CN109706185A - The method and application of gene knockout are realized based on base editing system mutation initiation codon - Google Patents

Abstract

The method and application of gene knockout are realized based on base editing system mutation initiation codon.The gene knockout method is based on accurate single base editing technique, is mutated for encoding gene initiation codon ATG, prevents it from normally playing translation Initiation Function, to reach the knockout purpose to target gene.It specifically includes: it is selected to be located at gene to be knocked out translation initiation region 20bp-NGG or CCN-20bp target sequence, make that it includes complete initiation codon ATG；ABE or BE4 are navigated into target sequence using sgRNA sequence, makes the target single base in target gene initiation codon ATG that A to G, T to C or G to A mutation occur, initiation codon is destroyed, to realize gene knockout.Knockout technique of the invention is more efficient, more accurate and less undershooting-effect, realizes the knockout that gene can not be knocked out to part conventional method.

Description

Based on base editing system mutation initiation codon realize gene knockout method and Using

Technical field

The present invention relates to gene knockout strategies, more specifically, being related to the clpp gene based on initiation codon base editor Except method and its application.

Background technique

Gene knockout is so that the gene of an organism is lost or is lost by various molecule manipulations using molecular genetic techniques It is living, and then the function that cannot bring into normal play.

In traditional gene knockout, mainly completed by methods of homologous recombination.This method first has to building and includes institute The DNA vector that need to be mutated, the construct include and the target sequence at least homology of 2kb (Hall et al., 2009).It can lead to It crosses electrotransfection or building is mentioned and is delivered to stem cell by microinjection.Then itself repairing in time by DNA construct by cell It recombinates in cell DNA.However, this is an inefficient process, because intracellular homologous recombination only accounts for the 10 of DNA recombination^-2It arrives 10^-3(Hall et al.,2009)。

Then, the editing technique based on site specific nucleic acid enzyme has been developed.It include at present by Zinc finger nuclease technology (zinc finger nucleases, ZFNs) (Santiago et al., 2008), transcription activation factor sample effect nucleic acid zymotechnic (Transcription activator-like effector nucleases, TALENs) (Joung and Sander, 2012) and aggregation the short palindrome repetitive sequence system of aturegularaintervals (Clustered regularly interspaced short Palindromic repeats, CRISPRs) (Gaj et al., 2013).These three methods be related to accurately targeting DNA sequence dna with It introduces double-strand DNA cleavage (double-strand breaks, DSB), and then attempts to repair double-strand using the repair mechanism of cell Fracture is usually repaired by non-homologous end joining (non-homologous end joining, NHEJ).This reparation may Lead to the insertion or missing of base-pair, and then causes frameshift mutation.This mutation can make the gene lose function, to reach pair Knockout (knockout) purpose (Gaj et al., 2013) of specific gene.In addition, CRISPR under conditions of having template, leads to It crosses homologous recombination to be repaired (homology-directed repair, HDR), realize gene knock-in (knockin).The process It is more efficient compared with homologous recombination, and then the knockout for the realization allele that can be more easier.

CRISPR/Cas9 system, it is easy, efficient, inexpensive in view of it the features such as, become gene editing field it is with fastest developing speed, Most widely used technology has caused the revolution in gene editing field.Currently, CRISPR/Cas9 system has been successfully used to The researchs such as the knockout of DNA is knocked in, substituted, modifying, marking, and RNA modification and genetic transcription are adjusted (Hsu et al., 2014； Komor et al.,2017a).And be successfully applied to multiple species gene editing (Barrangou and Doudna, 2016；Komor et al.,2017a).But since HDR low efficiency (is integrated and is seldom occurred), and nonhomologous end jointing machine System is easy to produce radom insertion and deletes (indel), so that new base may be randomly incorporated near breaking point, so as to cause Inaccurate gene editing.In addition, CRISPR/Cas9 mediate gene editing have certain undershooting-effect (Gorski et al., 2017)。

Summary of the invention

An object of the present invention is to provide a kind of efficient accurately gene knockout strategy.

Base editing technique is based on novel gene edit tool constructed by CRISPR/Cas9 technology.There are born of the same parents phonetic at present Pyridine base editing machine (CBEs, cytosine base editors) and guanine base editing machine (ABEs, Adenine base editors).Cytosine base editing machine is to merge dCas9 or Cas9 nickase with rat cytidine deaminase APOBEC1, it Uracil (U) is converted by cytimidine (C) by deamination, later, by DNA replication dna or reparation, uracil (U) is turned It is melted into thymidine (T).Similarly, guanine (G) can be also converted to adenine (A).It can be smart in target gene It really is effectively introduced into point mutation, without double-strand DNA cleavage or any donor template, it is latent to show very big gene editing Power (Komor et al., 2016).Cytosine base editing machine is not required to cutting DNA and DSB, the indel of formation is caused to be lower than 1%, The gene editing of realization is more accurate (Komor et al., 2016)；Moreover, this mode will miss the target, efficiency falls below nature 10 times of background, the gene editing of realization are safer (Nishida et al., 2016).Most common is BE3 (Komor Et al., 2016), more efficient BE4 (Komor et al., 2017b).

Adenine base editing machine induces the nucleotide variation of extensive target gene group locus in human cell (Gaudelli et al.,2017).In ABE system, TadA:TadA* heterodimer is by the mono- guidance RNA (sgRNA) of Cas9n/ Compound is guided to target site, and engineered TadA* is used as activity tRNA adenosine deaminase in single-stranded genomic DNA Inosine (I) is converted by adenine (A), then causes the guanine (G) in genome prominent during DNA reparation or DNA replication dna Become (Gaudelli et al., 2017).Similarly, thymidine (T) can be also converted to cytimidine (C).So far, BE and ABE system can programmably introduce all four conversions (C to T, G to A, A to G and T to C) on the target gene of genome, Greatly extend base editting function.

Accurate and specific single base editor of the inventor based on above-mentioned CBEs or ABEs System-mediated, ingehious design A kind of gene knockout strategy: it is mutated using BE4 system by G to A and target gene initiation codon ATG is sported into ATA；Or benefit ATG is sported by ACG or A to G mutation by T to C mutation with ABE system, ATG is sported into GTG.Pass through mutation initiation password Son, prevents the translation initiation of gene, to realize that gene silencing knocks out purpose.

According to the first aspect of the invention, a kind of gene knockout method is provided comprising:

Determine the initiation codon of gene to be knocked out, find design 20bp-NGG target sequence (PAM sequence), make it includes Complete initiation codon ATG；ABE is navigated into target sequence using sgRNA sequence, so that the target in initiation codon Single base A becomes G, so that initiation codon ATG is sported GTG, to realize gene knockout purpose.

Alternatively, it determines the initiation codon of gene to be knocked out, finds design CCN-20bp target sequence (PAM sequence Column), make that it includes complete initiation codon ATG；ABE, BE4 are navigated into target sequence using sgRNA sequence, so as to rise Target single base T in beginning codon becomes C or G becomes A, so that initiation codon ATG is sported ACG or ATA, to realize Gene knockout purpose.

If target single base A or T are preferably placed at 4-7 of target sequence using ABE, if using BE4, target list alkali Base G is preferably placed at 4-8 of target sequence；

The sgRNA sequence is and the complementary corresponding 20bp sequence of target sequence.

According to the present invention, ABE can be selected from pCMV-ABEmax SEQ ID NO.1

BE4 is selected from: pCMV-AncBE4max SEQ ID NO.2；pCMV-BE4max SEQ ID NO.3

Can be used for knocking out following 11 target genes according to the method for the present invention: people APOE, PD1, LAG3, TIGIT, VISTA, CD224, CD160, SOX9, HDAC1 and mouse TIM3 and LAG3, its corresponding sgRNA sequence respectively with sequence Target gene sequence shown in one to ten is complementary.

According to the second aspect of the invention, provide the above method cell line HEK293T carry out people APOE, PD1, The application of LAG3, TIGIT, VISTA, CD224, CD160, SOX9, HDAC1 gene knockout.

According to the third aspect of the invention we, provide the above method human T-cell carry out people APOE, PD1, LAG3, The application of TIGIT, VISTA, CD224, CD160, SOX9, HDAC1 gene knockout.

According to the fourth aspect of the invention, provide the isolated T cell or cell line obtained according to above-mentioned application or Their subculture.

According to the fifth aspect of the invention, a kind of kit for gene knockout is provided, including (with gene to be knocked out Above-mentioned sgRNA, ABE, BE3, BE4 and corresponding amplifing reagent accordingly).

The present invention utilizes the base editing technique developed on the basis of CRISPR/Cas9, passes through accurately A to G, T to C or G Single base mutation to A changes and destroys initiation codon ATG, thus establish it is more more efficient than CRISPR/Cas9, more accurate and The gene knockout strategy of less undershooting-effect.

Bibliography

Barrangou,R.,Doudna,J.A.,2016.Applications of CRISPR technologies in research and beyond.Nature Biotechnology 34,933.

Gaj,T.,Gersbach,C.A.,Barbas,C.F.,2013.ZFN,TALEN,and CRISPR/Cas-based methods for genome engineering.Trends in Biotechnology 31,397-405.

Gaudelli,N.M.,Komor,A.C.,Rees,H.A.,Packer,M.S.,Badran,A.H.,Bryson, D.I.,Liu,D.R.,2017.Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage.Nature551,464.

Gorski,S.A.,Vogel,J.,Doudna,J.A.,2017.RNA-based recognition and targeting:sowing the seeds of specificity.Nature Reviews Molecular Cell Biology 18,215.

Hall,B.,Limaye,A.,Kulkarni,A.B.,2009.Overview:generation of gene knockout mice.Curr Protoc Cell Biol Chapter 19,Unit 19 12 19 12 11-17.

Hsu,Patrick D.,Lander,Eric S.,Zhang,F.,2014.Development and Applications of CRISPR-Cas9for Genome Engineering.Cell 157,1262-1278.

Joung,J.K.,Sander,J.D.,2012.TALENs:a widely applicable technology for targeted genome editing.Nature Reviews Molecular Cell Biology 14,49.

Komor,A.C.,Badran,A.H.,Liu,D.R.,2017a.CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.Cell 168,20-36.

Komor,A.C.,Kim,Y.B.,Packer,M.S.,Zuris,J.A.,Liu,D.R.,2016.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature 533,420.

Komor,A.C.,Zhao,K.T.,Packer,M.S.,Gaudelli,N.M.,Waterbury,A.L.,Koblan, L.W.,Kim,Y.B.,Badran,A.H.,Liu,D.R.,2017b.Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.Sci Adv 3,eaao4774.

Nishida,K.,Arazoe,T.,Yachie,N.,Banno,S.,Kakimoto,M.,Tabata,M., Mochizuki,M.,Miyabe,A.,Araki,M.,Hara,K.Y.,Shimatani,Z.,Kondo,A.,2016.Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.Science 353.

Ren,B.,Yan,F.,Kuang,Y.,Li,N.,Zhang,D.,Zhou,X.,Lin,H.,Zhou,H., 2018.Improved Base Editor for Efficiently Inducing Genetic Variations in Rice with CRISPR/Cas9-Guided Hyperactive hAID Mutant.Molecular Plant 11,623-626.

Santiago,Y.,Chan,E.,Liu,P.-Q.,Orlando,S.,Zhang,L.,Urnov,F.D.,Holmes, M.C.,Guschin,D.,Waite,A.,Miller,J.C.,Rebar,E.J.,Gregory,P.D.,Klug,A., Collingwood,T.N.,2008.

Targeted gene knockout in mammalian cells by using engineered zinc- finger nucleases.Proceedings of the National Academy of Sciences 105,5809- 5814.

Detailed description of the invention

Fig. 1 is that according to the present invention be mutated using ATG realizes that target gene knocks out schematic diagram；

Fig. 2 is pCMV-AncBE4max structural schematic diagram used in the present invention；

Fig. 3 is pCMV-BE4max structural schematic diagram used in the present invention；

Fig. 4 is pCMV-ABEmax structural schematic diagram used in the present invention；

Fig. 5 is present invention transfection sgRNA and ABE or BE4 plasmid, FCM analysis GFP positive exemplary diagram；

Fig. 6 is gene editing efficiency and editing sites analysis chart in the present invention, by taking HDAC1 gene as an example；

Fig. 7 is gene editing efficiency and editing sites analysis chart in the present invention, by taking SOX9 gene as an example；

Fig. 8 is to obtain the monoclonal cell editor effect that target gene initiation codon ATG is edited by sorting in the present invention Rate and editing sites analysis, by taking HDAC1 gene as an example；

Fig. 9 is to obtain the monoclonal cell editor effect that target gene initiation codon ATG is edited by sorting in the present invention Rate and editing sites analysis, by taking SOX9 gene as an example；

Figure 10 is to obtain the monoclonal cell purpose base that target gene initiation codon ATG is edited by sorting in the present invention The Western blot of cause is analyzed, by taking HDAC1 gene as an example.

Specific embodiment

Clear, complete description is carried out to embodiment of the present invention below in conjunction with embodiment, it is clear that described reality It applies example and is merely to illustrate a part of the embodiments of the present invention, and be not construed as limiting the scope of the invention.It is not specified in embodiment Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, It is considered as the conventional products that can be obtained by commercially available purchase.

The foregoing is merely present pre-ferred embodiments, are not intended to limit the invention, all in spirit of the invention Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Base fixed point editor is to be navigated to ABE or BE4 or site-specific target site using sgRNA, target gene specific The selection and design of sgRNA is the key that place of the invention.The present invention selects design sgRNA as follows:

20bp-NGG CCN-20bp target sequence (the PAM sequence comprising initiation codon ATG that is selected then knocking out gene Column), if using ABE, target single base A or T are preferably placed at 4-7 of target sequence, if target single base G is excellent using BE4 Bit selecting is in 4-8 (Fig. 1-Fig. 4) of target sequence.

Preparation 20bp sgRNA sequence corresponding with target sequence complementation.

For 11 target genes --- people APOE, PD1, LAG3, TIGIT, VISTA, CD224, CD160, SOX9, HDAC1 and mouse TIM3 and LAG3, the present invention selectes following target gene sequences, and to design corresponding sgRNA, (runic is indicated PAM；Lower stroke of expression Candidate Mutant coding of italic):

One .hAPOE

ABE T to C

Sg-1:SEQ ID NO.4

ABE A to G

Sg-2:SEQ ID NO.5

Two .hPD1

ABE A to G

Sg-1:SEQ ID NO.6

Three .hLAG3

BE4 G to A

Sg-1:SEQ ID NO.7

ABE T to C

Sg-2:SEQ ID NO.8

Four .hTIGIT

ABE T to C

Sg-1:SEQ ID NO.9

BE4 G to A

Sg-2:SEQ ID NO.10

Five .hVISTA

ABE A to G

Sg-1:SEQ ID NO.11

Six .hCD224

ABE A to G

Sg-1:SEQ ID NO.12

Seven .hCD160

ABE A to G

Sg-1:SEQ ID NO.13

Eight .hSOX9

BE4 G to A

Sg-1:SEQ ID NO.14

Nine .mTIM3

BE4 G to A

Sg-1:SEQ ID NO.15

ABE T to C

Sg-2:SEQ ID NO.16

Ten .mLAG3

BE4G to A

Sg-1:SEQ ID NO.17

ABE T to C

Sg-2:SEQ ID NO.18

11 .HDAC1

ABE A to G

Sg-1:SEQ ID NO.19

For above-mentioned selected target gene sequence, people APOE (2), PD1, LAG3 (2), TIGIT (2), VISTA, CD224, CD160, SOX9, HDAC1 and mouse TIM3 (2) and LAG3 (2) construct corresponding sgRNA expression Different sgRNA is directed respectively into pGL3-U6-sgRNA carrier, SEQ ID NO.20 by carrier.

Embodiment 1

The base editor that ABE or BE4 is mediated is carried out on cell strain, mutation initiation codon realizes gene knockout.

The present invention carries out the culture and transfection of eukaryotic cells, carries out the gene knockout of cell strain (by electricity turn or rouge Plasmids).Below with HEK293T cell, to people APOE, PD1, LAG3, TIGIT, VISTA, CD224, CD160, SOX9, It demonstrates for the isogenic liposome transfection method of HDAC1.

(1) in DMEM culture solution of the HEK293T cell inoculation culture containing 10%FBS (HyClone, SH30022.01B), In contain penicillin (100U/ml) and streptomycin (100 μ g/ml).

(2) day before transfection divides cell to 6 orifice plates.Next day is transfected when density is to 70%-80%.

(3) according to Lipofectamine^TM2000Transfection Reagent's (Invitrogen, 11668-019) Operation manual mixes 2 μ g ABE or BE4 plasmids pGL3-U6-sgRNA plasmid corresponding with 2 μ g, and cotransfection is into cell, 6- Liquid is changed after 8 hours, cell is collected after 72 hours, and cell transfecting efficiency is determined (Fig. 5) by flow type analyzer.

(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) The cracking digestion of 100 μ g/ml Proteinase Ks, phenol-chloroform extracting are dissolved into 50 μ l ddH₂In O.

B, PCR amplification is carried out using pair of primers N-For and N-Rev, purifies acquisition with AxyPrep PCR cleanup PCR recovery product takes 200ng to be uniformly diluted to 20 μ l and is denaturalized, is annealed, program such as: 95 DEG C, 5min；95-85℃at-2 ℃/s；85-25℃at-0.1℃/s；hold at 4℃.

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 37 DEG C incubate 30 minutes, 1 μ l product and pMD19-T vector (TAKARA, 3271) is taken to connect and convert DH5 Competent cell (TransGen, CD201).

D, each mutant target gene is sequenced with universal primer M13-F in picking monoclonal, and sequencing result is following, and (runic indicates PAM；Italic indicates mutation coding；Lower stroke of expression mutating alkali yl of italic):

1.hAPOE

Sg-1:

Mut:

Sg-2:

Mut:

2.hPD1

Sg-1:

Mut:

3.hLAG3

Sg-1:

Mut:

Sg-2:

Mut:

4.hTIGIT

Sg-1:

Mut:

Sg-2:

Mut:

5.hVISTA

Sg-1:

Mut:

6.hCD224

Sg-1:

Mut:

(7.hCD160 Fig. 6, Fig. 8)

Sg-1:

Mut:

8.hSOX9

Sg-1:

Mut:

(9.HDAC1 Fig. 7, Fig. 9)

Sg-1:

Mut:

(5) Western blot detection knocks out effect

Further, we carry out western blot detection to the monoclonal cell of select HDAC1, HDAC1's Albumen can't detect (Figure 10), illustrates to be mutated ATG from protein level, can successfully realize the knockout of target gene.

The result shows that: the base mutation of sgRNA targeting has occurred in target gene initiation codon, translates it from playing Beginning effect is realized to the success of people's APOE, PD1, LAG3, TIGIT, VISTA, CD224, CD160, SOX9, HDAC1 gene knockout.

Embodiment 2

The base editor that ABE or BE4 is mediated is carried out on primary cell, mutation initiation codon realizes gene knockout.

Routinely operate, with the T cell of people into the gene knockout (passing through electrotransfection or liposome transfection) of primary cell, with For electrotransfection.

(1) PBMC cell isolates and purifies:

A, peripheral blood is acquired with anticoagulant tube, rocking in acquisition is sufficiently mixed peripheral blood with anti-coagulants；

B, peripheral blood cells mix in equal volume with lymphocyte separation medium, centrifugation, the tunica albuginea confluent monolayer cells after drawing centrifugation；

C, it is centrifuged after mixing obtained tunica albuginea confluent monolayer cells with serum-free cell culture medium 1640, collects sedimentation cell, i.e., For PBMC cell.

Above-mentioned separation process repeats three times.

(2) enrichment of CD3 positive cell

A, PBMC cell concentration is adjusted to 50x10⁶cell/ml。

B, 50 μ l of CD3+enriched antibodies cocktail is added by every 1ml, 5 points is stored at room temperature after mixing Clock.

C, 150 μ l of magnet is added by every 1ml, is stored at room temperature after mixing 10 minutes,

D, centrifuge tube is placed on magnetic frame and stands 5 minutes, draw upper cell suspension into new 15ml centrifuge tube.

E, it is primary to repeat the operation.

F, room temperature collects cell with 300g centrifugation 10 minutes.

G, cell count.

(3) electricity of CD3 positive cell turns

A, configuration electricity swivel system

8 μ g BE3, BE4 or ABE plasmid pGL3-U6-sgRNA matter corresponding with 8 μ g are separately added into 1.5ml centrifuge tube Grain, and turn kit specification requirement according to Lonza Amaxa electricity, 82 μ l electricity are added and turn buffer and 18 μ l Supplement1 is mixed.

B, 20X10 is collected⁶Into 15ml centrifuge tube, 300g is centrifuged 10 minutes a cell, abandons supernatant.

C, buffer solution mixture is turned with the plasmid electricity prepared in A and cell is resuspended, and be transferred in electric revolving cup.

D, electricity is carried out using instrument Lonza 2B, U-014 program to turn.

E, the cell after electricity turns is transferred quickly in the AIM-V culture medium added with 10%FBS preheated in advance, and 37 degree It is cultivated 2 hours in 5% carbon dioxide incubator.

F, the cell after electricity turns changes liquid entirely, with 1X10⁶Cell, overnight incubation is resuspended in the density of a/ml.

(4) the activation culture of T cell

A, after electricity turns culture 24 hours, 100U/ml IL-2 is added into culture medium, and CD3/ is added according to the ratio of 1:1 CD28dynabeads activates T cell.

B, liquid is partly changed to cell, or adds IL-2 within every two day, cell density maintains 1X10 always⁶A/ml.

C, after activating 5 days, T cell is collected into 15ml centrifuge tube, and centrifuge tube is placed in magnetic frame, it slowly will be upper It is transferred to clearly in another clean 15ml centrifuge tube, it is primary to repeat this step.

D, room temperature is centrifuged 300g, 10 minutes, abandons supernatant, and with 10%FBS, 300U/ml IL-2AIM-V culture medium is resuspended thin Born of the same parents, density domination is in 1X10⁶A/ml.

E, liquid is partly changed to cell, or adds IL-2, and count within every two day, cell maintains 1X10⁶A/ml.

(5) genotyping

A, receiving portions cell is in lysate (10 μM of Tris-HCl, 0.4M NaCl, 2 μM of EDTA, 1%SDS) with 100 μ After the cracking digestion of g/ml Proteinase K, 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, purifies acquisition with AxyPrep PCR cleanup PCR recovery product takes 200ng to be uniformly diluted to 20 μ l and is denaturalized, is annealed, program such as: 95 DEG C, 5min；95-85℃at-2 ℃/s；85-25℃at-0.1℃/s；hold at 4℃.

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 37 DEG C incubate 30 minutes, 1 μ l product and pMD19-T vector (TAKARA, 3271) is taken to connect and convert DH5 Competent cell (TransGen, CD201).

D, each mutant target gene of T cell, following (the runic table of sequencing result is sequenced with universal primer M13-F in picking monoclonal Show PAM；Italic indicates Candidate Mutant coding；Lower stroke of expression mutating alkali yl of italic):

1.hAPOE

Sg-1:

Mut:

Sg-2:

Mut:

2.hPD1

Sg-1:

Mut:

3.hLAG3

Sg-1:

Mut:

Sg-2:

Mut:

4.hTIGIT

Sg-1:

Mut:

Sg-2:

Mut:

5.hVISTA

Sg-1:

Mut:

6.hCD224

Sg-1:

Mut:

7.hCD160

Sg-1:

Mut:

8.hSOX9

Sg-1:

Mut:

9.HDAC1

Sg-1:

Mut:

The result shows that: the base mutation of sgRNA targeting has occurred in target gene, and gene start codon ATG introduces mutation, real Now to the success of the gene knockouts such as people APOE, PD1, LAG3, TIGIT, VISTA, CD224, CD160, SOX9, HDAC1.

Embodiment 3

Construct the knock out mice that ABE or BE4 is mediated

Routinely operation carries out embryo collection, microinjection, Embryo Culture and the embryo transfer etc. of mouse.With TIM3 and Knock out mice is constructed for LAG3 gene.

(1) microinjection: mRNA the and TIM3 specificity sgRNA that fertilized eggs inject ABE or BE4 respectively (corresponds to above-mentioned Sequence nine) or ABE or BE4 mRNA and LAG3 specificity sgRNA (correspond to above-mentioned sequence ten).It is conventional to carry out embryo transfer；

(2) genotyping: conventional mouse cuts tail and extracts genomic DNA, and PCR distinguishes amplification coding region, and Sanger is surveyed Sequence, sequencing result is following, and (runic indicates PAM；Italic indicates mutation coding；Lower stroke of expression mutating alkali yl of italic):

10.mTIM3

Sg-1:

Mut:

Sg-2:

Mut:

11.mLAG3

Sg-1:

Mut:

Sg-2:

Mut:

The above results confirm TIM3 and LAG3 initiation codon introduce mutation, building TIM3 and LAG3 knock-out mice at Function.

Sequence table

<110>national health State Family Planning Commission Institute Of Science And Technology

<120>method and application of gene knockout are realized based on base editing system mutation initiation codon

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8811

<212> DNA

<213>artificial sequence ()

<400> 1

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420

gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga agtcgagttt 480

agccacgagt attggatgag gcacgcactg accctggcaa agcgagcatg ggatgaaaga 540

gaagtccccg tgggcgccgt gctggtgcac aacaatagag tgatcggaga gggatggaac 600

aggccaatcg gccgccacga ccctaccgca cacgcagaga tcatggcact gaggcaggga 660

ggcctggtca tgcagaatta ccgcctgatc gatgccaccc tgtatgtgac actggagcca 720

tgcgtgatgt gcgcaggagc aatgatccac agcaggatcg gaagagtggt gttcggagca 780

cgggacgcca agaccggcgc agcaggctcc ctgatggatg tgctgcacca ccccggcatg 840

aaccaccggg tggagatcac agagggaatc ctggcagacg agtgcgccgc cctgctgagc 900

gatttcttta gaatgcggag acaggagatc aaggcccaga agaaggcaca gagctccacc 960

gactctggag gatctagcgg aggatcctct ggaagcgaga caccaggcac aagcgagtcc 1020

gccacaccag agagctccgg cggctcctcc ggaggatcct ctgaggtgga gttttcccac 1080

gagtactgga tgagacatgc cctgaccctg gccaagaggg cacgcgatga gagggaggtg 1140

cctgtgggag ccgtgctggt gctgaacaat agagtgatcg gcgagggctg gaacagagcc 1200

atcggcctgc acgacccaac agcccatgcc gaaattatgg ccctgagaca gggcggcctg 1260

gtcatgcaga actacagact gattgacgcc accctgtacg tgacattcga gccttgcgtg 1320

atgtgcgccg gcgccatgat ccactctagg atcggccgcg tggtgtttgg cgtgaggaac 1380

gcaaaaaccg gcgccgcagg ctccctgatg gacgtgctgc actaccccgg catgaatcac 1440

cgcgtcgaaa ttaccgaggg aatcctggca gatgaatgtg ccgccctgct gtgctatttc 1500

tttcggatgc ctagacaggt gttcaatgct cagaagaagg cccagagctc caccgactcc 1560

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 1620

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 1680

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 1740

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 1800

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 1860

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 1920

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1980

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 2040

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 2100

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 2160

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 2220

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 2280

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 2340

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 2400

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 2460

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 2520

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 2580

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 2640

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 2700

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 2760

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 2820

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 2880

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 2940

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 3000

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 3060

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 3120

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 3180

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 3240

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 3300

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 3360

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 3420

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 3480

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 3540

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 3600

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 3660

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 3720

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 3780

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 3840

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 3900

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3960

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 4020

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 4080

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 4140

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 4200

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 4260

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 4320

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 4380

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 4440

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 4500

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 4560

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 4620

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 4680

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 4740

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 4800

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 4860

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 4920

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4980

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 5040

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 5100

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 5160

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 5220

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 5280

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 5340

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 5400

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 5460

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 5520

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 5580

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 5640

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 5700

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 5760

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa 5820

ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt 5880

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 5940

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 6000

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 6060

atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 6120

ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 6180

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 6240

gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 6300

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 6360

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 6420

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 6480

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 6540

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 6600

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 6660

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 6720

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 6780

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 6840

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 6900

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 6960

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 7020

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 7080

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 7140

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga 7200

acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 7260

tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 7320

ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 7380

catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 7440

ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 7500

caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 7560

ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 7620

tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 7680

cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 7740

aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 7800

tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 7860

gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 7920

cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 7980

aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 8040

tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 8100

tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 8160

gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 8220

atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 8280

taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc 8340

gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa 8400

gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 8460

aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 8520

gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta 8580

gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 8640

ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 8700

cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 8760

gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat c 8811

<210> 2

<211> 8961

<212> DNA

<213>artificial sequence ()

<400> 2

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420

gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcagcag tgaaaccgga 480

ccagtggcag tggacccaac cctgaggaga cggattgagc cccatgaatt tgaagtgttc 540

tttgacccaa gggagctgag gaaggagaca tgcctgctgt acgagatcaa gtggggcaca 600

agccacaaga tctggcgcca cagctccaag aacaccacaa agcacgtgga agtgaatttc 660

atcgagaagt ttacctccga gcggcacttc tgcccctcta ccagctgttc catcacatgg 720

tttctgtctt ggagcccttg cggcgagtgt tccaaggcca tcaccgagtt cctgtctcag 780

caccctaacg tgaccctggt catctacgtg gcccggctgt atcaccacat ggaccagcag 840

aacaggcagg gcctgcgcga tctggtgaat tctggcgtga ccatccagat catgacagcc 900

ccagagtacg actattgctg gcggaacttc gtgaattatc cacctggcaa ggaggcacac 960

tggccaagat acccacccct gtggatgaag ctgtatgcac tggagctgca cgcaggaatc 1020

ctgggcctgc ctccatgtct gaatatcctg cggagaaagc agccccagct gacatttttc 1080

accattgctc tgcagtcttg tcactatcag cggctgcctc ctcatattct gtgggctaca 1140

ggcctgaagt ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc 1200

gagtcagcaa caccagagag cagtggcggc agcagcggcg gcagcgacaa gaagtacagc 1260

atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 1320

gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 1380

ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 1440

accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 1500

agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 1560

gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1620

gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1680

gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1740

cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1800

cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 1860

gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 1920

gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 1980

ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 2040

agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 2100

gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 2160

agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 2220

gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 2280

tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 2340

gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 2400

gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 2460

aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 2520

gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 2580

cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2640

agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2700

tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2760

gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 2820

gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 2880

gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 2940

gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 3000

ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 3060

ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 3120

gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 3180

gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 3240

atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 3300

ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 3360

gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 3420

ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 3480

ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 3540

aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 3600

ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 3660

cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 3720

gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 3780

ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 3840

agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 3900

ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 3960

ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 4020

atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 4080

gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 4140

cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 4200

gcctacctaa acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 4260

gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 4320

caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 4380

aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 4440

ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 4500

ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 4560

aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 4620

gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 4680

gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 4740

accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 4800

tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 4860

gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 4920

gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 4980

ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 5040

gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 5100

ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 5160

gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 5220

tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 5280

ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 5340

ggtgacagcg gcgggagcgg cgggagcggg gggagcacta atctgagcga catcattgag 5400

aaggagactg ggaaacagct ggtcattcag gagtccatcc tgatgctgcc tgaggaggtg 5460

gaggaagtga tcggcaacaa gccagagtct gacatcctgg tgcacaccgc ctacgacgag 5520

tccacagatg agaatgtgat gctgctgacc tctgacgccc ccgagtataa gccttgggcc 5580

ctggtcatcc aggattctaa cggcgagaat aagatcaaga tgctgagcgg aggatccgga 5640

ggatctggag gcagcaccaa cctgtctgac atcatcgaga aggagacagg caagcagctg 5700

gtcatccagg agagcatcct gatgctgccc gaagaagtcg aagaagtgat cggaaacaag 5760

cctgagagcg atatcctggt ccataccgcc tacgacgaga gtaccgacga aaatgtgatg 5820

ctgctgacat ccgacgcccc agagtataag ccctgggctc tggtcatcca ggattccaac 5880

ggagagaaca aaatcaaaat gctgtctggc ggctcaaaaa gaaccgccga cggcagcgaa 5940

ttcgagccca agaagaagag gaaagtctaa ccggtcatca tcaccatcac cattgagttt 6000

aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 6060

cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 6120

aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 6180

aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 6240

ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga 6300

gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 6360

cacacaacat acgagccgga agcataaagt gtaaagccta ggatgcctaa tgagtgagct 6420

aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 6480

agctgcatta atgaatcggc caacgcgcgg gaagaggcgg tttgcgtatt gggcgctctt 6540

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 6600

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 6660

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 6720

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 6780

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 6840

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 6900

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 6960

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 7020

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 7080

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 7140

actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 7200

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 7260

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 7320

tcttttctac ggggtctgac actcagtgga acgaaaactc acgttaaggg attttggtca 7380

tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 7440

caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 7500

cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 7560

agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 7620

acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 7680

gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 7740

ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 7800

tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 7860

ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 7920

tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 7980

attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 8040

agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 8100

ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 8160

ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 8220

cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 8280

gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 8340

tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 8400

tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 8460

tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca 8520

gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg 8580

aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca 8640

attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca 8700

gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 8760

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 8820

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 8880

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 8940

tggcagtaca tcaagtgtat c 8961

Claims (7)

1. a kind of gene knockout method, comprising:

According to the translation initiation region sequence characteristic of gene to be knocked out, 20bp-NGG or CCN-20bp target sequence is designed, its packet is made Containing complete initiation codon ATG；

ABE or BE4 navigated into target sequence using sgRNA sequence so that target single base A, T or G in initiation codon It is mutated into G, C or A, so that initiation codon be made to lose function, reaches and target gene is knocked out；

If PAM is NGG, ATG is sported into GTG using ABE, if PAM is CCN, ATG is sported into ACG using ABE, or ATG is sported into ATA using BE4.According to specific catastrophe, if target single base A or T are preferably placed at target sequence using ABE 4-7 of column, if target single base G is preferably placed at 4-8 of target sequence using BE4；The sgRNA sequence be with The complementary corresponding 20bp sequence of target sequence.

2. according to the method described in claim 1, wherein ABE is selected from pCMV-ABEmax

BE4 is selected from: pCMV-AncBE4max, pCMV-BE4max.

3. according to the method described in claim 1, for knocking out following 11 target genes: people APOE, PD1, LAG3, TIGIT, VISTA, CD224, CD160, SOX9, HDAC1 and mouse TIM3 and LAG3, its corresponding sgRNA sequence respectively with sequence Target gene sequence shown in one to ten is complementary.

4. claim 1 the method cell line HEK293T carry out people APOE, PD1, LAG3, TIGIT, VISTA, CD224, The application of CD160, SOX9, HDAC1 gene knockout.

5. claim 1 the method human T-cell carry out people APOE, PD1, LAG3, TIGIT, VISTA, CD224, CD160, The application of SOX9, HDAC1 gene knockout.

6. the isolated T cell or cell line or their subculture obtained according to the application of claim 4 or 5.

7. a kind of kit for gene knockout, including sgRNA, ABE, BE4 described in claims 1 or 2 and amplification Reagent.