.edu/ website, enter the CDS sequence to find available sgRNA, and select 250 bp around the position of the MARF1-D272A point mutation.

(4) Select the sgRNA according to the score given by the website. The higher the score, the lower the off-target effect and the better the specificity of the sgRNA. At the same time, the position of the sgRNA on the CDS should also be considered comprehensively. Use the domain architecture retrieval tool of NCBI to predict which important structural domains the protein encoded by the CDS of the gene has, and try to ensure that the sgRNA is located before the domain.

1.1.2 px330-sgRNA plasmid construction

(1) There is a U6 promoter on the px330 plasmid, and its downstream is immediately adjacent to a 20 base pair (base pair, bp) sgRNA, followed by a general sgRNA backbone sequence of about 80 bp. The original plasmid can be used to construct a new plasmid after single digestion with BbsI.

(2) Synthesis of oligodeoxynucleotides (DNA oligo, 5'→3')

sg-F: CACC-agtgatcttaggcaccggca

sg-R:AAAC-tgccggtgcctaagatcact

T7-sg-F: TAATACGACTCACTATAGGGagtgatcttaggcaccggca

T7-sg-R: AAAAGCACCGACTCGGTGCC

(3) Annealing of oligo (Anneal)

Mix well, 37°C for 30min in PCR instrument; 95°C for 5min; 0.1°C/s to cool down to 25°C

(4) The annealed fragments are connected to the carrier

Mix well, react at room temperature for 3 hours, transform 6 μL into E. coli competent, and plate

(5) Pick the bacteria after 16 hours, add 200 μL 2×YT medium (Amp ⁺ ) to shake the bacteria for 2 hours, and perform PCR on the bacterial solution

Mix well, PCR reaction: hot start at 95°C, anneal at 58°C, extend for 15s, 35×Cycles.

(6) Run 1% agarose gel, select 3 positive bacteria identified by PCR and send them for sequencing, and the sequencing primer is U6 promoter.

(7) Select the bacterial liquid micro-shake plasmid with the correct sequencing result.

1.1.3 Preparation of sgRNA in vitro transcription template

(1) High-fidelity enzyme PCR amplifies the DNA fragment (about 120bp) encoding the sgRNA sequence, and each sgRNA is equipped with 2 tubes of the same system

Mix well, PCR reaction: hot start at 98°C, anneal at 58°C, extend for 12s, 35×Cycles.

(2) Prepare 1% agarose gel, run the gel, cut off the gel block with a bright band size of about 120bp, and use DNA was recovered with the GelExtraction kit.

a. Weigh the gel block, estimate 100 mg as 100 μL, add Buffer QG 3 times the volume of the gel.

b. Place in an oven at 65°C for 10 minutes until the glue block melts.

c. Add 1 times the gel volume of isopropanol, invert and mix well.

d. Transfer the mixture to an adsorption column, centrifuge at 13,000 rpm for 1 min, and discard the filtrate.

e. Add 500 μL Buffer QG, centrifuge at 13000 rpm for 1 min, and discard the filtrate.

f. Add 750 μL Buffer PE, centrifuge at 13000 rpm for 1 min, and discard the filtrate.

g. Repeat step f.

h. Centrifuge the empty tube for 2 minutes.

i. Transfer the adsorption column to a clean 1.5mL imported centrifuge tube, open the cap and blow dry for 5 minutes in the RNA ultra-clean bench.

j. Add 30 μL of RNase-free ddH ₂ O preheated at 65°C to the center of the adsorption membrane, and let stand at room temperature for 5 minutes.

k. Centrifuge at 13000rpm for 2min to elute, suck out 2μL to measure the concentration (DNA50).

1.1.4 In vitro transcription of sgRNA

(1) (The following are all completed in the RNA clean bench) using the MEGAshortscript ^TM T7 transcription kit, ligand in vitro transcription reaction system

Mix well and react at 37°C for 4h.

(2) Add 0.5 μL TURBO DNase, mix well, and react at 37°C for 15 minutes.

(3) Transfer the system to a 0.6mL small tube, add 125μL RNase-free ddH ₂ O and 15μL AmmoniumAcetate Stop Solution, and mix well.

(4) Add an equal volume of phenol-chloroform to the lower layer, mix by inversion for 10 minutes, and centrifuge at room temperature at 12000 rpm for 10 minutes.

(5) Transfer the upper aqueous phase to a new tube, add an equal volume of chloroform, invert and mix for 10 minutes, and centrifuge at room temperature at 12,000 rpm for 10 minutes.

(6) Transfer the upper aqueous phase to a new tube, add 2 times the volume of absolute ethanol, mix well, and place at -20°C for 30 minutes.

(7) Prepare 2% agarose gel, mix 75% ethanol with RNase-free ddH ₂ O, and pre-cool the centrifuge at 4°C.

(8) Centrifuge at 12000 rpm for 15 minutes at 4°C.

(9) Discard the supernatant, rinse the precipitate once with 500 μL of 75% ethanol, and centrifuge at 12,000 rpm at 4° C. for 5 minutes.

(10) Discard the supernatant, open the lid of the RNA platform and blow dry until the edge of the precipitate becomes transparent.

(11) Depending on the size of the precipitate, add 50-80 μL RNase-free ddH ₂ O to dissolve, and place on ice for 2-3 minutes.

(12) Mix evenly, keep 3 μL, aliquot the remaining 6 μL/tube, and freeze in a -80°C refrigerator.

(13) Measure the concentration (ssDNA33), record 260/280 (2.0-2.2 is normal).

(14) Take 1 μL and run 2% agarose gel at 130V for 8 minutes to check whether the band brightness is consistent with the concentration and whether the band size is around 100 bp.

1.1.5 In vitro transcription of Cas9 mRNA

(1) Single digestion of pSpCas9(BB) (pX330; Addgene plasmid ID: 42230) plasmid as a template for in vitro transcription

Mix well and react overnight at 37°C.

(2) Prepare 1% agarose gel, run the gel, cut off the gel block where the linearized DNA is located, and use GelExtraction kit recovery.

(3) Use mMESSAGE T7 Transcription Kit for in vitro transcription of Cas9 mRNA.

① (The following are all completed in the RNA clean bench) Ligand in vitro transcription reaction system

Mix well and react at 37°C for 2h.

② Add 1 μL TURBO DNase, mix well, and react at 37°C for 15 minutes.

③Reaction system with polyA tail

Mix well, aspirate 2.5 μL and store on ice.

④Add 4μL E-PAP Mix, mix well, react at 37°C for 30min, and put it on ice.

⑤Add 10μL Ammonium Acetate Stop Solution and mix well.

⑥Phenol-chloroform extraction method for RNA purification, the steps are the same as 1.1.4(4)～(10).

⑦ Depending on the size of the precipitate, add Nuclease-free water to dissolve, and place on ice for 2 to 3 minutes.

⑧ Mix well, take 1.5 μL to measure the concentration (RNA40), 260/280 is about 2.0 is normal, and temporarily put the remaining mRNA on ice.

⑨ Dilute mRNA to about 1 μg/μL according to the measured concentration, take 1 μL and 2.5 μL before adding polyA tail and run on 2% agarose gel. The bands should be larger (maybe slightly blurred, but not affecting use).

⑩If the concentration and gel running results are normal, then 1 μg of each tube is aliquoted and quickly frozen in a -80°C refrigerator to obtain Cas9 mRNA.

1.2 MARF1-sgRNA efficiency and Cas9 mRNA quality detection

In one experiment, one sgRNA with high efficiency can be used to detect the quality of Cas9 mRNA, and high-quality Cas9 mRNA can also be used to detect the efficiency of one sgRNA.

1.2.1 Obtaining fertilized eggs from mice

Embryos used for efficiency testing are generally taken from BDF1 strain mice, which are the first generation of hybrid offspring bred after mating of inbred C57BL/6J and DBA/2 mice, and are sexually mature in 6 to 8 weeks. The mice used were raised in a specific-pathogen-free (SPF) environment.

(1) 0d, 13:00, grab a BDF1 female mouse about 8 weeks old, and inject 50 U/mL (about 60 μL) of pregnant horse serum gonadotropin (pregnant mare's serum gonadotropin, PMSG) intraperitoneally, imitating endogenous The role of follicle-stimulating hormone (FSH) in promoting egg maturation.

(2) 2d, 13:00, grab BDF1 female mice injected with PMSG, intraperitoneally inject 50 U/ml (about 70 μL) of human chorionic gonadotropin (human chorionic gonadotropin, hCG), imitating luteinizing hormone (luteinizinghormone, Ovulation induction effect of LH). And each female mouse was mated with one BDF1 male mouse separately.

(3) Make G1 embryo culture drops: 20 μL/drop on the bottom of a 35mm Petri dish, neatly arranged, and finally seal the liquid surface with Mineral oil, and equilibrate overnight in a 37°C, 5% CO ₂ incubator.

(4) On 3d, at 8:00, check whether the female rat has a vaginal suppository (the trace of secretion left by the male rat after mating), and if so, take out the rat room.

(5) Make CZB embryo culture drops, and equilibrate in a 37° C., 5% CO ₂ incubator for at least 15 minutes.

(6) Execute the female mouse by cervical dislocation: put the mouse on the iron frame of the mouse cage, make its front paws grasp the steel wire above it, press its neck (behind the skull) tightly, and at the same time move backward horizontally Stretch the tail so that it breaks the neck.

(7) Put the sacrificed mice back up in the enamel jar, and immediately spray the backs of the mice with 75% alcohol to minimize the pollution of the mouse hairs.

(8) Pinch the back skin of the mouse, cut a small opening in the middle and lower area with surgical scissors, firmly grasp the cut skin, stretch to the head and tail so that the back skin is completely opened, and the hair leaves the internal tissue completely.

(9) Use ophthalmic tweezers and sharp scissors to open the dorsal membrane, use tweezers to pick up the white fat tissue, gently pull the uterus, fallopian tubes, ovaries and fat pad out of the body cavity as a whole, use tweezers to hold the uterus below the ovaries, and use scissors Puncture a hole in the mesentery close to the fallopian tube, cut the mesentery, then separate the ovary from the fallopian tube with scissors (to ensure the integrity of the fallopian tube), and finally cut off the uterus close to the fallopian tube, and put the fallopian tube into HCZB in vitro operation solution.

(10) Piercing the dilated ampulla of the fallopian tube with the needle point of the syringe under the dissecting microscope to obtain the fertilized ovum mass wrapped by cumulus cells.

(11) All agglomerates were collected, digested in 0.1% hyaluronidase at 37°C for 3-5 minutes, and then fertilized eggs were separated from cumulus cells.

(12) Burn the capillary glass tube on the flame of the alcohol lamp to pull the needle and break the needle, the inner diameter is about 150-200 μm, and assemble the suction pipe. Eggs were transferred with a mouth pipette, and the separated fertilized eggs were washed 6 times in HCZB drops to remove residual cumulus cells and hyaluronidase.

(13) Transfer the fertilized eggs into CZB drops and wash them 3 times, and recover in a 5% CO ₂ incubator at 37°C.

1.2.2 Preparation of sgRNA and Cas9 mRNA mixture

Mixtures were prepared at RNA Desk. The mixture used to detect the efficiency was 100 ng/μL of Cas9 mRNA and 50 ng/μL of sgRNA. Generally, 5 μL of the mixture should be prepared for injection

Cas9 mRNA 500ng

sgRNA 250ng

Make up to 5μL with ddH ₂ O

Centrifuge at 10,000 rpm at 4°C for 10 minutes, and inject pronuclei into about 20 fertilized eggs.

1.2.3 Detection of sgRNA cleavage sites

A. Lysis of Embryos

(1) Fertilized eggs were cultured in G1 medium for 3 days, and after developing into blastocysts, transfer the embryos into 5 μL of G1 lysate with a mouth pipette.

(2) The embryos were lysed at 95°C for 20 minutes in a PCR machine to release DNA.

(3) Add 5 μL of G2 solution, mix well, and it can be used as a PCR template.

B.PCR amplification

(1) Use NCBI's Primer-BLAST tool to design primers with high specificity. The PCR product is a fragment of about 500 bp containing the sgRNA site on the mouse genomic DNA, and the sgRNA site should be located in the center of the fragment as much as possible.

(2) The specificity of the primers was tested with common mouse genomic DNA, and the temperature gradient PCR was used to obtain the optimal annealing temperature of the primers.

(3) The first round of PCR reaction

Mix well, PCR reaction: 94°C hot start, extension 36s, 30×Cycles.

(4) For the second round of PCR reaction, each sample is equipped with 2 tubes of the same system; at the same time, 2 tubes of the system using wild type (wild type, WT) mouse genomic DNA as template

Mix well, PCR reaction: 95°C hot start, extension 36s, 35×Cycles.

(5) Prepare 1% agarose gel, run the gel, cut off the gel block with the correct band, and recover DNA using the DNA purification and recovery kit.

① Weigh the gel block, estimate 100 mg as 100 μL, add Buffer PC of equal gel volume.

② Place in an oven at 65°C for about 10 minutes until the rubber block melts.

③Equilibrium adsorption column: add 500μL Buffer BL, centrifuge at 13000rpm for 1min, and discard the filtrate.

④ Add the sol solution to the adsorption column, let it stand at room temperature for 5 minutes, centrifuge at 13000rpm for 1 minute, and discard the filtrate.

⑤Add 600μL Buffer PW, centrifuge at 13000rpm for 1min, and discard the filtrate.

⑥Repeat step ⑤.

⑦ Centrifuge the empty tube for 2 minutes.

⑧Transfer the adsorption column to a clean 1.5mL centrifuge tube, open the cap and blow dry for 5 minutes in an ultra-clean bench.

⑨ Add 30 μL 65°C preheated Buffer EB to the center of the adsorption membrane, and let stand at room temperature for 5 minutes.

⑩ Centrifuge at 13000rpm for 2min to elute, and suck out 1.7μL to measure the concentration (DNA50).

C. T7 enzyme digestion test of gel back product

(1) With annealing reaction system

Gel recovery product DNA 250～300ng

NEB Buffer 2 1.5μL

Make up to 14.5μL with ddH ₂ O

Mix well, denaturation and annealing reaction in PCR instrument: 95°C for 5min, slowly cool down to 25°C at 0.1°C/s, 25°C for 10min.

(2) with 2% agarose gel.

(3) Add 0.5 μL T7 endonuclease I to the DNA annealing system, mix well, and react in a PCR instrument at 37°C for 15-17 minutes.

(4) Quickly add 10×Loading Buffer, mix well, run the gel for 30 minutes at 135V, and check the results. If there are obvious small bands in addition to the main band of about 500bp, but there is no WT, it means that the recovered product of the original gum contains If there is a mutated fragment, the corresponding sgRNA may be cleaved to cause a mutation.

D. Ligation of the sequencing vector

(1) The gel-backed fragments that were positive in the T7 enzyme digestion test were connected to the sequencing vector. There are two commercially available vectors.

a.pLB vector

①With end-filling reaction system

Mix evenly, in a PCR machine at 20°C for 5min, at 70°C for 5min, and place on ice briefly.

② Matching connection reaction system

Fill-in product 4μL

pLB vector 0.5μL

T4 _Ligase 0.5 μL

Mix well, and place at room temperature for 10 minutes, until the E. coli is competent and plated.

b. Zero Cloning

Ligation Reaction System

Gum recovery product 25ng

T5 Zero Cloning Vector 1μL

Make up to 4 μL with ddH ₂ O

Mix well, place at room temperature for 15 minutes, transform all into E. coli competent, and plate.

(2) Pick the bacteria after 16 hours, add 200 μL 2×YT medium (Amp ⁺ ) to shake the bacteria for 2 hours, and perform PCR on the bacterial solution

Mix well, PCR reaction: 95°C hot start, extension 36s, 35×Cycles.

(3) Run 1% agarose gel, select 8 positive bacteria identified by PCR and send them for sequencing, and the sequencing primer is T7 promoter. Looking at the results, the proportion of results containing insertion/deletion (indel) mutations to the total represents the efficiency of the corresponding sgRNA.

(4) For samples that are negative in the T7 enzyme digestion test, directly send the gel-backed fragments for sequencing, and check the peak diagram of the sequencing. If there is a single peak before the sgRNA cutting site, and there are peaks near the sgRNA from the beginning to the end , then the glue back product may contain a mutated fragment (T7 enzyme digestion test is false negative), and the presence of the mutation is confirmed by ligation sequencing vector.

1.3 Design of Donor DNA and pronuclear injection of MARF1 point mutant mice

1.3.1 Design of Donor DNA

Centering on the cleavage position between 3-4bp in the 5' end direction of the sgRNA1 PAM region, 65 bp in the 5' end direction is the left homology arm, and 65 bp in the 3' end direction is the right homology arm. Order single-stranded ssDNA ( Sangong, Shanghai China). The purity of the single-stranded ssDNA must be purified by HPLC.

The comparison of cutting efficiency of MARF1-272-sgRNA-1 and MARF1-272-sgRNA-2 is shown in Figure 4.

The design map of MARF1-272donor DNA is shown in Figure 5.

1.3.2 Donor DNA, sgRNA and Cas9 mRNA mixture preparation

Mixtures were prepared at RNA Desk. The mixture used to detect the efficiency is 100ng/μL of Cas9 mRNA, 50ng/μL of sgRNA, and 100ng/μL of donor DNA. Generally, 5 μL of the mixture should be prepared for injection

Centrifuge at 10,000 rpm at 4°C for 10 minutes, and inject the cytoplasm into approximately fertilized eggs.

1.3.3 Pronuclear injection

The acquisition of fertilized eggs and pronuclear injection have been described, as above, see sgRNA efficiency and Cas9 mRNA quality detection.

1.3.4 Embryo transfer

A. Preparation for ligation of male mice

(1) After weighing, anesthetize 6-8 week-old male mice at a dose of 100ml/kg, fix the mice in the dorsal position, and apply 75% ethanol to disinfect the surgical incision site to prevent hair from polluting the incision.

(2) Cut the skin of the lower abdomen horizontally at about 1.3 cm in front of the genitals, make an incision of about 1 cm, and wipe the incision site with gauze soaked in 75% ethanol to clean the hair.

(3) Cut the peritoneum and locate the bladder. A duct can be seen running on each side. The left duct was gently held with tweezers, and the part was lifted to make the surgical field of view clearly visible, and it was determined to be the vas deferens.

(4) Use tweezers to pinch the vas deferens into a U shape, heat the other tweezers on an alcohol lamp, scald the vas deferens between the two ligation points, put the ligated vas deferens into the body and then ligate the other side.

(5) After the operation on both sides is completed, the internal and external surgical openings are sutured with No. 0-5 sutures. The mice were placed on a constant temperature platform at 37°C until they woke up. After the operation, the mice were fed for 2 weeks to determine whether the operation was successful.

(6) Experimental feeding: 1-2 female mice were housed together with vasectomized mice, and the vaginal suppository was checked the next morning. Female mice with vaginal suppositories can use the suppository as a smear, and observe under the microscope whether there is sperm. It can also be cultured until the next day to take the oviduct to check whether there are two-cell embryos. If there are spermatozoa or two-cell embryos, male mice will not be ligated successfully.

B. Preparation of recipient mice

(1) Simultaneously select the ICR or CD-1 in estrus to house the ligated male mice.

(2) In the early morning of the next day, check whether the donor mice and recipient mice see thrombi, and use them for embryo transfer on the day when the thrombus is seen or 2.5 days later.

C. Fallopian tube transplantation

(1) Prepare sterilized surgical instruments, sutures, etc.

(2) After weighing, the mice were anesthetized with 1.25% Avertin solution at a dose of 200ml/kg, the back coat of the mice was cut off, and after disinfection with 75% alcohol, it was placed 1 cm from the side of the spine, about the same level as the last rib. Cut the skin, use tweezers to bluntly separate the skin and muscles along the incision, use tweezers to gently pull out the ovaries, fallopian tubes and uterus, clamp part of the fat, and place them on the skin on the back of the mouse.

(3) Use a transfer needle to inhale 20 fertilized eggs that survived the injection. The inhalation sequence is: M16 culture medium & air & M16 culture medium & air & fertilized eggs. Under the condition of ensuring that the embryos are completely inhaled, try to reduce the amount of inhalation of the culture medium .

(4) Put the mouse under the dissecting microscope, pinch the fat pad with a small hemostatic forceps, tear off a small opening in the membrane covering the ovary with ophthalmic tweezers, expose the fimbria of the oviduct, insert the egg transfer needle, and blow the fertilized eggs Into the fallopian tube, there are obvious air bubbles in the ampulla of the fallopian tube, indicating that the transplantation is successful.

(5) Send the fat pad, ovary, uterus and fallopian tube back to the abdominal cavity with blunt forceps, suture the peritoneal and skin incisions, place the transplanted recipient mice on the warm platform for 10 minutes to recover, and then transfer them to the barrier environment for rearing.

(6) Mark the mice after transplantation, and indicate the time of transplantation, the name of the strain, and the number of embryos. After giving birth, the date of birth and the number of births were marked, and the survival of the born cubs was observed every day.

1.4. Genotype identification of MARF1 point mutant mice

After the fertilized eggs are injected with RNA and transplanted into the fallopian tubes of pseudopregnant mother mice, mice are usually born within 19 to 20 days. After 6 days of birth, the toes of the mice are separated, and the toes can be cut for numbering and marking, and a small piece of tissue at the tip of the tail can be used for the extraction of genomic DNA.

1.4.1 Phenol-chloroform extraction of genomic DNA

(1) Cut the tissue into a centrifuge tube, add 300 μL of mouse tail lysate containing proteinase K, and lyse overnight at 65°C.

(2) Add 300 μL of phenol chloroform, mix by inversion for 10 minutes, and centrifuge at room temperature at 12000 rpm for 10 minutes.

(3) Carefully absorb the supernatant and transfer it to a new tube, add twice the volume of absolute ethanol, mix by inverting to see the flocculent precipitate, and centrifuge at 12000rpm at 4°C for 5min.

(4) Discard the supernatant, add 500 μL of 70% ethanol to rinse the precipitate, and centrifuge at 12,000 rpm at 4° C. for 5 minutes.

(5) Discard the supernatant, and blow dry for 5 minutes on a clean bench until the bottom precipitate becomes transparent. Depending on the size of the precipitate, add ddH ₂ O preheated at 65°C.

(6) Place at room temperature for 5 minutes to fully dissolve the DNA.

(7) Mix evenly by inverting, and suck out 2 μL to measure the concentration (DNA50).

1.4.2 PCR amplification of fragments containing predicted mutation sites

(1) The primers used are the same as those used in the detection of sgRNA efficiency, and each DNA sample is prepared with 2 to 3 tubes of reaction system depending on the efficiency of the primers

(2) Glue recovery, the steps are the same as 3.2.3B.(5).

1.4.3 Genomic PCR product direct StyI digestion test and sequencing confirmation

(1) PCR product direct StyI digestion test:

(2) 37 degrees, water bath, digestion for 3 hours, electrophoresis.

(3) Direct sequencing of PCR products.

Due to the change of site-directed mutagenesis, if precise recombination occurs, a restriction site StyI will be added. Wild-type mice cannot be dissected. In the mice born in the present invention, #1 can be digested by StyI, but #2, #3, #4, and #5 cannot be digested by enzymes. See Figure 6 for details. In order to further confirm that precise recombination occurs, sequencing can reveal a significant double peak at the recombined point mutation position. In contrast, wild-type mice do not exhibit bimodality. Of the mice born in the present invention, #1 has bimodality, but #2, #3, #4, and #5 have no bimodality. See Figure 7 for details.

1.4.4 StyI digestion test and sequencing confirmation of PCR products after cloning

After the PCR product was recovered by QIANGEN kit gel, it was ligated with PLB vector kit, cloned, picked bacteria, monoclonal, and performed bacterial liquid PCR to screen out positive bacteria. On the one hand, the PCR product is directly digested with StyI, electrophoresis, the monoclonal strains L5 and L9 can be digested by StyI, but the monoclonal strains L1, L2, L3, L4, L6, L7, and L8 cannot be digested by StyI , see Figure 8. On the other hand, the monoclonal positive bacteria were sent to the company for sequencing, and the sequencing results are shown in Figure 9.

1.5 The application of MARF1 point mutant mice as a stable genetic model to study female infertility

1.5.1 Female mice with MARF1 point mutation are infertile and oocytes cannot develop normally

As shown in Figure 10. The oocytes of MARF1 point mutant mice are arrested at the germinal vesicle (GV) stage, unable to resume meiosis, and fail to mature normally. Further lead to female infertility.

1.5.2 MARF1 point mutation mouse oocyte series detection index RNA significantly increased

As shown in Figure 11. A large number of detection index RNAs in the oocytes of MARF1 point mutant mice increased significantly, while a large number of detection index RNAs in normal oocytes remained at a low level. These can provide an effective research platform with stable genetics for prenatal diagnosis, third-generation "test-tube baby" PGD (preimplantation genetic diagnosis), the advancement of assisted reproductive technology, and the development of contraceptive drugs.

SEQUENCE LISTING

<110> Nanjing Medical University

<120> A method and application of CRISPR/Cas9-based mouse model for site-directed mutation of MARF1

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<170> PatentIn version 3.3

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Claims (5)

1. a kind of construction method that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9, its feature include following step Suddenly：

1st step：The sgRNA sequences in the efficient identification specific gene group PAM regions of design, the sgRNA sequences such as SEQ ID Shown in NO.1；And Donor DNA are designed in sgRNA sequence basis, shown in the Donor DNA sequence dnas SEQ ID NO.2；

2nd step：By Donor DNA, sgRNA and Cas9 mRNA mixtures are to embryonated egg procaryotic injection, embryo transfer, strain base Because of identification, founder mouse, and cloning and sequencing are obtained, the genetic mouse for the MARF1-D272A point mutation for confirming to finally give is dashed forward Varying model.

Exist 2. what construction method described in claim 1 obtained obtains MARF1 rite-directed mutagenesis mouse models based on CRISPR/Cas9 Screen the application in contraceptive.

Exist 3. what construction method described in claim 1 obtained obtains MARF1 rite-directed mutagenesis mouse models based on CRISPR/Cas9 Application in screening preventing and treating female infertility medicine.

4. obtain the kit of MARF1 rite-directed mutagenesis mouse models, it is characterised in that contain the Donor shown in SEQ ID NO.2 DNA sequence dna and the sgRNA as shown in SEQ ID NO.1.

5. kit according to claim 4, it is characterised in that also containing Cas9 mRNA.