CN107043787A - A kind of construction method and application that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9 - Google Patents

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

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CN107043787A
CN107043787A CN201710355368.3A CN201710355368A CN107043787A CN 107043787 A CN107043787 A CN 107043787A CN 201710355368 A CN201710355368 A CN 201710355368A CN 107043787 A CN107043787 A CN 107043787A
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sgrna
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rite
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苏友强
曹广义
李明哲
王昊
时兰英
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Nanjing Medical University
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Abstract

A kind of construction method and application that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9, the sgRNA sequences in the efficient identification specific gene group PAM regions of design；And Donor DNA are designed in sgRNA sequence basis；By Donor DNA, sgRNA and Cas9 mRNA mixtures obtain founder mouse, and cloning and sequencing to embryonated egg procaryotic injection, embryo transfer, strain identified for genes, and confirmation finally gives genetic mouse catastrophic model.The present invention can be to improving human assistance reproductive technology, prevent and treat female infertility and female reproduction relevant disease, and the effective female contraception medicine of development of new all there are important references to be worth, to promoting China or even the healthy reproduction of global women to be significant.

Description

A kind of structure side that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9 Method and application
Technical field
The present invention relates to gene code technical field, and in particular to uses CRISPR/Cas9 gene code technique constructions The methods and applications of MARF1 rite-directed mutagenesis mouse models.
Background technology
MRAF1 proteins. Introductions
Soviet Union etc. is found that the Riken genes of the brand-new albumen of a coding past Unknown Function recently (4921513D23Rik).The mouse for carrying the Gene unit point mutation and gene trap (gene trap) mutation shows together The female sterile of sample and the normal table of male.Further study showed that, the immediate cause for causing female sterile is that female mice ovum is female Cell can not recover meiosis and discharge immature egg mother cell.Accordingly, the unnamed gene is Marf1 by we (meiosis arrest female 1).Clinically, MARF1 albumen also has great importance.The basis tested at us On, once the crucial extron of maturation of ovum impaired patients gene is sequenced by Chen Zijiang etc., inquires into mankind's maturation of ovum obstacle It is whether related to MARF1 genetic mutations.
MARF1 is a rna binding protein, and RRM (RNA recognition are typically of except possessing the albuminoid Motif) outside, it possesses the domain (domain) of 1 similar ribalgilase (RNase) at N- ends, and 1 is possessed at C- ends newly It is being found, be named as OST (oskar, tudor) or LOTUS (limkain, oskar and tudor domain The and 7 of containing proteins 5) domain.OST/LOTUS domains exist in drosophila protein Oskar and the food in one's mouth In newborn animal protein TDRD5 and 7 (and 7 of tudor domaincontaining proteins 5), and can by speculating With reference to double-stranded RNA be particularly those through with piRNA hybridization after formed by double-stranded RNA, they are then carried to reproduction cell Interior such as nuage or germ granule privileged site.Oskar and TDRD5,7 determine (germ in drosophila reproduction cell respectively Cell specification) and the meiosis of mouse male sex-cell and retrotransposon silence in play an important role (Su.et.al.PNAS.2012).It is specifically shown in Fig. 1.
MARF1 albumen rite-directed mutagenesises
Protein structure databases NCBI-Structure (https://www.ncbi.nlm.nih.gov/ Structure/cdd/) the protein structure domain analogy guarded, compare NYN and equally conservative XNI, 5 '- EXONUCLEASE, TAQ DNA POLYMERASE, RIBONUCLEASE H, it is found that they possess 4 conservative Asp nucleases Catalytic residue.It is corresponding in turn in the NYN of MARF1 albumen as D178, D215, D246, D272.In protein structure research, the third ammonia Acid is side chain most short chiral amino acid in amino acid., can be by investigating by the way that certain amino acid residue is sported into alanine Therefore whether the function of the mutain be mutated and lose or change, and has crucial effect to the protein function so as to position Amino acid residue.Alanine is replaced as, the active group on side chain is eliminated, small volume, the first without other functional groups has been changed into Base, while the influence to protein structure is smaller.Therefore, the present invention have selected No. 272 sites of mutation, and aspartic acid (D) is prominent It is changed into alanine (A).MARF1 No. 272 aspartic acids (D) are sported after alanine (A), can be used for specifically studying MARF1 NYN domains inactivation after influence to whole MARF1 albumen, further careful research MARF1 albumen lose NYN enzyme activity Property after, to egg mother cell transcribe after mRNA steady-state adjustments influence.Particular sequence, which compares, sees Fig. 2.
CRISPR/Cas9 SYSTEM SUMMARYs
CRISPR (clustered, regularly interspaced, short palindromic repeats) is one Plant the viral DNA invaded from bacterial degradation or the immunologic mechanism of other exogenous DNAs.In bacterium and archeobacteria, CRISPR systems Altogether it is divided into 3 classes, wherein I class and III class need a variety of CRISPR GAP-associated protein GAPs (Cas albumen) to play a role jointly, and II class system System only needs to a kind of Cas albumen, and this can be extensively using the condition of providing convenience for it.At present, come from Streptococcus pyogenes CRISPR/Cas9 systems are most widely used.Cas9 albumen (contains two nuclease knots Structure domain, can be respectively cut DNA two single-stranded.Cas9 is combined into compound with crRNA and tracrRNA first, then passes through PAM sequences are combined and invade DNA, form RNA-DNA composite constructions, and then target DNA double-strand is cut, and make DNA double chain Fracture.Because PAM sequential structures are simple (5 '-NGG-3 '), a large amount of target spots can be almost found in all genes, because This is widely used.It is thin that CRISPR/Cas9 technologies have been successfully applied to plant, bacterium, yeast, fish and mammal Born of the same parents, are current most efficient genome editing techniques.CrRNA and tracrRNA are transformed by genetic engineering means, will It, which links together, obtains sgRNA (single guide RNA).RNA after fusion has the vigor similar with wild type rna, But because structure is simplified, more convenient researcher uses.It is specifically shown in Fig. 3.
The content of the invention
The technical problem of solution：The present invention provides a kind of based on CRISPR/Cas9 acquisition MARF1 rite-directed mutagenesis mouse models Construction method and application, animal level carry out egg mother cell predominant expression gene M ARF1 albumen research.Especially may be used To probe into when occurring point mutation, whether NYN domains can lose work, how to regulate and control Growth of Oocytes development to MARF1 During 3 key links-" acquisition of Meiotic resumption ability ", " mRNA post-transcriptional controls " and " retrotransposon sink It is silent " expansion more in-depth study.Present invention contemplates that MARF1 NYN domain action principles can be disclosed first, and will be deeply Disclose the key mechanism that regulation and control Growth of Oocytes is reached maturity.This prevents and treats female acyesis to improving human assistance reproductive technology Infertility and female reproduction relevant disease, and the effective female contraception medicine of development of new all there are important references to be worth, to promoting The healthy reproduction for entering China or even global women is significant.
Technical scheme：A kind of construction method that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9, including Following steps：1st step：The sgRNA sequences in the efficient identification specific gene group PAM regions of design, the sgRNA sequences such as SEQ Shown in ID NO.1；And Donor DNA, the Donor DNA sequence dnas SEQ ID NO.2 institutes are designed in sgRNA sequence basis Show；2nd step：By Donor DNA, sgRNA and Cas9 mRNA mixtures are to embryonated egg procaryotic injection, embryo transfer, strain gene Identification, obtains founder mouse, and cloning and sequencing, confirms the genetic mouse mutation of MARF1-D272A point mutation finally given Model.
What above-mentioned construction method was obtained obtains MARF1 rite-directed mutagenesises mouse model in screening contraception based on CRISPR/Cas9 Application in medicine.
What above-mentioned construction method was obtained obtains MARF1 rite-directed mutagenesises mouse model in screening preventing and treating based on CRISPR/Cas9 Application in female infertility or female genital disorders medicine.
The kit of MARF1 rite-directed mutagenesis mouse models is obtained, contains the sgRNA as shown in SEQ ID NO.1.
Mentioned reagent box is also containing the Donor DNA sequence dnas shown in SEQ ID NO.2.
Mentioned reagent box also contains Cas9 mRNA.
The specific building process of transgene mouse model is as follows：
1st, the structure of CRISPR-Cas system elements
In UCSC websites, MARF1 genomic dna sequence is downloaded.Intohttp://crispr.mit.edu/Website, it is defeated Enter CDS sequences and search available sgRNA, 250bp is selected before and after MARF1-D272A point mutation position.Build px330- SgRNA plasmids, and sgRNA in-vitro transcription templates are prepared, in-vitro transcription obtains sgRNA.Carry out Cas9 mRNA in-vitro transcriptions.
2nd, MARF1-sgRNA efficiency and Cas9 mRNA quality testings
Make after mouse superfecundation and public mouse is closed up, examine bolt, obtain more embryonated eggs.Prepare sgRNA and Cas9 mRNA Mixture.By PCR, clone, sequencing, to determine the detection of sgRNA cleavage sites.
3rd, Donor DNA design, the procaryotic injection of MARF1 point mutation mouse and embryo transfer
Donor DNA are designed according to sgRNA cleavage sites, ssDNA is synthesized.Prepare donor DNA, sgRNA and Cas9 MRNA mixtures, procaryotic injection, embryo transfer.
4th, the genotype identification of MARF1 point mutation mouse
Newborn small son is numbered, and clip part toe extracts genomic DNA.By PCR, clone, sequencing, to determine to be The no accurate restructuring that there occurs MARF1-D272A point mutation.It is final to set up the small of MARF1-D272A point mutation in mouse genome Mouse model, the head of birth is on behalf of founder mouse；Each founder will be considered as an independent strain and be bred, right Each founder strains are cut toe, the secondary PCR identifications for cutting tail first, and clone respectively and verification is sequenced.MARF1 272 asparagus fern amino acid mutations of albumen NYN domains are alanine, the amino acid sequence such as SEQ ID after the mutation Shown in NO.1.
Beneficial effect：(1) structure that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9 that the present invention is provided Construction method, facilitates the multi-functional MARF1 of research in terms of mouse propagation develops especially Oocyte Development in actual applications Mechanism of action, and the model can stablize passage.(2) mankind's ovum it is rare and by Medical Ethics limited can not easily as experiment Object, " genetic modification mouse " is the important models for studying mankind's ovum.Research, production of the present invention for clinical female acyesis disease Preceding diagnosis, the third generation " test-tube baby " PGD (preimplantation genetic diagnosis), that is, gene is examined before transplanting The disconnected, progress of auxiliary procreation technology and the exploitation of contraceptive, which are provided, can stablize effective research platform of heredity.
Brief description of the drawings
Fig. 1 .MARF1-NYN conservative domain site sequences compare figure.
Fig. 2 .MARF1-NYN conservative domain site sequences compare figure.
Fig. 3 .CRISPR-Cas9 SYSTEM SUMMARY figures.
Fig. 4 .MARF1-272-sgRNA-F1, MARF1-272-sgRNA-F2 are in genome shear efficiency figure.
Fig. 5 .MARF1-272 rite-directed mutagenesis Donor DNA sequence dnas, MARF1-272-sgRNA-F1, MARF1-272- SgRNA-F2 is in the position of genome and corresponding PAM regions, and corresponding same sense mutation schematic diagram.
Fig. 6 .MARF1-D272A rite-directed mutagenesis mouse identification-PCR and StyI cleavage maps.
Fig. 7 .MARF1-D272A rite-directed mutagenesises mouse identification-sequencer map.
Identification-PCR and StyI cleavage maps after Fig. 8 .MARF1-D272A rite-directed mutagenesis cloned mouses.
Identification-sequencer map after Fig. 9 .MARF1-D272A rite-directed mutagenesis cloned mouses.
Figure 10 .MARF1 point mutation female mice egg mother cells and normal cell comparison diagram.
A left side for every group of data in Figure 11 .MARF1 point mutation oocyte of mouse and normal cell Testing index comparison diagram, figure Side data post is normal cell, and right side data post is point mutation cell.
Embodiment
The experiment material needed in this experiment is specially：
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)
plasmid miniprep plus purification kit(genemark,Dp01-plus/Dp01-plus- 300)
Embodiment 1
A kind of construction method and application that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9, with reference to Specific embodiment, is expanded on further the present invention.
The structure of 1.1 CRISPR-Cas system elements
1.1.1 it is used for the design of the sgRNA sequences of gene knock-in
(1) enter UCSC websites, MARF1 genomic dna sequence is downloaded, with SnapGene software editing sequences.
(2) enter NCBI websites, according to gene information in sequence annotated exon, introne, promoter, coded sequence (coding sequence, CDS) feature.
(3) enterhttp://crispr.mit.edu/Website, input CDS sequences search available sgRNA, surround Selection 250bp before and after MARF1-D272A point mutation position.
(4) score value provided according to website selects sgRNA, and fraction is higher to represent that its effect of missing the target is lower, and sgRNA's is special Property is better.Also to consider positions of the sgRNA on CDS simultaneously, use NCBI domain architecture Retrieval instruments predict which important feature domain the albumen of gene C DS codings has, and ensure that sgRNA is located at domain as far as possible Before.
1.1.2 px330-sgRNA plasmid constructions
(1) U6 promoters are carried on px330 plasmids, downstream close to 20 base-pairs (base pair, bp) sgRNA, its It is the about 80bp general frame sequences of sgRNA afterwards.By original plasmid with after the single endonuclease digestions of Bbs I can be used for build new plasmid.
(2) synthesis oligodeoxynucleotide (DNA oligo, 5 ' → 3 ')
sg-F：CACC-agtgatcttaggcaccggca
sg-R：AAAC-tgccggtgcctaagatcact
T7-sg-F：TAATACGACTCACTATAGGGagtgatcttaggcaccggca
T7-sg-R：AAAAGCACCGACTCGGTGCC
(3) oligo annealing (Anneal)
Mix, 37 DEG C of 30min in PCR instrument；95℃ 5min；0.1 DEG C/s is cooled to 25 DEG C
(4) fragment after annealing is connected to carrier
Mix, react at room temperature 3h, 6 μ L of conversion to E. coli competent, coated plate
(5) bacterium, plus 200 μ L 2 × YT culture mediums (Amp are chosen after 16h+) shake bacterium 2h, bacterium solution PCR
Mix, PCR reactions：95 DEG C of thermal startings, 58 DEG C of annealing, extend 15s, 35 × Cycles.
(6) 1% Ago-Gel is run, the positive bacterium of PCR identifications selects 3 to send sequencing, and sequencing primer is U6 promoter.
(7) the selection correct bacterium solution of sequencing result is small shakes upgrading grain.
1.1.3 the preparation of sgRNA in-vitro transcriptions template
(1) DNA fragmentation (about 120bp) of high-fidelity enzyme PCR amplification codings sgRNA sequences, every sgRNA is identical with 2 pipes System
Mix, PCR reactions：98 DEG C of thermal startings, 58 DEG C of annealing, extend 12s, 35 × Cycles.
(2) match somebody with somebody 1% Ago-Gel, run glue, cut bright band size 120bp or so blob of viscose, useGel Extraction kits reclaim DNA.
A. blob of viscose is weighed, and 100mg is estimated as 100 μ L, adds the Buffer QG of 3 times of colloid products.
B.65 DEG C baking oven places 10min until blob of viscose melts.
C. the isopropanol of 1 times of colloid product is added, overturns and mixes.
D. mixture is transferred to adsorption column, 13000rpm centrifugation 1min abandon filtrate.
E. 500 μ L Buffer QG, 13000rpm centrifugation 1min are added, filtrate is abandoned.
F. 750 μ L Buffer PE, 13000rpm centrifugation 1min are added, filtrate is abandoned.
G. repeat step f.
H. blank pipe centrifuges 2min.
I. adsorption column is transferred to clean 1.5mL imports centrifuge tube, uncap drying 5min in RNA super-clean benches.
J. the RNase-free ddH of 30 μ L, 65 DEG C of preheatings are added to adsorbed film center2O, is stored at room temperature 5min.
K.13000rpm centrifugation 2min elutions, suction out 2 μ L and survey concentration (DNA50).
1.1.4 sgRNA in-vitro transcription
(1) (complete) to use MEGAshortscript in RNA super-clean benches belowTMT7 transcript reagent boxes, with external Responsive transcription system
Mix, 37 DEG C of reaction 4h.
(2) 0.5 μ L TURBO DNase are added, are mixed, 37 DEG C of reaction 15min.
(3) system is transferred to 0.6mL tubules, adds 125 μ L RNase-free ddH2O and 15 μ L Ammonium Acetate Stop Solution, are mixed.
(4) isometric lower floor's phenol chloroform is added, overturns and mixes 10min, room temperature 12000rpm centrifugations 10min.
(5) upper strata aqueous phase is transferred to new pipe, plus isometric chloroform, overturns and mix 10min, room temperature 12000rpm centrifugations 10min。
(6) upper strata aqueous phase is transferred to new pipe, plus 2 times of volume absolute ethyl alcohols, mixed, -20 DEG C of placement 30min.
(7) match somebody with somebody 2% Ago-Gel, use RNase-free ddH2O matches somebody with somebody 75% ethanol, 4 DEG C of centrifuges of precooling.
(8) 4 DEG C of 12000rpm centrifuge 15min.
(9) supernatant is abandoned, a precipitation is rinsed with the ethanol of 500 μ L 75%, 4 DEG C of 12000rpm centrifuge 5min.
(10) abandoning drying to the edge of precipitation of being uncapped in most supernatant, RNA platforms tends to be transparent.
(11) precipitation size is regarded, 50~80 μ L RNase-free ddH are added2O is dissolved, and 2~3min is placed on ice.
(12) mix, stay 3 μ L, remaining 6 μ L/ pipes packing is frozen in -80 DEG C of refrigerators.
(13) concentration (ssDNA33) is surveyed, 260/280 (2.0~2.2 be normal) is recorded.
(14) 1 μ L are taken to run 2% Ago-Gel, 130V 8min check whether band brightness is consistent with concentration, and band is big It is small whether in 100bp or so.
1.1.5 Cas9 mRNA in-vitro transcriptions
(1) single endonuclease digestion pSpCas9 (BB) (pX330；Addgene plasmid ID:42230) plasmid, is used as in-vitro transcription Template
Mix, 37 DEG C of reactions are stayed overnight.
(2) match somebody with somebody 1% Ago-Gel, run glue, blob of viscose where cutting linearisation DNA is usedGel Extraction kits are reclaimed.
(3) mMESSAGE is usedT7 transcript reagent box in-vitro transcription Cas9 mRNA.
1. (complete) to match somebody with somebody external responsive transcription system in RNA super-clean benches below
Mix, 37 DEG C of reaction 2h.
2. 1 μ L TURBO DNase are added, are mixed, 37 DEG C of reaction 15min.
3. with addition of the reaction system of polyA tails
Mix, suction out 2.5 μ L and preserve on ice.
4. 4 μ L E-PAP Mix are added, are mixed, 37 DEG C of reaction 30min are put on ice.
5. 10 μ L Ammonium Acetate Stop Solution are added, are mixed.
6. phenol chloroform method carries out RNA purifying, and step is same as 1.1.4 (4)~(10).
7. precipitation size is regarded, Nuclease-free water dissolvings is added, 2~3min is placed on ice.
8. mix, take 1.5 μ L to survey concentration (RNA40), 260/280 is normal 2.0 or so, and remaining mRNA is temporarily put in ice On.
9. basis measures concentration and mRNA is diluted into about 1 μ g/ μ L, takes 1 μ L and adds 2.5 μ L before polyA tails to run 2% fine jade Sepharose, should have under normal circumstances plus before polyA tails the band after obvious bright band, plus polyA tails should it is bigger (may slightly mould Paste, does not influence to use).
If 10. concentration, the cementing fruit of race are normal, often pipe is dispensed 1 μ g, is frozen rapidly in -80 DEG C of refrigerators, is obtained Cas9 mRNA。
1.2 MARF1-sgRNA efficiency and Cas9 mRNA quality testings
In once testing, 1 quality for determining efficient sgRNA detection Cas9 mRNA can be used, it is also possible to high-quality Cas9 mRNA detect 1 sgRNA efficiency.
1.2.1 the acquisition of mouse fertilized egg
Embryo for Efficiency testing is typically derived from BDF1 Strains of Mouse, and it is inbred strais C57BL/6J and DBA/2 mouse The first generation filial generation that post-coitum is cultivated, sexal maturity in 6~8 weeks.The mouse used is raised in no-special pathogen (specific-pathogen-free,SPF)。
(1) 0d, 13:00, capture the week old BDF1 female mices of 8 weeks or so, intraperitoneal injection 50U/mL (about 60 μ L) pregnant horse blood Clear promoting sexual gland hormone (pregnant mare ' s serum gonadotropin, PMSG), imitates endogenous follicle-stimulating hormone The promotion maturation of ovum effect of (follicle-stimulating hormone, FSH).
(2) 2d, 13:00, PMSG BDF1 female mices, intraperitoneal injection 50U/ml (about 70 μ L) human chorionic were injected in crawl Promoting sexual gland hormone (human chorionic gonadotropin, hCG), imitates luteotropin (luteinizing Hormone, LH) induced ovulation effect.And every female mice is individually mated with the public mouse of 1 BDF1 mated.
(3) G1 Embryo Culture drops are done：35mm Petri culture dishes bottom, about 20 μ L/ drops, proper alignment is finally used Mineral oil seal liquid level, 37 DEG C, 5%CO2Balance is stayed overnight in incubator.
(4) 3d, 8:00, check female mice whether there is vaginal plug (the secretion vestige that public mouse post-coitum leaves), if any then taking out Mouse room.
(5) CZB Embryo Culture drops, 37 DEG C, 5%CO are done215min is at least balanced in incubator.
(6) cervical dislocation puts to death female mice：Mouse is placed on the brandreth of mouse cage, its fore paw is caught steel wire above Bar, tightly pushes down its neck (after skull), while level stretches afterbody backward, makes its disconnected neck.
(7) mouse back of execution is put in Enamel jar upward, sprays mouse back with 75% alcohol immediately, subtract as far as possible The pollution of few mouse hair.
(8) mouse back skin is pinched, an osculum is cut off in Zhong Xiaqu with surgery scissors, firmly catches the skin cut off Skin, makes skin of back open completely, hair also leaves interior tissue completely to head and afterbody stretching.
(9) open notacoria with ophthalmic tweezers and sharp scissors, white adipose tissue picked up with tweezers, lightly uterus, Fallopian tubal, ovary and fat pad integrally pull out body cavity, and the uterus below ovary is clamped with tweezers, with scissors close to fallopian tubal A hole is pierced on mesometrium, mesometrium is cut off, then ovary is separated with fallopian tubal with scissors (ensures the complete of fallopian tubal Property), finally cut close to the uterus of fallopian tubal, fallopian tubal is put into HCZB manipulation in vitro liquid.
(10) ampulla that fallopian tubal expands is punctured with the needle point of syringe under anatomical lens, cumulus cell parcel is obtained Embryonated egg group.
(11) all agglomerates are collected, in 0.1% hyaluronidase after 37 DEG C of 3~5min of digestion, embryonated egg is thin with ovarian cumulus Born of the same parents separate.
(12) calcination glass capillary draws pin and broken needle, about 150~200 μm of internal diameter, assembling on the flame of alcolhol burner Mouth suction pipe.Ovum is moved with mouth suction pipe, the embryonated egg separated washes 6 times in HCZB drops, wash away the cumulus cell of residual and transparent Matter acid enzyme.
(13) embryonated egg is moved into and washed 3 times in CZB drops, 37 DEG C, 5%CO2Recover in incubator.
1.2.2 sgRNA and Cas9 mRNA mixtures are prepared
Mixture is prepared in RNA platforms.The Cas9 mRNA and 50ng/ μ L that mixture for detection efficiency is 100ng/ μ L SgRNA.General injection need to prepare 5 μ L mixtures
Cas9 mRNA 500ng
sgRNA 250ng
ddH2O is mended to 5 μ L
4 DEG C of 10000rpm centrifuge 10min, and procaryotic injection is into about 20 pieces of embryonated eggs.
1.2.3 the detection of sgRNA cleavage sites
A. the cracking of embryo
(1) embryonated egg is cultivated 3 days in G1 nutrient solutions, is developed to after blastaea and is cracked 5 μ L G1 of embryo's immigration with mouth suction pipe In liquid.
(2) 95 DEG C of 20min cracking embryo's released dnas in PCR instrument.
(3) 5 μ L G2 solution are added, mixes, pcr template can be used as.
B.PCR is expanded
(1) with the high primer of NCBI Primer-BLAST tool designs specificity, PCR primer is mouse gene group DNA The upper 500bp comprising sgRNA sites or so fragment, sgRNA sites should try one's best in fragment center.
(2) primer specificity is tested with common mouse gene group DNA, thermograde PCR is to obtain the annealing of primer most preferably Temperature.
(3) first round PCR reacts
Mix, PCR reactions：94 DEG C of thermal startings, extend 36s, 30 × Cycles.
(4) second wheel PCR reactions, each sample matches somebody with somebody 2 pipe same systems；Simultaneously with 2 pipes with wild type (wild type, WT) mouse gene group DNA is the system of template
Mix, PCR reactions：95 DEG C of thermal startings, extend 36s, 35 × Cycles.
(5) match somebody with somebody 1% Ago-Gel, run glue, cut the correct blob of viscose of band, reclaimed using DNA purifying QIAquick Gel Extraction Kits DNA。
1. blob of viscose is weighed, and 100mg is estimated as 100 μ L, adds the Buffer PC of equimultiple colloid product.
2. 65 DEG C of baking ovens place about 10min until blob of viscose melts.
3. equilibrium adsorption post：500 μ L Buffer BL, 13000rpm centrifugation 1min are added, filtrate is abandoned.
4. sol solutionses are added in adsorption column, are stored at room temperature 5min, 13000rpm centrifugation 1min, abandon filtrate.
5. 600 μ L Buffer PW, 13000rpm centrifugation 1min are added, filtrate is abandoned.
6. repeat step is 5..
7. blank pipe centrifuges 2min.
8. adsorption column is transferred to clean 1.5mL centrifuge tubes, uncap drying 5min in super-clean bench.
9. the Buffer EB of 30 μ L, 65 DEG C of preheatings are added to adsorbed film center, 5min is stored at room temperature.
10. 13000rpm centrifuges 2min elutions, suctions out 1.7 μ L and surveys concentration (DNA50).
C. glue returns the T7 digestions experiment of product
(1) annealing reaction system is matched somebody with somebody
250~300ng of glue reclaim product DNA
NEB Buffer 2 1.5μL
ddH2O is mended to 14.5 μ L
Mix, denaturation, annealing reaction in PCR instrument：95 DEG C of 5min, 0.1 DEG C/s slow coolings to 25 DEG C, 25 DEG C of 10min.
(2) 2% Ago-Gel is matched somebody with somebody.
(3) 0.5 μ L T7 restriction endonucleases I are added to DNA annealing systems, mixed, 37 DEG C of 15~17min of reaction in PCR instrument.
(4) 10 × Loading Buffer are rapidly added, are mixed, 135V voltages run glue 30min, result are checked, if removed There is obvious band less than normal outside about 500bp master tapes, and WT is without then illustrating the piece that mutation is contained in virgin rubber recovery product Section, corresponding sgRNA may have cutting to cause mutation.
D. sequencing vector is connected
(1) positive glue is tested into T7 digestions and returns fragment connection sequencing vector, there are two kinds of commercial vectors can use.
a.pLB vector
1. end-filling reaction system is matched somebody with somebody
Mix, 20 DEG C of 5min, 70 DEG C of 5min in PCR instrument, it is of short duration on ice.
2. coupled reaction system is matched somebody with somebody
The μ L of filling-in product 4
pLB vector 0.5μL
T4Ligase 0.5μL
Mix, room temperature places 10min, all to E. coli competent, coated plate.
b.Zero Cloning
With coupled reaction system
Glue reclaim product 25ng
T5 Zero Cloning Vector 1μL
ddH2O is mended to 4 μ L
Mix, room temperature places 15min, is totally converted to E. coli competent, coated plate.
(2) bacterium, plus 200 μ L 2 × YT culture mediums (Amp are chosen after 16h+) shake bacterium 2h, bacterium solution PCR
Mix, PCR reactions：95 DEG C of thermal startings, extend 36s, 35 × Cycles.
(3) 1% Ago-Gel is run, the positive bacterium of PCR identifications selects 8 to send sequencing, and sequencing primer is T7 promoter. Result is checked, the result being mutated comprising insertion/deletion (insertion/deletion, indel) accounts for the proportional representation of sum Corresponding sgRNA efficiency.
(4) for the sample of T7 digestion negatives, glue is directly returned into fragment and send sequencing, the peak figure of sequencing is checked, if All it is single peak before sgRNA cleavage sites, and starts to ending have set peak in sgRNA vicinity, then the glue returns product The fragment (false negative is tested in T7 digestions) of mutation is may included, the presence that sequencing vector confirms mutation is attached.
1.3 Donor DNA design and the procaryotic injection of MARF1 point mutation mouse
1.3.1 Donor DNA design
It is by sgRNA1 PAM regions 5 ' between extreme direction 3-4bp centered on cutting position, respectively to 5 ' extreme direction 65bp Left side homology arm, 3 ' extreme direction 65bp is right side homology arm, orders single-stranded ssDNA (raw work, Shanghai China).The single-stranded ssDNA Purity have to using HPLC purifying.
MARF1-272-sgRNA-1, MARF1-272-sgRNA-2 cutting efficiency, which compare, sees Fig. 4.
MARF1-272donor DNA design collection of illustrative plates is shown in Fig. 5.
1.3.2 donor DNA, sgRNA and Cas9 mRNA mixtures are prepared
Mixture is prepared in RNA platforms.The Cas9 mRNA and 50ng/ μ L that mixture for detection efficiency is 100ng/ μ L SgRNA, 100ng/ μ L donor DNA.General injection need to prepare 5 μ L mixtures
4 DEG C of 10000rpm centrifuge 10min, and kytoplasm is injected in about embryonated egg.
1.3.3 procaryotic injection
The acquisition of embryonated egg, and procaryotic injection have been described, ibid, see sgRNA efficiency and Cas9 mRNA quality testings.
1.3.4 embryo transfer
A, the preparation of the public mouse of ligation
(1) the public mouse of 6-8 week old is anaesthetized by 100ml/kg dosage after weighing, mouse is fixed in back of the body position, and 75% ethanol, which is applied, to be put on the skin Sterile surgical cutting part, to prevent hair contamination of incision.
(2) about 1.3cm places row cuts off lower abdomen skin in front of the genitals, work about 1cm otch, with soaking 75% second The gauze of alcohol is wiped over cutting part to clear up hair.
(3) peritonaeum is cut, bladder positioning is carried out.There is a pipeline traveling per side is all visible, left side is gently clamped with tweezers Pipeline, lifting part makes surgical field of view high-visible, determines that it is vas deferens.
(4) vas deferens is pinched into tweezers it is U-shaped, it is another that tweezers are heated on alcolhol burner, scald disconnected defeated between 2 ligation points Spermaduct, the vas deferens of ligation is put into and carries out opposite side ligation again in vivo.
(5) after both sides operation is finished, inside and outside art mouthful is sutured with 0-5 sutures.Mouse is put on 37 DEG C of thermostatic platforms directly To revival.Post operation Mouse feeder determines whether operation succeeds after 2 weeks.
(6) it is experimental to raise：1-2 dams and vasectomy mouse are mated into raising, secondary morning carries out vaginal plug inspection. The dams for having vaginal plug, which can be made of bolt under smear, microscope, has seen whether sperm.Also it can cultivate to next day and take fallopian tubal inspection Whether two cell stages are had.If sperm or there are two cell stages then public mouse ligation is unsuccessful.
B, the preparation of acceptor mouse
(1) while selecting the ICR or CD-1 of heat and the public mouse of ligation mates.
(2) m seq checks whether donor mouse and acceptor mouse see bolt, and embryo transfer is used on the day of seeing bolt or after 2.5.
C, common oviduct transplantation
(1) operating theater instruments, suture of sterilization etc. are got out.
(2) implement the anesthesia of 1.25% avertin solution to mouse by 200ml/kg dosage after weighing, cut off mouse back It is about concordant with last 1 rib to locate in backbone side 1cm after hair, 75% alcohol disinfecting, skin is cut off, with tweezers along notch bluntness Integumentary musculature is separated, ovary, fallopian tubal and uterus are gently pulled out with tweezers, partial fat is clamped, is placed on the skin of mouse behind.
(3) embryonated egg survived after 20 pieces of injections is sucked with transplanting pin, suction order is:M16 nutrient solution ＆ Kong Qis ＆M16 is trained Nutrient solution ＆ air ＆ embryonated eggs, in the case where ensureing that embryo is fully entrained, reduce the soakage of nutrient solution as far as possible.
(4) mouse is placed under anatomical lens, fat pad is pinched with small size haemostatic clamp, the film of ovary will be wrapped up with ophthalmology tweezers An osculum is torn, fimbriae tubae portion is exposed, insertion moves ovum pin, embryonated egg is blown into fallopian tubal, ampulla of uterine tube has bright Aobvious bubble shows to transplant successfully.
(5) fat pad, ovary, uterus and fallopian tubal are sent back to abdominal cavity, peritoneal suture and skin incision in the lump with blunt tweezers, The acceptor mouse transplanted is placed to be transplanted in barrier environment after 10min recovers on warm platform and raised.
(6) mouse after transplanting makes marks, and indicates transplant time, family name, embryo's quantity.Date of birth is indicated after farrowing Phase and birth quantity, the survival condition of the young baby of observation birth daily.
1.4.MARF1 the genotype identification of point mutation mouse
After RNA injects embryonated egg and is transplanted in false pregnancy dams fallopian tubal, general 19~20 days fertility mouse.Mouse is born After 6 days, separated between its toe, toe can be cut and make numbering mark, cutting tail point fritter tissues is used for the extracting of genomic DNA.
1.4.1 the phenol chloroform of genomic DNA
(1) clip tissue is put into centrifuge tube, adds the rat-tail lysate that 300 μ L contain Proteinase K, and 65 DEG C of cracking are stayed overnight.
(2) 300 μ L phenol chloroforms are added, overturns and mixes 10min, room temperature 12000rpm centrifugations 10min.
(3) it is careful to draw supernatant and move to new pipe, the absolute ethyl alcohol of two volumes is added, mixing is overturned visible cotton-shaped heavy Form sediment, 4 DEG C of 12000rpm centrifuge 5min.
(4) supernatant is abandoned, the ethanol of 500 μ L 70% rinsing precipitation is added, 4 DEG C of 12000rpm centrifuge 5min.
(5) most supernatant is abandoned, super-clean bench dries up 5min to bottom precipitation bleach, depending on precipitation size, 65 DEG C of preheatings of addition ddH2O。
(6) room temperature, which places 5min, makes DNA fully dissolve.
(7) overturn and mix, suction out 2 μ L and survey concentration (DNA50).
1.4.2 the PCR amplifications comprising prediction mutational site fragment
(1) identical when the primer used is with sgRNA Efficiency testings, each DNA sample regards primer efficiency and prepares 2~3 pipes Reaction system
(2) glue reclaim, the same 3.2.3B. of step (5).
1.4.3 the direct StyI digestions experiment of Genomic PCR products and sequencing confirm
(1) the direct StyI digestions experiment of PCR primer：
(2) 37 degree, water-bath, digestion 3h, electrophoresis.
(3) PCR primer direct Sequencing.
Due to the change of rite-directed mutagenesis, in the event of accurate restructuring, it will one restriction enzyme site StyI of increase.Wild type Mouse can not then be cut open.The mouse that the present invention is born, #1 can be by StyI digestions, but #2, #3, #4, #5 cannot It is digested.See Fig. 6 in detail.Occur accurate restructuring, sequencing it can be found that the point mutation position meeting of restructuring to further confirm that Appearance one is significant bimodal.And the mouse of wild type is not in then bimodal.The mouse that the present invention is born, #1 has bimodal, but # 2, #3, #4, #5 be not bimodal.See Fig. 7 in detail.
1.4.4 StyI digestions experiment and sequencing confirm after PCR primer clone
After PCR primer is by QIANGEN kit glue reclaims, connected with PLB vector kits, clone chooses bacterium, Monoclonal, is bacterium solution PCR, filters out positive bacteria.On the one hand, PCR primer is directly used to StyI digestions, electrophoresis, monoclonal bacterial strain L5, L9 can be by StyI digestions, but monoclonal bacterial strain L1, L2, L3, L4, L6, L7, L8 cannot be shown in Fig. 8 by StyI digestions. On the other hand, monoclonal positive bacteria is sent into company's sequencing, sequencing result is shown in Fig. 9.
The application infertile as genetic model research female is stablized of 1.5 MARF1 point mutation mouse
1.5.1 MARF1 point mutation female mice is infertile, and egg mother cell is unable to normal development
As shown in Figure 10.MARF1 point mutation oocyte of mouse is arrested in germ-vesicle (GV) period, it is impossible to recover subtrahend Division, it is impossible to normal mature.Further result in female infertile.
1.5.2 MARF1 point mutation oocyte of mouse series Testing index RNA significantly rises
As shown in figure 11.Substantial amounts of Testing index RNA significantly rises in MARF1 point mutation oocyte of mouse, and normal Substantial amounts of Testing index RNA maintains reduced levels in egg mother cell.These can be pre-natal diagnosis, the third generation " test-tube baby " PGD (preimplantation genetic diagnosis), that is, transplant before gene diagnosis, the progress of auxiliary procreation technology with And contraceptive exploitation provide can stablize heredity effective research platform.
SEQUENCE LISTING
<110>Nanjing Medical University
<120>A kind of construction method and application that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9
<130>
<160> 10
<170> PatentIn version 3.3
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Claims (6)

1. a kind of construction method that MARF1 rite-directed mutagenesis mouse models are obtained based on CRISPR/Cas9, its feature includes 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, confirms that the genetic mouse of the MARF1-D272A point mutation finally given is dashed forward Varying model.
Exist 2. what construction method described in claim 1 was 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 was obtained obtains MARF1 rite-directed mutagenesis mouse models based on CRISPR/Cas9 Application in screening preventing and treating female infertility or female genital disorders medicine.
4. obtain the kit of MARF1 rite-directed mutagenesis mouse models, it is characterised in that containing as shown in SEQ ID NO.1 sgRNA。
5. kit according to claim 4, it is characterised in that also containing the Donor DNA sequences shown in SEQ ID NO.2 Row.
6. kit according to claim 5, it is characterised in that also containing Cas9 mRNA.
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