CN109234316A - Realize that same gene effectively knocks out by a plurality of sgRNA co-injection - Google Patents

Realize that same gene effectively knocks out by a plurality of sgRNA co-injection Download PDF

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CN109234316A
CN109234316A CN201811113109.0A CN201811113109A CN109234316A CN 109234316 A CN109234316 A CN 109234316A CN 201811113109 A CN201811113109 A CN 201811113109A CN 109234316 A CN109234316 A CN 109234316A
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sgrna
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CN109234316B (en
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黄行许
王新
贾堃
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Beijing Huaxia Cage Biotechnology Co Ltd
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Abstract

Realize that same gene effectively knocks out by a plurality of sgRNA co-injection.Knockout technique includes: at least two target sequences of the code area of selected gene to be knocked out, it is made to separately include complete target codon CAA, CAG or CGA；BE3 is navigated into corresponding target sequence so that the target single base C in target codon becomes T to introduce terminator codon TAA or TAG, TGA accordingly to realize gene knockout using at least two sgRNA sequences；At least two sgRNA sequence is sequence corresponding at least two target sequences complementation respectively, wherein at least two sgRNA is realized effective knockout of same gene by co-injection.The present invention provides a kind of more efficient, and the gene knockout method with high precision and low undershooting-effect.

Description

Realize that same gene effectively knocks out by a plurality of sgRNA co-injection
Technical field
The present invention relates to the improvement and its application of the gene knockout method strategy based on base editor.
Background technique
Gene editing technology refer to target gene carry out " editor ", it would be desirable to variation introducing genomic DNA target site Process.The gene editing of early stage is realized by discovery endogenous homologous recombination.However, traditional eucaryote targets Genetic manipulation has gene targeting efficiency low, the disadvantages of being of limited application.II type CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated Cas9 endonuclease) Original limitation has been broken in the discovery and application of system, which is proved to be a kind of tool [the Le et of multipurpose gene editing al.,2013；Patrick et al.,2014].
It is to utilize sgRNA (single guided that CRISPR/Cas9 System-mediated specific gene, which knocks out (knockout), RNA Cas9 albumen position shearing double-stranded DNA) is guided by target sequence complementation, non-homologous end joining (non-occurs Homologous end joining, NHEJ) it repairs, frameshift mutation (frameshift mutation) is caused, clpp gene is caused Remove, this method mainly has the disadvantage that in practical application: nonhomologous end engagement mechanisms first are easy to produce radom insertion With deletion (indel) so that new base may be randomly incorporated into, near breaking point so as to cause inaccurate gene editing. Secondly, the gene editing that CRISPR/Cas9 is mediated always has some undershooting-effects [Gorski et al., 2017].
It recent studies have shown that, the fusion protein based on Cas9 constructed by CRISPR/Cas9 technology and deaminase can be used as " base editing machine (Base editor, BE) ".First generation base editor (BE1) is by by rat cytidine deaminase ApOBEC1 It is fused to dCas9, C/G base-pair is changed into A/T by deamination by it.It hereafter is to improve its editorial efficiency to BE system System has carried out various modifications, and current widely used BE3 can be in the window of the position 4-8 base of the non-binding chain of sgRNA C-T nucleotide replacement [Komor et al., 2016] is introduced in mouthful.This provides new thinking for realization gene knockout --- It is mutated by CT and introduces terminator codon, such as CAA, CAG, CGA are mutated into terminator codon TAA, TAG, TGA, or passed through TGG is mutated into terminator codon TAA, TGA, TAG by GA mutation, the translation of encoding gene is terminated, to realize gene knockout.It Provide it is more safer than the NHEJ that Cas9 is mediated and more it is accurate knock out it is tactful [Komor et al., 2017；Kim et al., 2017].However due to being limited at present by factors such as BE3 editorial efficiencies, there is no obtain gene by this method effectively to knock out Example.
Summary of the invention
An object of the present invention is to provide a kind of efficient accurately gene knockout strategy.
According to the first aspect of the invention, a kind of gene knockout method is provided comprising:
At least two 19~21bp-NGG target sequence of the code area of selected gene to be knocked out, separately includes it completely Target codon CAA, CAG or CGA, target single base C therein is located at 4-8 of target sequence, target codon with The interval NGG 12-14bp；
BE3 navigated into corresponding target sequence using at least two sgRNA sequences so that mesh in target codon Mark single base C becomes T to introduce terminator codon TAA or TAG, TGA accordingly to realize gene knockout；
At least two sgRNA sequence is 19~21bp corresponding at least two target sequences complementation respectively Sequence,
Wherein at least two sgRNA is realized effective knockout of same gene by co-injection.
In alternative solution, CCN-19~21bp target sequence of the code area of gene to be knocked out can be selected, its packet is made Containing complete target codon TGG, target single base G is preferably placed at (right end) 4-8 of target sequence, target codon With the interval NGG 12-14bp.
According to the present invention, BE3 can be rAPOBEC-SpCas9-NLS-UGI-NLS.
It can be used for knocking out following two target genes: mouse Pcdc1 and Tyr according to the method for the present invention.
According to the second aspect of the invention, it provides the above method and carries out mouse Pcdc1 and Tyr gene in cell line N2a The application of knockout.
According to the third aspect of the invention we, the effective introducing terminator codon obtained according to above-mentioned application is provided to realize The sgRNA of gene knockout carries out the application of mouse Pcdc1 and Tyr gene knockout in mouse embryo cell.
According to the fourth aspect of the invention, a kind of kit for gene knockout is provided, including above-mentioned sgRNA, BE3 and amplifing reagent.
The present invention utilizes the base editing technique developed on the basis of CRISPR/Cas9, passes through accurately CT or GA single base Terminator codon is created in mutation, and a plurality of sgRNA co-injection is combined to improve gene knockout efficiency, thus establish it is a kind of efficiently, The gene knockout strategy of accurate and low undershooting-effect.
Detailed description of the invention
Fig. 1 is schematic diagram (the lower stroke of expression PAM of runic being knocked according to the present invention using CT mutation realization target gene； Italic indicates mutation coding；Lower stroke of expression mutating alkali yl of italic overstriking)；
Fig. 2 is the structural schematic diagram of BE3；
Fig. 3 is the schematic diagram of the gene knockout efficiency on mice embryonic；
Fig. 4 is the schematic diagram of the gene knockout efficiency on mouse.
Specific embodiment
Firstly, carrying out the design of sgRNA.Base fixed point editor is that BE3 is navigated to target site, target gene using sgRNA The selection and design of specific sgRNA is the key that place of the invention.The present invention selects design sgRNA as follows:
19~21bp-NGG target sequence of the code area of selected gene to be knocked out, makes that it includes complete target codons CAA, CAG or CGA, wherein target single base C is located at 4-8 of target sequence, target codon and the interval NGG 12-14bp；
BE3 is navigated into target sequence so that the target single base C in target codon becomes T using sgRNA sequence To accordingly introduce terminator codon TAA or TAG, TGA；
In alternative solution, then select gene to be knocked out code area CCN-19~21bp target sequence, make it includes Complete target codon TGG, target single base G are preferably placed at (right end) 4-8 of target sequence, and target codon is excellent Choosing is with CCN compartment every 12-14bp.
For two target genes --- mouse Pcdc1 and Tyr, it is corresponding to design that the present invention selectes following target gene sequences SgRNA (lower stroke of expression PAM of runic；Lower stroke of expression Candidate Mutant coding of italic):
1.Pcdc1
Sg-1:
Sg-2:
Sg-3:
2.Tyr
Sg-1:
Sg-2:
Sg-3:
For above-mentioned selected target gene sequence, mouse Pcdc1 (3), Tyr (3) construct corresponding sgRNA expression Different sgRNA is directed respectively into pGL3-U6-EGFP-sgRNA by carrier.
Embodiment 1
The base editor that BE3 mediation is carried out in cell line, introduces terminator codon, realizes gene knockout.Routinely grasp Make, the gene knockout (by electricity turn or liposome transfection) of cell strain is carried out, by taking liposome transfection as an example.
(1) by taking N2a cell as an example, the present invention carries out the culture and transfection of eukaryotic cells: N2a cell inoculation is incubated at It adds in the sugared culture solution of DMEM high of 10%FBS (HyClone, SH30022.01B), wherein (100U/ml) containing penicillin With streptomycin (100 μ g/ml).
(2) divide before transfection into 6 orifice plates, transfected when density reaches 70%-80%.
(3) transfection is by taking liposome transfection as an example.According to LipofectamineTM2000Transfection Reagent The operation manual of (Invitrogen, 11668-019), by taking SpCas9nickase as an example, by 2 μ g BE3 plasmids and 1 μ g pGL3- U6-EGFP-sgRNA plasmid mixes, and cotransfection changes liquid, airflow classification is collected carefully after 72 hours into every hole cell after 6-8 hours Born of the same parents.
(4) genotyping
A, part cell is collected to use in lysate (10 μM of Tris-HCl, 0.4M NaCl, 2 μM of EDTA, 1% SDS) After the cracking digestion of 100 μ g/ml Proteinase Ks, it is dissolved into 50 μ l deionized waters after phenol-chloroform extracting.
B, PCR amplification is carried out using pair of primers N-For and N-Rev, with AxyPrep PCR cleanup kit (AXYGEN, AP-PCR-250G) purifying obtains PCR recovery product, PCR reaction system are as follows:
300-400ng genomic DNA
25μl 2X Buffer
1μl dNTP
2μl N-For(10μm)
2μl N-Rev(10μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Moisturizing is to 50 μ l systems.
C, the PCR recovery product of acquisition is carried out with rTaq plus A reacts.Add A reaction system are as follows:
700-800ng PCR recovery product
5μl 10X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001AM)
Moisturizing is to 50 μ l systems.
After 72 DEG C incubate 30 minutes, 2 μ l products and pMD19-T vector (TAKARA, 3271) is taken to connect, connection reaction System are as follows:
2 μ l add A product
0.5μl pMD19-T vector
2.5μl SolutionΙ(TAKARA,6022)
16 DEG C be incubated for 30 minutes after take 2 μ l products convert DH5 competent cell (TransGen, CD201).
D, each mutant target gene, following (the lower stroke of table of runic of sequencing result is sequenced with universal primer M13-F in picking monoclonal Show PAM；Italic indicates mutation coding；Lower stroke of expression mutating alkali yl of italic overstriking):
1.Pcdc1
Sg-1:
Mut:
Sg-2:
Mut:
Sg-3:
Mut:
2.Tyr
Sg-1:
Mut:
Sg-2:
Mut:
Sg-3:
Mut:
The result shows that: the base mutation of sgRNA targeting has occurred in target gene, introduces terminator codon, Pcdc1, Tyr Gene knockout success.
Embodiment 2
The base editor that BE3 mediation is carried out on mice embryonic, introduces terminator codon, realizes gene knockout.
(1) it is transcribed in vitro: BE3 plasmid being linearized with AgeI enzyme (NEB, R3552L), and uses T7ULTRA (Ambion, AM1345) is transcribed in vitro.BE3mRNA is purified with RNeasy Mini kit (QIAGEN, 74104).By sgRNA Annealing oligonucleotide is the pUC57 sgRNA expression vector (Addgene, 51132) with T7 promoter.Then sgRNA is expanded And it is transcribed with MEGAshorttranscript T7 kit (Ambion, AM1345).With MEGAclear kit (Ambion, AM1908) purifies sgRNA, is recycled with ethanol precipitation.
(2) microinjection: the female C57BL/6J mouse through Superovluation mates with C57BL/6J male mice, 0.5 It collects fertilized eggs from fallopian tubal.Fertilized eggs inject BE3mRNA (50ng/ μ l) and specificity sgRNA (25ng/ μ l) respectively.Note It is as follows to penetrate situation:
1.Pcdc1
combination 1:BE3mRNA+PD1-sg1
combination 2:BE3mRNA+PD1-sg1+2
combination 3:BE3mRNA+PD1-sg1+2+3
2.Tyr
combination 1:BE3mRNA+Tyr-sg1
combination 2:BE3mRNA+Tyr-sg1+2
(3) genotyping
A, by the fertilized eggs culture of injection to blastaea carry out genome analysis: using rapid fractionation method (Luxige, QE09050 embryonic gene group DNA) is extracted, 10 μ L rapidly extracting liquid are added in embryo and are digested.
B, PCR amplification is carried out using pair of primers N-For and N-Rev, with AxyPrep PCR cleanup kit (AXYGEN, AP-PCR-250G) purifying obtains PCR recovery product, PCR reaction system are as follows:
300-400ng genomic DNA
25μl 2X Buffer
1μl dNTP
2μl N-For(10μm)
2μl N-Rev(10μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Moisturizing is to 50 μ l systems.
C, the PCR recovery product of acquisition is carried out with rTaq plus A reacts.Add A reaction system are as follows:
700-800ng PCR recovery product
5μl 10X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001AM)
Moisturizing is to 50 μ l systems.
After 72 DEG C incubate 30 minutes, 2 μ l products and pMD19-T vector (TAKARA, 3271) is taken to connect, connection reaction System are as follows:
2 μ l add A product
0.5μl pMD19-T vector
2.5μl SolutionΙ(TAKARA,6022)
16 DEG C be incubated for 30 minutes after take 2 μ l products convert DH5 competent cell (TransGen, CD201).
D, picking monoclonal is sequenced each mutant target gene with universal primer M13-F, counts gene knockout by sequencing result Successful embryo number, the embryo number that editor occurs but is not compiled as the embryo number of i-stop and does not edit, the results are shown in attached figure 3 (black portions indicate the successful embryo number of gene knockout, and dark gray section indicates that editor occurs but is not compiled as the embryo of i-stop Tire number, bright gray parts indicate the embryo number that do not edit).
The result shows that: the base mutation of sgRNA targeting has occurred in target gene, introduces terminator codon, Pcdc1, Tyr Gene knockout success；In addition, the gene knockout efficiency on embryo can be improved in a plurality of sgRNA co-injection.
Embodiment 3
Construct the knock out mice that BE3 is mediated.
Routinely operation carries out embryo collection, microinjection, Embryo Culture and the embryo transfer etc. of mouse.
(1) microinjection: fertilized eggs inject BE3mRNA and specificity sgRNA (Experiment 1:PD1-sg1+ respectively 2+3, Tyr-sg1+2) or BE3mRNA and specificity sgRNA (Experiment 2:PD1-sg1+2+3, Tyr-sg1+2+3). It is conventional to carry out embryo transfer；
(2) genotyping:
A, conventional mouse cuts tail and extracts genomic DNA: lysate (10 μM of Tris-HCl, 0.4M NaCl, 2 μM EDTA, 1%SDS) in 100 μ g/ml Proteinase Ks cracking digestion after, phenol-chloroform extract after be dissolved into 50 μ l deionized waters.
B, PCR amplification is carried out using pair of primers N-For and N-Rev, with AxyPrep PCR cleanup kit (AXYGEN, AP-PCR-250G) purifying obtains PCR recovery product, PCR reaction system are as follows:
300-400ng genomic DNA
25μl 2X Buffer
1μl dNTP
2μl N-For(10μm)
2μl N-Rev(10μm)
1μl DNA Polymerase(Vazyme,P505-d3)
Moisturizing is to 50 μ l systems.
C, the PCR recovery product of acquisition is carried out with rTaq plus A reacts.Add A reaction system are as follows:
700-800ng PCR recovery product
5μl 10X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001AM)
Moisturizing is to 50 μ l systems.
After 72 DEG C incubate 30 minutes, 2 μ l products and pMD19-T vector (TAKARA, 3271) is taken to connect, connection reaction System are as follows:
2 μ l add A product
0.5μl pMD19-T vector
2.5μl SolutionΙ(TAKARA,6022)
16 DEG C be incubated for 30 minutes after take 2 μ l products convert DH5 competent cell (TransGen, CD201).
D, picking monoclonal is sequenced each mutant target gene with universal primer M13-F, counts gene knockout by sequencing result Successful mouse, the mouse that editor occurs but is not compiled as the mouse of i-stop and does not edit, the results are shown in attached figure 4 (black Part indicates the successful mouse of gene knockout, and dark gray section indicates that editor occurs but is not compiled as the mouse of i-stop, light gray Color part indicates the mouse that do not edit).
The base mutation of sgRNA targeting has occurred in the above results confirmation target gene, introduces terminator codon, Pcdc1, The success of Tyr gene knockout；In addition, the gene knockout efficiency on embryo can be improved in a plurality of sgRNA co-injection, and on mouse Realize the complete knockout of gene.
Sequence table
<110>Kai Qi Bioisystech Co., Ltd of Beijing China
<120>realize that same gene effectively knocks out by a plurality of sgRNA co-injection
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 1
aaaacaggcc gccttctgta atgg 25
<210> 2
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 2
cggtttcaag gcatggtcat tgg 23
<210> 3
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 3
cctggtcatt cacttaagct gtg 23
<210> 4
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 4
tgcggccagc tttcaggcag agg 23
<210> 5
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 5
ccttcttctc ctcctggcag gta 24
<210> 6
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 6
ccagggtttc tgccttggca cag 24

Claims (6)

1. a kind of gene knockout method, comprising:
At least two 19~21bp-NGG target sequence of the code area of selected gene to be knocked out, makes it separately include complete mesh Codon CAA, CAG or CGA are marked, target single base C therein is located at 4-8 of target sequence, between target codon and NGG Every 12-14bp；
BE3 navigated into corresponding target sequence using at least two sgRNA sequences so that target list in target codon Base C becomes T to introduce terminator codon TAA or TAG, TGA accordingly to realize gene knockout；
At least two sgRNA sequence is 19~21bp sequence corresponding at least two target sequences complementation respectively,
Wherein at least two sgRNA is realized effective knockout of same gene by co-injection.
2. according to the method described in claim 1, wherein BE3 is rAPOBEC-SpCas9-NLS-UGI-NLS.
3. according to the method described in claim 1, for knocking out following two target genes: mouse Pdcd1 and Tyr.
4. the application that claim 1 the method carries out mouse Pdcd1, Tyr gene knockout in cell line N2a.
5. the application of mouse Pdcd1, Tyr gene knockout is carried out in mouse embryo cell method according to claim 1.
6. a kind of kit for gene knockout, including sgRNA, BE3 described in claims 1 or 2 and amplifing reagent.
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CN109929846A (en) * 2019-03-28 2019-06-25 南京北恒生物科技有限公司 A kind of double sites sgRNA knock out the CRISPR/Cas9 system and application of LRRC20 gene
CN112029769A (en) * 2020-09-11 2020-12-04 中国人民解放军陆军特色医学中心 Construction method of Cyp1a1 gene knockout mouse model and application of model in sepsis
CN112029769B (en) * 2020-09-11 2022-03-22 中国人民解放军陆军特色医学中心 Construction method of Cyp1a1 gene knockout mouse model and application of model in sepsis
CN117844811A (en) * 2024-03-08 2024-04-09 上海恒润达生生物科技股份有限公司 sgRNA composition for targeted knockout of CD70 gene and application thereof

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