CN110305909A - The method for constructing sterile mouse model based on CRISPR-Cas9 system - Google Patents
The method for constructing sterile mouse model based on CRISPR-Cas9 system Download PDFInfo
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- 238000002347 injection Methods 0.000 claims abstract description 11
- 239000007924 injection Substances 0.000 claims abstract description 11
- 108091033409 CRISPR Proteins 0.000 claims abstract description 6
- 238000010363 gene targeting Methods 0.000 claims abstract description 6
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 4
- 238000000520 microinjection Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 16
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 11
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Abstract
The invention belongs to genetic engineering fields, and in particular to the method for constructing sterile mouse model based on CRISPR-Cas9 system.The method that sterile mouse model is constructed based on CRISPR-Cas9 system of the invention, comprising the following steps: determine mouse Piwil1 gene targeting site sequence；Prepare gRNA；External microinjection: collecting mouse fertilized egg, gRNA and Cas9 RNA injection is entered to the fertilized eggs of mouse, carries out transplant to the fertilized eggs of injection.The present invention constructs MIWI D633A mutant mouse using CRISPR-Cas9 system, the sub- sexuality ability of male heterozygosis is normal, there are a certain number of sperms, it can mate with female mice, normally form smart bolt, but it can lead to infertility, mutant mouse constructed by the present invention can be used for the preparation of genetic engineering mouse.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to construct sterile mouse model based on CRISPR-Cas9 system
Method.
Background technique
Mouse is one of model animal important in clinic study, has been widely used in life science and biology doctor
In research.It is Chinese to use about 10,000,000 or more every year.Currently, the genetic engineering technology based on mouse mainly passes through false pregnancy
Mouse transplants xenogenous fertilization ovum.This technology need to prepare the public mouse of ligation in advance, and test ligation effect, can use.
Piwil1 (piwi-like RNA-mediated gene silencing 1) is reproduction cell specifically expressing base
Cause is mainly expressed in male sex-cell.Combine to PIWI protein specific a kind of non-volume of small molecule for being referred to as piRNA
Code rna regulation, forms piRNA/PIWI function and service object, by transposable element in silencing germ cell and regulation it is other under
Target RNA molecule is swum, germ cells gene group is maintained to stablize, it is required to break up for animals' reproduction cell development.Research card both at home and abroad
Real, this gene mutation can lead to male mouse infertility.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for constructing sterile mouse model based on CRISPR-Cas9 system.
The method that sterile mouse model is constructed based on CRISPR-Cas9 system of specific embodiment according to the present invention,
Method the following steps are included:
(1) mouse Piwil1 gene targeting sequence is determined；
(2) sgRNA is prepared；
(3) cas9mRNA is prepared；
(4) external microinjection: collecting mouse fertilized egg, and gRNA and Cas9RNA injection is entered to the fertilized eggs of mouse；
(5) transplant is carried out to the fertilized eggs of injection.
The method that sterile mouse model is constructed based on CRISPR-Cas9 system of specific embodiment according to the present invention, step
Suddenly in (1), mouse Piwil1 gene targeting site sequence as shown in SEQ ID No.1,
SEQ ID No.1:
GTTACCATGACACCACAGCT
Specific embodiment according to the present invention, the nucleotide sequence of wild type Piwil1 gene such as SEQ ID No.2 institute
Show:
ATGACTGGCCGAGCCCGAGCTCGGGCCCGCGGCAGGGCACGAGGTCAGGAGACGGTGCAGCATGTTGGG
GCTGCTGCGAGCCAGCAACCTGGGTACATCCCACCGAGACCTCAACAGTCCCCCACAGAGGGGGACTTGGTTGGCCG
AGGACGACAGAGGGGGATGGTAGTCGGAGCCACATCCAAGTCACAAGAACTGCAGATCTCAGCTGGGTTTCAGGAGC
TGTCACTGGCAGAGAGAGGAGGGCGTCGCCGAGACTTCCATGACCTTGGTGTGAACACCAGACAGAACCTTGACCAT
GTCAAAGAGTCAAAGACAGGCTCCTCTGGCATCATTGTGAAGCTGAGCACCAACCACTTCCGGCTGACCTCGCGCCC
ACAGTGGGCCCTGTATCAGTACCACATCGACTACAATCCCCTGATGGAGGCCCGAAGGCTTCGCTCCGCACTGCTCT
TCCAGCATGAAGACCTCATTGGAAGGTGTCATGCTTTCGATGGGACAATATTGTTTTTACCTAAGAGACTACAGCAC
AAGGTCACAGAAGTATTCAGTCAGACTCGGAATGGGGAACACGTGAGGATCACCATCACCCTGACCAACGAGCTGCC
GCCCACCTCGCCCACCTGCCTGCAGTTCTATAACATCATCTTCAGGAGGCTCTTGAAAATCATGAATTTGCAACAAA
TTGGACGGAATTATTACAATCCAAGTGACCCGATTGATATTCCAAACCACAGGTTGGTGATCTGGCCCGGCTTCACC
ACCTCCATCCTTCAGTATGAGAACAACATCATGCTCTGCACAGACGTCAGCCACAAGGTGCTCCGCAGCGAGACTGT
CCTAGACTTCATGTTCAATCTATACCAGCAGACAGAGGAGCACAAGTTCCAGGAGCAAGTGTCGAAGGAGCTCATAG
GCCTCATCGTTCTCACCAAGTACAATAACAAGACCTACCGGGTGGATGACATTGACTGGGACCAGAATCCAAAGAGC
ACCTTCAAGAAGGCGGATGGCTCGGAGGTCAGCTTCCTGGAGTACTACAGGAAGCAATACAACCAGGAGATCACGGA
CCTGAAGCAGCCGGTGCTGGTGAGCCAACCCAAGCGGAGGAGAGGCCCCGGCGGCACCCTGCCTGGCCCAGCTATGC
TCATCCCTGAACTCTGCTATCTCACAGGCCTGACTGATAAAATGCGCAATGATTTCAATGTGATGAAGGACCTGGCA
GTGCACACGCGGCTGACCCCTGAGCAGCGGCAGCGGGAGGTGGGCCGCCTCATCGACTACATCCACAAGGATGACAA
TGTGCAGAGAGAGCTTCGAGACTGGGGCCTGAGCTTCGACTCAAACTTGCTGTCCTTCTCTGGAAGAATCTTACAAT
CTGAGAAGATCCACCAGGGCGGAAAGACGTTTGATTACAACCCACAATTTGCAGACTGGTCCAAAGAAACAAGAGGC
GCGCCGCTGATCAGCGTGAAGCCATTGGATAACTGGCTGCTGATCTATACCCGCAGGAATTATGAAGCAGCCAACTC
ACTGATACAGAACCTGTTCAAAGTGACTCCAGCCATGGGCATCCAGATGAAAAAGGCAATCATGATCGAGGTGGATG
ACAGAACAGAAGCTTATCTGAGAGCCTTGCAGCAGAAGGTGACGTCAGACACTCAGATAGTTGTCTGTCTCTTGTCA
AGTAATCGGAAGGACAAATATGATGCCATCAAGAAGTACTTGTGTACAGACTGCCCCACCCCAAGTCAGTGTGTGGT
GGCCCGGACCCTGGGCAAGCAGCAAACAGTCATGGCCATTGCCACCAAGATCGCCCTGCAGATGAACTGCAAGATGG
GAGGCGAGCTCTGGCGGGTGGACATGCCCCTGAAACTGGCAATGATCGTGGGCATCGACTGTTACCATGACACCACA
GCTGGGCGGAGGTCCATCGCAGGATTCGTCGCCAGCATCAATGAAGGGATGACCCGCTGGTTCTCCCGCTGCGTCTT
TCAGGACCGCGGGCAGGAGCTGGTGGATGGTCTCAAGGTGTGCTTGCAAGCTGCTCTGAGGGCTTGGAGTGGCTGCA
ATGAATACATGCCCAGCCGTGTCATCGTGTACCGAGACGGTGTGGGGGACGGGCAGCTGAAGACCCTGGTCAATTAT
GAGGTCCCACAGTTCCTAGATTGCCTCAAGTCAGTCGGGAGAGGTTACAACCCAAGACTGACTGTAATCGTGGTGAA
GAAGCGTGTCAATGCCAGGTTTTTTGCTCAGTCTGGGGGAAGACTTCAGAACCCTCTTCCAGGGACAGTCATCGATG
TGGAAGTCACCAGACCAGAGTGGTATGACTTTTTCATCGTGAGCCAGGCAGTGAGAAGCGGGAGTGTGTCCCCAACA
CACTACAATGTCATCTATGACAGCAGTGGCCTGAAGCCCGACCACATCCAGCGGCTGACATACAAGCTCTGCCACGT
GTACTATAATTGGCCTGGAGTCATCCGAGTCCCTGCACCTTGCCAGTATGCACACAAGCTGGCCTTCCTCGTGGGCC
AGAGCATCCACAGAGAGCCAAACCTCTCCCTGTCCAACCGCCTCTACTACCTCTAA
The nucleotide sequence of Piwil1 gene is mutated as shown in SEQ ID No.3,
ATGACTGGCCGAGCCCGAGCTCGGGCCCGCGGCAGGGCACGAGGTCAGGAGACGGTGCAGCATGTTGGG
GCTGCTGCGAGCCAGCAACCTGGGTACATCCCACCGAGACCTCAACAGTCCCCCACAGAGGGGGACTTGGTTGGCCG
AGGACGACAGAGGGGGATGGTAGTCGGAGCCACATCCAAGTCACAAGAACTGCAGATCTCAGCTGGGTTTCAGGAGC
TGTCACTGGCAGAGAGAGGAGGGCGTCGCCGAGACTTCCATGACCTTGGTGTGAACACCAGACAGAACCTTGACCAT
GTCAAAGAGTCAAAGACAGGCTCCTCTGGCATCATTGTGAAGCTGAGCACCAACCACTTCCGGCTGACCTCGCGCCC
ACAGTGGGCCCTGTATCAGTACCACATCGACTACAATCCCCTGATGGAGGCCCGAAGGCTTCGCTCCGCACTGCTCT
TCCAGCATGAAGACCTCATTGGAAGGTGTCATGCTTTCGATGGGACAATATTGTTTTTACCTAAGAGACTACAGCAC
AAGGTCACAGAAGTATTCAGTCAGACTCGGAATGGGGAACACGTGAGGATCACCATCACCCTGACCAACGAGCTGCC
GCCCACCTCGCCCACCTGCCTGCAGTTCTATAACATCATCTTCAGGAGGCTCTTGAAAATCATGAATTTGCAACAAA
TTGGACGGAATTATTACAATCCAAGTGACCCGATTGATATTCCAAACCACAGGTTGGTGATCTGGCCCGGCTTCACC
ACCTCCATCCTTCAGTATGAGAACAACATCATGCTCTGCACAGACGTCAGCCACAAGGTGCTCCGCAGCGAGACTGT
CCTAGACTTCATGTTCAATCTATACCAGCAGACAGAGGAGCACAAGTTCCAGGAGCAAGTGTCGAAGGAGCTCATAG
GCCTCATCGTTCTCACCAAGTACAATAACAAGACCTACCGGGTGGATGACATTGACTGGGACCAGAATCCAAAGAGC
ACCTTCAAGAAGGCGGATGGCTCGGAGGTCAGCTTCCTGGAGTACTACAGGAAGCAATACAACCAGGAGATCACGGA
CCTGAAGCAGCCGGTGCTGGTGAGCCAACCCAAGCGGAGGAGAGGCCCCGGCGGCACCCTGCCTGGCCCAGCTATGC
TCATCCCTGAACTCTGCTATCTCACAGGCCTGACTGATAAAATGCGCAATGATTTCAATGTGATGAAGGACCTGGCA
GTGCACACGCGGCTGACCCCTGAGCAGCGGCAGCGGGAGGTGGGCCGCCTCATCGACTACATCCACAAGGATGACAA
TGTGCAGAGAGAGCTTCGAGACTGGGGCCTGAGCTTCGACTCAAACTTGCTGTCCTTCTCTGGAAGAATCTTACAAT
CTGAGAAGATCCACCAGGGCGGAAAGACGTTTGATTACAACCCACAATTTGCAGACTGGTCCAAAGAAACAAGAGGC
GCGCCGCTGATCAGCGTGAAGCCATTGGATAACTGGCTGCTGATCTATACCCGCAGGAATTATGAAGCAGCCAACTC
ACTGATACAGAACCTGTTCAAAGTGACTCCAGCCATGGGCATCCAGATGAAAAAGGCAATCATGATCGAGGTGGATG
ACAGAACAGAAGCTTATCTGAGAGCCTTGCAGCAGAAGGTGACGTCAGACACTCAGATAGTTGTCTGTCTCTTGTCA
AGTAATCGGAAGGACAAATATGATGCCATCAAGAAGTACTTGTGTACAGACTGCCCCACCCCAAGTCAGTGTGTGGT
GGCCCGGACCCTGGGCAAGCAGCAAACAGTCATGGCCATTGCCACCAAGATCGCCCTGCAGATGAACTGCAAGATGG
GAGGCGAGCTCTGGCGGGTGGACATGCCCCTGAAACTGGCAATGATCGTGGGAATAGCATGCTATCACGATACAACT
GCAGGTCGGAGGTCCATCGCAGGATTCGTCGCCAGCATCAATGAAGGGATGACCCGCTGGTTCTCCCGCTGCGTCTT
TCAGGACCGCGGGCAGGAGCTGGTGGATGGTCTCAAGGTGTGCTTGCAAGCTGCTCTGAGGGCTTGGAGTGGCTGCA
ATGAATACATGCCCAGCCGTGTCATCGTGTACCGAGACGGTGTGGGGGACGGGCAGCTGAAGACCCTGGTCAATTAT
GAGGTCCCACAGTTCCTAGATTGCCTCAAGTCAGTCGGGAGAGGTTACAACCCAAGACTGACTGTAATCGTGGTGAA
GAAGCGTGTCAATGCCAGGTTTTTTGCTCAGTCTGGGGGAAGACTTCAGAACCCTCTTCCAGGGACAGTCATCGATG
TGGAAGTCACCAGACCAGAGTGGTATGACTTTTTCATCGTGAGCCAGGCAGTGAGAAGCGGGAGTGTGTCCCCAACA
CACTACAATGTCATCTATGACAGCAGTGGCCTGAAGCCCGACCACATCCAGCGGCTGACATACAAGCTCTGCCACGT
GTACTATAATTGGCCTGGAGTCATCCGAGTCCCTGCACCTTGCCAGTATGCACACAAGCTGGCCTTCCTCGTGGGCC
AGAGCATCCACAGAGAGCCAAACCTCTCCCTGTCCAACCGCCTCTACTACCTCTAA
Compared with wild type Piwil1 gene order, mutation knocks in mouse Piwil1 gene order at+1891-1923
Original sequence GGC ATC GAC TGT TAC is replaced with GGa ATa gca TGc TAt CAc GAt ACa ACt GCa GGt
CAT GAC ACC ACA GCT GGG, wherein+1897-1899GAC is replaced by GCA, amino acid is replaced by aspartic acid
(Asp, D) becomes alanine (Ala, A), other to replace for degeneracy, destroys CAS9 target spot.
The method that sterile mouse model is constructed based on CRISPR-Cas9 system of specific embodiment according to the present invention, step
Suddenly in (2), Piwil1sgRNA1 and PX330TRAC R primer is designed, primer sequence difference is as follows:
Piwil1sgRNA1 primer:
ATAATACGACTCACTATAGgGTTACCATGACACCACAGCTGTTTTAGAGCTAGAAATAG；
PX330TRAC R primer:
AAAAGCACCGACTCGGTGCC。
The method that sterile mouse model is constructed based on CRISPR-Cas9 system of specific embodiment according to the present invention, step
Suddenly in (2), using Piwil1sgRNA1 and PX330TRAC R primer amplification PX330 plasmid, template DNA is obtained, then poly- by T7
Synthase synthesizes TK sgRNA.
The method that sterile mouse model is constructed based on CRISPR-Cas9 system of specific embodiment according to the present invention, step
Suddenly in (3), upstream primer CAS9-T7F and downstream primer CAS9-T7R is designed, primer sequence difference is as follows:
CAS9-T7F:
GAAATTAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC；
CAS9-T7R:
GCGAGCTCTAGGAATTCTTAC。
Beneficial effects of the present invention:
The sgRNA of the selectively targeted mouse Piwil1 gene of the present invention can accurately target Piwil1 gene, and realize
Gene site-directed insertion, sgRNA targeting is good, and the knockout of CRISPR-Cas9 system is high-efficient.
The present invention constructs MIWI D633A mutant mouse using CRISPR-Cas9 system, and display female can be male, male
Sex heterozygote sexuality ability is normal, there is a certain number of sperms, can mate with female mice, normally forms smart bolt, but can lead to not
It educates.Make sterile male mouse with this, with normal female mice with numerous, pseudopregnant mouse can be prepared, embryo transfer efficiency and ligatures male mouse preparation
Pseudopregnant mouse is identical.Therefore, constructed MIWI D633A mutant mouse can be used for the preparation of genetic engineering mouse, the skill
Art be also applied to some species for being difficult to prepare ligation buck or can not telogenesis essence bolt species genetic engineering mode it is raw
Preparation of object, such as rat, Golden Hamster or cavy etc..
Detailed description of the invention
Fig. 1 shows that the 17th exon of Piwil1 gene is gene editing target spot.
Specific embodiment
The type and raising of experimental animal
Experiment mice of the invention comes from the long-living biotechnology Fu Fen in Liaoning Co., Ltd, and raises in the said firm；Raising
Condition barrier environment, using 22-26 DEG C of temperature, humidity 40-60RH%, light application time 10:14.Experimental implementation obtains long-living biology
Experimental animal Ethics Committee examines and supervision.
The building of embodiment 1:CRISPR/Cas9 system
1.1 design mouse Piwil1 gene specific target practice sequences
In the 17th exon of mouse Piwil1 gene, designs gene specific target practice DNA and identify sequence, carry out sequence of missing the target
Column analysis.
Mouse Piwil1 gene targeting sequence is GTTACCATGACACCACAGCT.
Mouse Piwil1 gene D633A mutant nucleotide sequence is
tttTCACTAAAGCTGAAACTGGCAATGATCGTG GGa ATa gca TGc TAt CAc GAt ACa ACt
GCa GGt CGGAGGTCCATCGCAGGATTCGTCGCCAGCATCAA。
The preparation of 1.2Piwil1sgRNA
Piwil1sgRNA1 primer and PX330TRAC R primer are designed, directly amplification PX330 plasmid, obtain template DNA,
Again by T7 polymerase, TK sgRNA is synthesized.It is specific as follows:
Primer component: specific oligonucleotide sequence CRISPRF, the sequence include T7 promoter, sgRNA sequence and part
SgRNA skeleton part；
Piwil1sgRNA1 primer:
ATAATACGACTCACTATAGg GTTACCATGACACCACAGCT GTTTTAGAGCTAGAAATAG
PX330TRAC R primer:
AAAAGCACCGACTCGGTGCC。
Primer Piwil1sgRNA1, PX330TRAC R make 100 μ L PCR reaction systems, size 127bp, reaction respectively
Condition is (94 DEG C of 30s, 30cycles (94 DEG C of 20s, 58 DEG C of 30s, 72 DEG C of 20s), 72 DEG C of 10min, 4 DEG C of ∞).PCR product is direct
With PCR product Purification Kit DNA, the i.e. template DNA of sgRNA.
Transcript reagent box MEGAshortscriptTM(No.1345) it is transcribed in vitro.SgRNA is purified with ethyl alcohol after reaction
No RNase water dissolution makes 1 μ g/ μ L of its concentration.- 80 DEG C are stored in, for injecting.
1.3 preparation Cas9mRNA
Upstream primer CAS9-T7F and downstream primer CAS9-T7R, the primer sequence for designing the sequence containing T7 are as follows:
CAS9-T7F:
GAAATTAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC；
CAS9-T7R:
GCGAGCTCTAGGAATTCTTAC。
Make 100 μ L PCR reaction systems, size 4269bp, reaction condition be (94 DEG C of 30s, 30cycles (94 DEG C of 30s,
58 DEG C of 30s, 72 DEG C of 5min), 72 DEG C of 10min, 4 DEG C of ∞).PCR product agarose electrophoresis cuts glue purification, mMESSAGE
mMACHINETMThe transcription of T7ULTRA (Ambion, AM1345) kit, transcription product LiCl purify mRNA.It is stored in -80
DEG C, for injecting.
Embodiment 2: building Piwil1 D633A mouse
2.1 obtain Piwil1 fertilized eggs
3-4 week old ICR female mice is chosen, the 9 points one morning, is injected intraperitoneally pregnant mare serum gonadotrop(h)in (PMSG) (PMSG)
The super row of (15IU/100g) induction.3rd day morning HCG injection, at 4 points in afternoon, mates with male mouse and mates.2nd day 9 points of the morning,
Examine bolt.The female mice of mating, is euthanized with yellow Jackets.
Fallopian tubal is taken, fertilized eggs are collected in M2 culture solution, M2 cultivates fertilized eggs.100 pieces of fertilized eggs are directly used in micro-
Injection.In vitro operation is completed in 30 minutes.
2.2 protokaryons (PN) injection
20ng/ μ L sgRNA1,100ng/ μ L ssDNA and 50ng/ μ L cas9mRNA PN injection, fertilized eggs are straight after injection
It connects and is implanted into replace-conceive mouse rat body.
The 2.3 fertilization implantation of ovums
Replace-conceive mouse mates with male mouse on the same day for ovum mouse.2nd day, by the fertilized eggs injected from 5 mouse replace-conceives of fallopian tubal
In rat body.Every mouse is implanted into 14-20 pieces of fertilized eggs, and be born offspring after 21 days, and be born 11 hamsters altogether.
2.4 genotyping
7 days mouse cut toe and extract DNA after birth, are identified with PCR amplification.
1) PCR outer primer CAS9-T7R, Piwil1-TF amplifying genom DNA reacts 20 μ L systems, size 404bp
Piwil1-TF:GCAAGATGGGAGGCGAGCTC
Piwil1-TR:CAGCTGGGCTGTCACACTAAAA
Glue purification is cut after PCR primer agarose electrophoresis identification, is sequenced and is identified with CAS9-T7R primer, wherein through sequencing identification
2 knock out for homozygote, and 2 female mices are to knock in mouse.
Compared with wild type Piwil1 gene, the mouse Piwil1 gene of knocking in of mutation uses GGa at+1891-1923
ATa gca TGc TAt CAc GAt ACa ACt GCa GGt replaces original sequence GGC ATC GAC TGT TAC CAT GAC
ACC ACA GCT GGG, wherein+1897-1899GAC is replaced by GCA, amino acid is become by aspartic acid replacement (Asp, D)
Alanine (Ala, A), it is other to be replaced for degeneracy, destroy CAS9 target spot.
The phenotypic analysis of 2.5Piwil1 D633A gene knockout hamster
Female mice Piwil1 D633A is mated with Piwil1 (wt/wt), obtains Piwil1 D633A/WT hero mouse, this is small
Mouse mates with Piwil1 (wt/wt) female mice.The results show that Piwil1 D633A/WT hero mouse can normally mate, bolt rate and normal is seen
Mouse is not significantly different, and has a small amount of sperm, but 16 female mices of bolt are shown in mating, none offspring is born, thus, Piwil1
D633A/WT hero mouse is male sterility.
2.6Piwil1 D633A is used for the preparation of genetic engineering mouse as ligation mouse
The Piwil1 D633A hero mouse in 10 weeks -32 weeks, one day before the injection, wild-type mice mating in 8 weeks, seeing bolt, person made
For replace-conceive mouse.2nd day, the C57BL/6N mouse fertilized egg injected is implanted into replace-conceive mouse.Every replace-conceive mouse mouse is implanted into 14-20 pieces
Fertilized eggs.Offspring's birth in 21 days, can be distinguished after 3 days by color.
It is measured through continuous F1 generation to F3 generation, no Albino mice birth is transplanting mouse.Therefore, Piwil1 D633A can
For ligaturing the birth of mouse.
Although the embodiments of the invention are described in conjunction with the attached drawings, but patent owner can be in appended claims
Within the scope of make various deformations or amendments, as long as it does not exceed the scope of protection described in the claims to the invention, all should
Within protection scope of the present invention.
Sequence table
<110>long-living Biotechnology Ltd. in Liaoning
<120>method that sterile mouse model is constructed based on CRISPR-Cas9 system
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>mouse (Mus musculus)
<400> 1
gttaccatga caccacagct 20
<210> 2
<211> 2589
<212> DNA
<213>mouse (Mus musculus)
<400> 2
atgactggcc gagcccgagc tcgggcccgc ggcagggcac gaggtcagga gacggtgcag 60
catgttgggg ctgctgcgag ccagcaacct gggtacatcc caccgagacc tcaacagtcc 120
cccacagagg gggacttggt tggccgagga cgacagaggg ggatggtagt cggagccaca 180
tccaagtcac aagaactgca gatctcagct gggtttcagg agctgtcact ggcagagaga 240
ggagggcgtc gccgagactt ccatgacctt ggtgtgaaca ccagacagaa ccttgaccat 300
gtcaaagagt caaagacagg ctcctctggc atcattgtga agctgagcac caaccacttc 360
cggctgacct cgcgcccaca gtgggccctg tatcagtacc acatcgacta caatcccctg 420
atggaggccc gaaggcttcg ctccgcactg ctcttccagc atgaagacct cattggaagg 480
tgtcatgctt tcgatgggac aatattgttt ttacctaaga gactacagca caaggtcaca 540
gaagtattca gtcagactcg gaatggggaa cacgtgagga tcaccatcac cctgaccaac 600
gagctgccgc ccacctcgcc cacctgcctg cagttctata acatcatctt caggaggctc 660
ttgaaaatca tgaatttgca acaaattgga cggaattatt acaatccaag tgacccgatt 720
gatattccaa accacaggtt ggtgatctgg cccggcttca ccacctccat ccttcagtat 780
gagaacaaca tcatgctctg cacagacgtc agccacaagg tgctccgcag cgagactgtc 840
ctagacttca tgttcaatct ataccagcag acagaggagc acaagttcca ggagcaagtg 900
tcgaaggagc tcataggcct catcgttctc accaagtaca ataacaagac ctaccgggtg 960
gatgacattg actgggacca gaatccaaag agcaccttca agaaggcgga tggctcggag 1020
gtcagcttcc tggagtacta caggaagcaa tacaaccagg agatcacgga cctgaagcag 1080
ccggtgctgg tgagccaacc caagcggagg agaggccccg gcggcaccct gcctggccca 1140
gctatgctca tccctgaact ctgctatctc acaggcctga ctgataaaat gcgcaatgat 1200
ttcaatgtga tgaaggacct ggcagtgcac acgcggctga cccctgagca gcggcagcgg 1260
gaggtgggcc gcctcatcga ctacatccac aaggatgaca atgtgcagag agagcttcga 1320
gactggggcc tgagcttcga ctcaaacttg ctgtccttct ctggaagaat cttacaatct 1380
gagaagatcc accagggcgg aaagacgttt gattacaacc cacaatttgc agactggtcc 1440
aaagaaacaa gaggcgcgcc gctgatcagc gtgaagccat tggataactg gctgctgatc 1500
tatacccgca ggaattatga agcagccaac tcactgatac agaacctgtt caaagtgact 1560
ccagccatgg gcatccagat gaaaaaggca atcatgatcg aggtggatga cagaacagaa 1620
gcttatctga gagccttgca gcagaaggtg acgtcagaca ctcagatagt tgtctgtctc 1680
ttgtcaagta atcggaagga caaatatgat gccatcaaga agtacttgtg tacagactgc 1740
cccaccccaa gtcagtgtgt ggtggcccgg accctgggca agcagcaaac agtcatggcc 1800
attgccacca agatcgccct gcagatgaac tgcaagatgg gaggcgagct ctggcgggtg 1860
gacatgcccc tgaaactggc aatgatcgtg ggcatcgact gttaccatga caccacagct 1920
gggcggaggt ccatcgcagg attcgtcgcc agcatcaatg aagggatgac ccgctggttc 1980
tcccgctgcg tctttcagga ccgcgggcag gagctggtgg atggtctcaa ggtgtgcttg 2040
caagctgctc tgagggcttg gagtggctgc aatgaataca tgcccagccg tgtcatcgtg 2100
taccgagacg gtgtggggga cgggcagctg aagaccctgg tcaattatga ggtcccacag 2160
ttcctagatt gcctcaagtc agtcgggaga ggttacaacc caagactgac tgtaatcgtg 2220
gtgaagaagc gtgtcaatgc caggtttttt gctcagtctg ggggaagact tcagaaccct 2280
cttccaggga cagtcatcga tgtggaagtc accagaccag agtggtatga ctttttcatc 2340
gtgagccagg cagtgagaag cgggagtgtg tccccaacac actacaatgt catctatgac 2400
agcagtggcc tgaagcccga ccacatccag cggctgacat acaagctctg ccacgtgtac 2460
tataattggc ctggagtcat ccgagtccct gcaccttgcc agtatgcaca caagctggcc 2520
ttcctcgtgg gccagagcat ccacagagag ccaaacctct ccctgtccaa ccgcctctac 2580
tacctctaa 2589
<210> 3
<211> 2589
<212> DNA
<213>mouse (Mus musculus)
<400> 3
atgactggcc gagcccgagc tcgggcccgc ggcagggcac gaggtcagga gacggtgcag 60
catgttgggg ctgctgcgag ccagcaacct gggtacatcc caccgagacc tcaacagtcc 120
cccacagagg gggacttggt tggccgagga cgacagaggg ggatggtagt cggagccaca 180
tccaagtcac aagaactgca gatctcagct gggtttcagg agctgtcact ggcagagaga 240
ggagggcgtc gccgagactt ccatgacctt ggtgtgaaca ccagacagaa ccttgaccat 300
gtcaaagagt caaagacagg ctcctctggc atcattgtga agctgagcac caaccacttc 360
cggctgacct cgcgcccaca gtgggccctg tatcagtacc acatcgacta caatcccctg 420
atggaggccc gaaggcttcg ctccgcactg ctcttccagc atgaagacct cattggaagg 480
tgtcatgctt tcgatgggac aatattgttt ttacctaaga gactacagca caaggtcaca 540
gaagtattca gtcagactcg gaatggggaa cacgtgagga tcaccatcac cctgaccaac 600
gagctgccgc ccacctcgcc cacctgcctg cagttctata acatcatctt caggaggctc 660
ttgaaaatca tgaatttgca acaaattgga cggaattatt acaatccaag tgacccgatt 720
gatattccaa accacaggtt ggtgatctgg cccggcttca ccacctccat ccttcagtat 780
gagaacaaca tcatgctctg cacagacgtc agccacaagg tgctccgcag cgagactgtc 840
ctagacttca tgttcaatct ataccagcag acagaggagc acaagttcca ggagcaagtg 900
tcgaaggagc tcataggcct catcgttctc accaagtaca ataacaagac ctaccgggtg 960
gatgacattg actgggacca gaatccaaag agcaccttca agaaggcgga tggctcggag 1020
gtcagcttcc tggagtacta caggaagcaa tacaaccagg agatcacgga cctgaagcag 1080
ccggtgctgg tgagccaacc caagcggagg agaggccccg gcggcaccct gcctggccca 1140
gctatgctca tccctgaact ctgctatctc acaggcctga ctgataaaat gcgcaatgat 1200
ttcaatgtga tgaaggacct ggcagtgcac acgcggctga cccctgagca gcggcagcgg 1260
gaggtgggcc gcctcatcga ctacatccac aaggatgaca atgtgcagag agagcttcga 1320
gactggggcc tgagcttcga ctcaaacttg ctgtccttct ctggaagaat cttacaatct 1380
gagaagatcc accagggcgg aaagacgttt gattacaacc cacaatttgc agactggtcc 1440
aaagaaacaa gaggcgcgcc gctgatcagc gtgaagccat tggataactg gctgctgatc 1500
tatacccgca ggaattatga agcagccaac tcactgatac agaacctgtt caaagtgact 1560
ccagccatgg gcatccagat gaaaaaggca atcatgatcg aggtggatga cagaacagaa 1620
gcttatctga gagccttgca gcagaaggtg acgtcagaca ctcagatagt tgtctgtctc 1680
ttgtcaagta atcggaagga caaatatgat gccatcaaga agtacttgtg tacagactgc 1740
cccaccccaa gtcagtgtgt ggtggcccgg accctgggca agcagcaaac agtcatggcc 1800
attgccacca agatcgccct gcagatgaac tgcaagatgg gaggcgagct ctggcgggtg 1860
gacatgcccc tgaaactggc aatgatcgtg ggaatagcat gctatcacga tacaactgca 1920
ggtcggaggt ccatcgcagg attcgtcgcc agcatcaatg aagggatgac ccgctggttc 1980
tcccgctgcg tctttcagga ccgcgggcag gagctggtgg atggtctcaa ggtgtgcttg 2040
caagctgctc tgagggcttg gagtggctgc aatgaataca tgcccagccg tgtcatcgtg 2100
taccgagacg gtgtggggga cgggcagctg aagaccctgg tcaattatga ggtcccacag 2160
ttcctagatt gcctcaagtc agtcgggaga ggttacaacc caagactgac tgtaatcgtg 2220
gtgaagaagc gtgtcaatgc caggtttttt gctcagtctg ggggaagact tcagaaccct 2280
cttccaggga cagtcatcga tgtggaagtc accagaccag agtggtatga ctttttcatc 2340
gtgagccagg cagtgagaag cgggagtgtg tccccaacac actacaatgt catctatgac 2400
agcagtggcc tgaagcccga ccacatccag cggctgacat acaagctctg ccacgtgtac 2460
tataattggc ctggagtcat ccgagtccct gcaccttgcc agtatgcaca caagctggcc 2520
ttcctcgtgg gccagagcat ccacagagag ccaaacctct ccctgtccaa ccgcctctac 2580
tacctctaa 2589
<210> 4
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ataatacgac tcactatagg gttaccatga caccacagct gttttagagc tagaaatag 59
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
aaaagcaccg actcggtgcc 20
<210> 6
<211> 49
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gaaattaata cgactcacta tagggagaat ggactataag gaccacgac 49
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gcgagctcta ggaattctta c 21
Claims (5)
1. the method for constructing sterile mouse model based on CRISPR-Cas9 system, which is characterized in that the method includes following steps
It is rapid:
(1) mouse Piwil1 gene targeting sequence is determined；
(2) sgRNA is prepared；
(3) cas9 mRNA is prepared；
(4) external microinjection: collecting mouse fertilized egg, gRNA and Cas9 RNA injection is entered to the fertilized eggs of mouse；
(5) transplant is carried out to the fertilized eggs of injection.
2. the method according to claim 1 for constructing sterile mouse model based on CRISPR-Cas9 system, feature exist
In in step (1), Piwil1 gene targeting sequence is as shown in SEQ ID No.1.
3. the method according to claim 1 for constructing sterile mouse model based on CRISPR-Cas9 system, feature exist
In in step (2), design Piwil1 sgRNA1 and PX330 TRAC R primer, primer sequence is distinguished as follows:
Piwil1 sgRNA1 primer:
ATAATACGACTCACTATAGgGTTACCATGACACCACAGCTGTTTTAGAGCTAGAAA TAG；
PX330 TRAC R primer:
AAAAGCACCGACTCGGTGCC。
4. the method according to claim 3 for constructing sterile mouse model based on CRISPR-Cas9 system, feature exist
In, in step (2), using Piwil1 sgRNA1 and PX330 TRAC R primer amplification PX330 plasmid, template DNA is obtained, then
By T7 polymerase, TK sgRNA is synthesized.
5. the method according to claim 1 for constructing sterile mouse model based on CRISPR-Cas9 system, feature exist
In in step (3), design upstream primer CAS9-T7F and downstream primer CAS9-T7R, primer sequence is distinguished as follows:
CAS9-T7F:
GAAATTAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC；
CAS9-T7R:
GCGAGCTCTAGGAATTCTTAC。
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| US20090062228A1 (en) * | 2007-03-07 | 2009-03-05 | Hannon Gregory J | piRNA and uses related thereto |
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| US20090062228A1 (en) * | 2007-03-07 | 2009-03-05 | Hannon Gregory J | piRNA and uses related thereto |
| CN101864419A (en) * | 2010-04-27 | 2010-10-20 | 武汉大学 | Mouse Miwi promoter as well as carrier and application thereof |
| CN107603982A (en) * | 2016-07-06 | 2018-01-19 | 中国科学院上海生命科学研究院 | The method and kit of male sterility caused by diagnosing Piwil1 gene mutations |
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