CN109666684A - A kind of CRISPR/Cas12a gene editing system and its application - Google Patents

Abstract

The invention relates to a CRISPR/Cas12a gene editing system and its application. The invention comprehensively utilizes biochemistry, molecular biology, cell biology and other means to screen and identify a new LiCas12a protein with endonuclease activity from a variety of different bacteria. Based on LiCas12a, a set of CRISPR/LiCas12a gene editing systems with high editing efficiency and high specificity were established in eukaryotic cells and prokaryotic cells respectively, and genetic manipulations such as gene knockout, insertion and point mutation were realized; and revealed Molecular mechanism of TERT gene promoter mutation promoting bladder cancer and liver cancer cell proliferation. The present invention establishes a gene editing system based on CRISPR-LiCas12a, and provides a more precise and efficient genome editing method for research fields such as disease pathogenesis and metabolic engineering.

Description

A kind of CRISPR/Cas12a gene editing system and its application

Technical field

The present invention relates to gene editing system, in particular to a kind of completely new high specific CRISPR/Cas12a system.

Background technique

First generation gene editing technology is exactly that homologous recombination establishes animal gene knockout, the gene mutation mould of gene knock-in Type.Gene knockout and gene knock-in are completed by DNA homologous recombination technology.This is an extremely complex technology, one As for establishing the animal model of genetic disease, be difficult to clinical or agricultural zootechnical.Second generation gene editing technology is zinc Finger nuclease (zinc finger endonuclease, ZFN) and class activating transcription factor effector nuclease (transcription activator-like effector nuclease, TALEN) technology.The principle of the two technologies is all It is the gene editing system that foundation is combined together by DNA nucleic acid binding protein and endonuclease.Because these albumen can To identify specific nucleotide sequence, which can carry out gene knockout and gene mutation to specific gene.

Third generation gene editing technology is CRISPR-Cas system.CRISPR(clustered regularly Interspaced short palindromic repeats) be life concern in history, bacterium and virus are waged a struggle generation Immune weapon, be briefly exactly that virus can utilize the cellular machinery of bacterium oneself gene integration to the genome of bacterium For the gene duplication service of oneself.Bacterium evolves CRISPR system to remove the exotic invasive gene of virus.Utilize this System, bacterium can destroy the special excision of viral genome.CRISPR-Cas system has two major classes type: I class and II class.Its In, I class needs multiple Cas albumen to form complex cutting DNA double-strand, and II class CRISPR-Cas system only needs a Cas Albumen carrys out cutting DNA double-strand.Just because of this feature of II class CRISPR-Cas system, after being modified, is widely used in original The genome editor of core and eukaryocyte.

The existing prokaryotic cell genome edit methods period is long, and homologous recombination efficiency is low, and by resistance marker and together The limitation of source arm is not well positioned to meet the needs of genome editor；And the eukaryon based on artificial incision enzyme ZEN and TALAN The edit methods of gene, then need to construct huge and complicated library, and process takes time and effort and inefficient, can not be to any Gene carries out genetic manipulation.Compared with traditional genome edit methods, genome editor of the CRISPR-Cas as a new generation Technology has the advantage that one, more editable sites.Theoretically, every 8bp just has one to be suitble in genome The site that CRISPR-Cas is edited；And for ZFN and TALEN, average 500bp and 125bp just has one respectively in genome A suitable editing sites.Two, multiple sites are edited simultaneously.In prokaryotes, ssDNA is depended on, the gene mediated by λ-Red Method of modifying MAGE (multiplex automated genome engineering) can accomplish polygenes editor, but it is only There is some superiority in allele exchange, is inserted beyond the DNA fragmentation of certain length (> 1kb) with regard to highly difficult.CRISPR-Cas Technology once-through operation can obtain Escherichia coli or mammalian cell containing 3 mutational sites simultaneously, this is for ZFN and TALEN It is difficult to realize for two technologies.Three, vector construction is simpler.In CRISPR-Cas system, it is desirable to change target sequence Recognition site need to only change one section of short RNA sequence, achievable more wheel editing process in one week.And ZFN and TALEN are then needed According to different identification sequence assemblings and to construct sufficiently complex albumen identification domain.So CRISPR-Cas is because of its operation letter The advantages such as single, experimental period is short and at low cost, are used widely in different plant species.

But there is also deficiencies by CRISPR/Cas9: 1, undershooting-effect (off-target effect): Cas9 is to target sequence Identification is mainly by the short rna of one section of 20bp, and when there is single even up to 5 base mispairings, cutting remains to normally send out It is raw, cause undershooting-effect；2, the identification limitation of PAM: if the end of target sequence 3 ' does not have PAM sequence (3 '-NGG-5 '), even if target sequence Column are exactly matched with sgRNA sequence, and Cas9 albumen will not cut the sequence site；3, Cas9 albumen has some species bright Aobvious cytotoxicity, these directly affect the editorial efficiency and the scope of application of CRISPR/Cas9 system.4, DNA is not suitable for Point mutation: due to CRISPR/Cas9 undershooting-effect with higher, even if DNA mutation is formed, but Cas9 can still be cut Target sequence easily causes the unstable of DNA and mutation to lose.Therefore, existing CRISPR/Cas9 system is improved, identification and exploitation are new CRISPR system, be genome editor field need carry out research work.

CRISPR/Cas12a also belongs to II class CRISPR/Cas system, with streptococcus pyogenes Streptococcus Equally, Cas12a also has endonuclease activity to pyogenes Cas9 (SpCas9).At present people, mouse, rice with Genome editor is realized in tobacco and cyanobacteria.In addition to there is advantage identical with Cas9, Cas12a has a characteristic that 1, Cutting efficiency is high, and undershooting-effect is low: Cas12a is guided by single crRNA, to the more efficient, more accurate of DNA cutting, therefore It is more suitable for the point mutation of genome editor, especially DNA, and Cas9 needs two molecules of crRNA and tracrRNA, to DNA The efficiency of cutting is lower；2, identification is rich in the PAM sequence of T: Cas12a identification is rich in the PAM sequence (3 '-TTN-5 ') of T, and Cas9 preference is rich in the PAM sequence (3 '-NGG-5 ') of G, and the PAM site Preference different from Cas9 greatly expands CRISPR system It unites manipulable gene range；3, building is simple, and high conversion efficiency: Cas12a (3687bp) molecular weight is smaller, it is easier to structure Carrier is built, is also easier to be transferred into intracellular；4, cutting mode: the staggered end of Cas12a cutting double-stranded DNA generation, and Cas9 Cutting double-stranded DNA generates flat end.The short outstanding end (overhang) that notch offset leaves facilitates the fine cut insertion of target fragment. Currently, only Francisella novicida Cas12a (FnCas12a), Acidaminococcus sp.BV3L6Cas12a (AsCas12a) with three kinds of Cas12a albumen quilts of Lachnospiraceae bacterium ND2006Cas12a (LbCas12a) Report has the function of the ability of endonuclease and genome editor, be not able to satisfy to different plant species, difference target gene into The demand of row genome editor.Identify new Cas12a albumen, establishing different CRIPSPR/Cas12a systems facilitates CRISPR genome editing system it is perfect.

Summary of the invention

Low for traditional homologous recombination system recombination frequency, at high cost, the period is long in operation, heavy workload, with And Cas9 there are undershooting-effect, to the identification of PAM limitation, the problems such as not being suitable for point mutation, the present invention is to CRISPR/ Cas12a system is optimized, and has invented a kind of efficient, special genetic manipulation method, and it is thin to be applied to mammal The genome editor of born of the same parents and Escherichia coli provide powerful for the functional study and metabolic engineering of gene.

Present invention firstly provides a kind of LiCas12a genes for CRISPR/Cas12a system optimization, and sequence is such as In sequence table shown in SEQ ID No:1.

The present invention provides the recombinant vectors for constructing simple substance grain or double-mass model CRISPR/LiCas12a system, to take The recombinant vector phCas or pCas of LiCas12a gene with above-mentioned screening.The LiCas12a gene is coding LiCas12a The gene of albumen.

Above-mentioned recombinant vector phCas applies to mammalian cell, and the recombinant vector is inserted on pCas carrier The LiCas12a gene of the screening of CMV promoter driving expression, while including the crRNA+ of U6 promoter driving expression N23 sequence, sequence is as shown in SEQ ID No:2 in sequence table.

The carrier pCas applies to Escherichia coli, and the recombinant vector is inserted on pTarget carrier The crRNA+N22 sequence of pJ23119 promoter driving expression, sequence is as shown in SEQ ID No:3 in sequence table

The present invention constructs a kind of simple substance grain CRISPR/LiCas12a for carrying recombinant vector phCas and pcrRNA in turn System, and carry the double-mass model CRISPR/LiCas12a system of recombinant vector pCas and pcrRNA.

Above-mentioned simple substance grain and double-mass model CRISPR/LiCas12a system and completely new high specific CRISPR/ LiCas12a system, which is characterized in that it is comprising two or more digestion with restriction enzyme that the target sequence, which is connected into site, The multiple cloning sites in site.

Further, above-mentioned simple substance grain and double-mass model CRISPR/LiCas12a system, which is characterized in that the pCas Contain selectable marker gene on plasmid, and includes temperature sensitive replicon repA101ts.

The present invention constructs a kind of completely new high specific CRISPR/LiCas12a gene editing system, including in right The recombinant vector of the LiCas12a gene for the carrying screening stated.

Using completely new high specific CRISPR/LiCas12a system of the invention, can in mammalian cells into The gene editing that row CRISPR/LiCas12a is mediated, specifically includes the genetic manipulations such as knockout and the point mutation of gene.Above-mentioned application Target sequence is single stranded DNA or linear dsdna.

Using completely new high specific CRISPR/LiCas12a system of the invention, can be carried out in Escherichia coli Gene editing is realized in the homologous recombination operation of CRISPR/LiCas12a auxiliary, specifically includes knockout, insertion and the point mutation of gene Equal genetic manipulations.

The homologous recombination system for the CRISPR/LiCas12a auxiliary that the present invention is constructed is applied to mammalian cell and greatly In enterobacteria, either CRISPR/LiCas12a assists single strand dna oligonucleotide homologous recombination or CRISPR/Cas12a auxiliary Double chain DNA fragment homologous recombination is helped, can realize required mutation.The results show system can be in Escherichia coli and lactation High specific is worked normally and shown in animal and cannot detect undershooting-effect.

Detailed description of the invention

The phylogenetic tree of Fig. 1 .Cas12a albumen homology object.

The external functional verification of Fig. 2 .LiCas12a.

The DNMT1 gene knockout for the 293T cell that Fig. 3 .LiCas12a is mediated.

The DNMT1 albumen of Fig. 4 .Western Blot detection 293T cell.

The undershooting-effect detection of LiCas12a and SpCas9 in Fig. 5 293T cell.

The TERT gene promoter mutation for the tumour cell that Fig. 6 .LiCas12a is mediated

The expression of TERT gene and thin in TERT gene promoter mutation modulate tumor cell in Fig. 7 .T24 and Huh7 cell Born of the same parents' proliferation.

The double-mass model system of Fig. 8 .pLiCas12a and pcrRNA.

The PCR analysis of Fig. 9 .cadA, meaA, meaB and lacZ missing；LiCas12a edits cadA, the meaA in clone, The representative Sanger of meaB and lacZ is sequenced.

Figure 10 is surveyed in Escherichia coli by the miss the target Sanger in site of the lacZ of LiCas12a and the SpCas9 prediction mediated Sequence.

The PCR analysis of Figure 11 .gfpmut3a insertion；The flow cytometry that gfpmut3a is expressed in EC3004.

Figure 12 screens mutant strain on the LB agar containing streptomysin；It is tested by test streptomycin resistance and DNA sequencing Demonstrate,prove mutant strain EC3013.

Specific embodiment

The present invention is described in further detail below by embodiment, the range of but do not limit the invention in any way.

Experimental method in following embodiments is unless otherwise specified conventional method.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Human cell line 293T (CRL-3216) and bladder cancer cell lines T24 (HTB-4) is obtained from American Type Tissue Culture Center (ATCC, MA, USA).HCC cell line Huh7 (JCRB JCRB0403) comes from Health Science Research Resources Bank (HSRRB, Osaka, Japan).FBS, penicillin, DMEM culture medium, streptomysin come from Thermo Fisher Scientific (MA, USA).The scheme of separation quality grain and genomic DNA is from (the China north manufacturer Qiagen Capital).The Taq archaeal dna polymerase that PCR is used comes from Thermo Fisher Scientific (MA, USA), Q5High- Fidelity DNAPolymerase comes from New England Biolabs (MA, USA).Restriction endonuclease and T4DNA Ligase is purchased from Takara.NEBuilder comes from New England Biolabs (MA, USA).Plasmid pCas9 is Addgene Products, catalog number #62225.All crRNA and sgRNA for biochemical reaction are by HiScribe T7High Yield RNASynthesis Kit (New England Biolabs, MA, USA) synthesis.Corresponding to the reversed of target RNA sequence The ssDNA oligonucleotides of complementary series is synthesized by Invitrogen (Chinese Shanghai).MEGAclear Transcription Clean-Up Kit comes from Ambion (MA, USA).Lysis buffer from Cutsmart (New England Biolabs, MA, USA).PCR purification column comes from Qiagen (Beijing, China).FACSCalibur flow cytometer from BD (New York, USA).RNA separating kit is Qiagen product (Beijing, China).M-MLV reverse transcriptase comes from Promega (Madison, Wisconsin, USA).7300 analyzer of ABI comes from Applied Biosystems (MA, USA).SYBR Green comes from Qiagen (Beijing, China).TERT antibody (C-12) is the production of Santa Cruz Biotechnology company Product, production number sc-377511.DNMT1 antibody (D63A6) is Cell Signaling Technology Products, product Number be #5032.Pierce TM ECL Western Blotting Substrate comes from Thermo Fisher Scientific company (MA, USA).Cell counting Kit -8 (CCK-) comes from Dojindo (Kumamoto, Japan).Plate Reader (Multiskan GO Microplate Spectrophotometer) comes from Thermo Fisher Scientific (MA, USA).

Embodiment one

40 Cas12a protein sequences are collected from Uniprot, phylogenetic tree construction is analyzed, wherein LiCas12a It is proved to the cleavage activity with double-stranded DNA.The specific method is as follows:

In order to find the new functionalized Cas12a for genome editor, we from Uniprot (https: // Www.uniprot.org) have collected 40 Cas12a protein sequences, and by phylogenetic tree by these sequences with AsCas12a, FnCas12a and LbCas12a analyze (Fig. 1) together.In view of the Different Evolutionary origin of Cas12a albumen and length Degree comes from Arcobacter butzleri L348, Eubacterium eligens, Francisella philomiragia 5 kinds of Cas12a albumen of subsp.Select philomiragia, Helcococcus kunzii and Leptospira inadai Serovar Lyme is as candidate.In this five candidates, LiCas12a proves there is DNA lytic activity.

LiCas12a is cloned into pGEX-6p-1 carrier first.Use the Ni affinity chromatography from e. coli bl21 Express simultaneously protein purification LiCas12a.The crRNA and N22 that are guided by synthetic promoter pJ23119 are assembled into pTargetF To construct pcrRNA.5 kinds of crRNA of targeting pMB1 replication orgin are devised using Cas-Designer.Pass through HiScribe^TM T7 high yield RNA synthetic agent box transcribes crRNA and N22 in vitro.

In order to test Bacillus coli expression LiCas12a whether can with 37 DEG C in vitro cutting target DNAs, carry out plasmid and The cutting of PCR product measures.LiCas12a linearizes plasmid pcrRNA when can have crRNA in vitro.When providing in vitro When crRNA, LiCas12a can also cut the PCR product containing the region pMB1.Therefore, LiCas12a can be instructed with crRNA In 37 DEG C of effective cutting double-stranded DNA targets (Fig. 2).

Embodiment two

In order to verify the activity of LiCas12a in mammalian cells, i.e., whether have the function of shearing DNA, Wo Mentong Cross following verified.

The LiCas12a gene filtered out (sequence is shown in SEQ ID No:1 in sequence table) is cloned into crRNA first With selected in the plasmid pLiCas12a of N23 DNMT1 gene as knock out target spot (sequence is shown in SEQ ID No:4 in sequence table).It will The LiCas12a plasmid transfection of the targeting DNMT1 of building then sub-elects the training of GFP positive cell monoclonal into 293T cell It supports, extract the genomic DNA of monoclonal cell and is used as template to expand DNMT1, sequence verification.

In 10 monoclonal cell systems, the DNMT1 gene in 9 monoclonal cell systems is successfully destroyed (90%).Fig. 3 Show representative sequencing result.In western blot analysis, compared with wild-type cell (WT), DNMT1 deletion cells (Mut) expression of DNMT1 is totally disrupted (Fig. 4) in.It predicts that LiCas2a potentially misses the target site by website, has selected 13 A prediction bits point positioned at different chromosomes is detected, and does not detect apparent undershooting-effect (Fig. 5).Result above table Bright LiCas12a can effectively the gene in knock-out mammals cell without undershooting-effect.

Embodiment three

Point mutation and back mutation in the mammalian cell of CRISPR-LiCas12a System-mediated.

The TERT of encoding telomerase reverse transcriptase is the component of Telomerase, and Telomerase is repeated by addition telomere The ribonucleoprotein polymerase of TTAGGG21 maintenance telomerase.TERT promoter often changes in many tumor types, example Such as melanoma, hepatocellular carcinoma, glioma, bladder transitional cell carcinoma and clear-cell carcinoma.Chr5,1,295,228C in TERT promoter > T and 1,295,250 C > T mutation is most important mutation.We test TERT in bladder cancer and hepatocellular carcinoma cells system Genomic states, select T24 and Huh7 as editor target.

For TERT gene promoter region devise two independent crRNA (sequence is shown in SEQ ID No in sequence table: 5).Bladder cancer cell lines T24 (chr 5:1295228T) and hepatocellular carcinoma cells system Huh7 (chr 5:1295228C) is selected to make For edit object.By building targeting TERT promoter region pLiCas12s plasmid and DNA recovery template cotransfection to T24 with In Huh7, and GFP positive monoclonal cell is sub-elected by flow cytometry.PCR sequencing result shows that TERT promoter exists It is successfully corrected to 228C in T24 cell and is mutated into 228T (Fig. 6) in Huh7 cell.When TERT promoter C228T is mutated Afterwards, the expression quantity of TERT mRNA and albumen all increases, and promotes the proliferation (Fig. 7) of tumour cell.

Therefore, LiCas12a can be applied to the point mutation and back mutation of tumour cell, have for what gene therapy provided Power tool.

Example IV

Next whether verifying CRISPR/LiCas12a can be subjected to gene editing to Escherichia coli, which will LiCas12a and crRNA functional element is building up on two plasmids respectively and carries out.That is the CRISPR- of pLiCas12a and pcrRNA LiCas12a system.In plasmid pCas, LiCas12a is driven using from the promoter for making Streptococcus pyogenes (PspCas9) Cas9 Expression, be built into pLiCas12a.In plasmid pcrRNA, the crRNA+N22 sequence of pJ23119 promoter driving expression is inserted Column, target the target site of target genome.(Fig. 8).

4 genes cadA, lacZ, meaA and meaB are selected to be knocked out, respectively each gene design two independent CrRNA (sequence is shown in SEQ ID No:6 in sequence table) constructs pcrRNA plasmid respectively.Firstly, pLiCas12a electricity is gone to greatly Enterobacteria W3110 is prepared W3110-pLiCas12a competent cell (preparation method is as follows).Then electricity is transferred to pcrRNA plasmid, Secondary culture and identification.

Escherichia coli Electroporation-competent cells preparation method:

First day:

1. bacterial strain is placed on LB culture medium, it is incubated overnight at 37 DEG C,

2. the big centrifugal bottle of high-temperature sterilization (250-500ml) was used in case of second day shaking flask,

3. preparing several bottles of aqua sterilisas (about 1.5 liters of total amount), it is stored in freezing chamber in case second day resuspension cell is used.

Second day

4. shifting 0.2-1ml overnight culture to the 1-2 liter that 500ml LB (or other culture mediums full of nutrition) are housed Shaking flask,

5. violent shaken cultivation 2-6 hours at 37 DEG C

6. timing monitoring OD600 value (per half an hour measurement is primary after culture 1 hour),

7. take ouing of the shaker shaking flask when OD600 value reaches 0.4-0.6, it is placed in cooled on ice at least 15 minutes and (needs If this mode can store culture solution a few hours),

8. cell is centrifuged 15 minutes at 4 DEG C of 5000g, abandoning supernatant, (as needed, precipitating can be in 4 DEG C of 10% glycerol Save a couple of days),

9. first using vortex instrument or pipette resuspension cell (several in a small amount of volume with the ice water resuspension cell of sterilizing Milliliter), it is then diluted with water to 2/3 volume of centrifuge tube,

10. shining previous step repeated centrifugation, liquid is carefully discarded supernatant,

11. according to the ice water resuspension cell of previous step sterilizing,

12. supernatant is abandoned in centrifugation,

13. 10% glycerol resuspension cell after being sterilized with 20ml, ice-cold,

14. being centrifuged according to previous step, liquid (precipitating may be very loose) is carefully discarded supernatant,

15. it is 2-3ml with 10% glycerol resuspension cell to final volume,

16. cell is packed into microcentrifugal tube by 150 μ l equal portions, saved in -80 DEG C.

4 genes of cadA, lacZ, meaA and meaB are successfully deleted in three independent experiments in W3110, editor Efficiency is up to 94.45~100% (Fig. 9).

In the site of missing the target of prediction, be not detected by LiCas12a mediate miss the target DNA double chain fracture DSB (0/1, 0.0%) (Figure 10).

It tests while cadA in Escherichia coli, lacZ, meaA and 4 genes of meaB is knocked out using SpCas9 Experiment, average knockout efficiency is 38.89~52.78%.In 6 sites of missing the target of prediction, detect what SpCas9 was mediated One DNA double chain fracture (DSBs) (1/6,16.7%) of significantly missing the target.Therefore, in Escherichia coli, LiCas12a is one More efficient, more special gene editing tool.

Embodiment five

Design the feasibility that following experimental verification CRISPR-LiCas12a system carries out gene insertion in Escherichia coli.

Insertion of the site pseudogene ECK0694 for gfpmut3a in W3110 genome is selected, and design two is independent CrRNA (sequence is shown in SEQ ID No:7 in sequence table).By the gfpmut3a containing target position ECK0694 upstream and downstream homology arm Gene order is inserted into the downstream crRNA, constructs pcrRNA plasmid, and electricity is transferred to W3110-pLiCas12a competent cell.

By passing on and identifying, gfpmut3a gene is successively inserted into W3110- by CRISPR-LiCas12a system The site ECK0694, efficiency are up to 52.78%.Using similar method, while having detected SpCas9 and being mediated in Escherichia coli The editorial efficiency of gfpmut3a gene insertion, is 22.78%.It can be seen that the large intestine of CRISPR-LiCas12a System-mediated The efficiency of bacillus gene insertion is significantly higher than CRISPR-SpCas9 system (Figure 11).

Embodiment six

In order to assess the feasibility for using CRISPR-LiCas12a system to carry out accurate point mutation, by single base mutation (A128C) it is introduced into the rpsL gene of coding small subunit ribosomal protein S12, which assigns Escherichia coli streptomysin Resistance.For rpsL gene, two independent crRNA are designed, by the point mutation containing target position rpsL upstream and downstream homology arm Gene order is inserted into the downstream crRNA, constructs pcrRNA plasmid respectively, and electricity is transferred to W3110-pLiCas12a competent cell.

In W3110, by CRISPR-LiCas12a system by rpsL gene mutation be rpsL^A128C(sequence is shown in sequence table Middle SEQ ID No:8), editorial efficiency is 67.93% (Figure 12).However, by CRISPR-SpCas9 system, in W3110 The efficiency of A128C point mutation is 40.39%, significant to be lower than LiCas12a.Therefore, LiCas12a is lacked in the gene of Escherichia coli It loses, shows higher genome editorial efficiency more significant than SpCas9 in insertion and point mutation.

Sequence table

<110>Beijing University of Chemical Technology

<120>a kind of CRISPR/Cas12a gene editing system and its application

<141> 2018-12-25

<160> 19

<170> SIPOSequenceListing 1.0

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ttaaagcccg tgggaaagac tctagaacat atagagaaaa aaggattttt aaagaaagat 120

aaaattcgag ccgaagacta taaggccgtt aaaaaaatta tagataagta tcatagagca 180

tatatcgaag aagtctttga ttcagtattg catcaaaaaa agaaaaaaga taaaacaaga 240

ttctcgactc aatttataaa ggagataaaa gaattctccg aattgtatta caagacggaa 300

aagaatatac cggataaaga gaggcttgag gcattatccg agaaactaag aaagatgcta 360

gtaggggctt ttaaagggga atttagtgaa gaagtcgctg aaaaatataa aaatctattt 420

agtaaggaat taatcagaaa tgaaattgag aaattctgtg agacggatga ggaaaggaaa 480

caggtttcga attttaagtc atttacaact tactttactg gatttcatag caatcggcag 540

aatatatatt cggatgaaaa aaaatctacg gctatcggat atcgaatcat tcaccaaaat 600

cttccgaaat ttctcgataa cttaaaaatc atcgaatcta ttcaaaggcg atttaaagat 660

tttccctggt cggatttgaa aaagaattta aaaaaaatag ataagaacat taaattaaca 720

gaatattttt cgattgatgg gtttgttaat gtcttaaatc aaaaaggaat agatgcgtat 780

aatacgattt tgggagggaa atcggaagaa agtggagaga aaattcaggg gcttaatgaa 840

tatattaatt tatacagaca gaagaataat atagatcgga aaaatttacc gaatgtaaag 900

attttattca aacaaattct cggggatcgg gaaactaaat cctttattcc cgaagcattt 960

ccggacgatc aaagcgtctt aaattccatc acagaatttg caaagtatct gaaactcgat 1020

aaaaaaaaga agagcatcat agcagagctt aaaaagtttc tttcgagctt caatcggtat 1080

gagctagatg gaatttatct agcaaatgat aatagcctcg catcaatctc caccttcctc 1140

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gatcctaaaa aaaagataaa gtcgcctctc aaatatgaaa aagaaaaaga aaaatggtta 1260

aaacaaaaat actatacaat ttcattcttg aacgatgcca tagaatctta ttccaaaagt 1320

caggacgaaa aacgagttaa gattcgtctc gaagcctact ttgcggagtt caaatcgaag 1380

gatgatgcaa aaaaacaatt cgatctactc gaaaggatag aagaggctta tgcgatagta 1440

gaaccgcttt tgggagccga atacccgagg gatcgaaatc ttaaagctga taaaaaggaa 1500

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ggaaaaatat attcgaaaga aaaattcaaa ttaaactttg aaaactctac attgctaaag 1740

ggctgggatg agaatcgcga agttgctaac ttgtgtgtaa tttttcggga agatcaaaaa 1800

tattatctcg gggtaatgga taaggaaaat aatacaattt tatcggatat tcctaaagta 1860

aaacccaatg aattgtttta tgaaaagatg gtgtataaac ttattcccac tcctcatatg 1920

cagcttccaa gaataatctt ttcttcagat aatttgagta tttataatcc gtctaaatct 1980

attttaaaga tccgagaggc taagagtttt aaagaaggca agaactttaa gctaaaggac 2040

tgccacaagt tcattgattt ctataaggaa tccatttcta aaaatgaaga ttggagtagg 2100

ttcgacttta aattctcaaa aacctccagt tacgaaaata ttagcgaatt ttatcgagaa 2160

gtagaaagac aaggttacaa tctcgacttt aagaaagtat caaagtttta tatcgattcg 2220

ttagtagagg atggtaagct ttatctattt caaatttata ataaggattt ttcgatcttt 2280

tctaaaggta agccgaacct tcacacgatt tattttaggt cattgttctc caaggaaaat 2340

ttaaaggatg tatgcctgaa gctaaacggc gaagcggaaa tgttctttcg taagaaatca 2400

attaactacg atgaaaaaaa gaaacgagag ggtcaccatc ctgaattatt cgagaaatta 2460

aaatatccga ttttaaagga taaacgttat tcggaagata aattccaatt tcacttacct 2520

atttctttaa acttcaaatc aaaggaacga ctcaatttta atctcaaagt aaacgaattt 2580

cttaaaagga ataaggatat aaatattatt gggatcgatc gtggagagcg taaccttctc 2640

tatttagtca tgatcaatca gaagggggag atccttaaac aaaccttgct agattcaatg 2700

caaagtggga aaggccgtcc tgaaataaat tacaaagaga agttacaaga aaaagaaatt 2760

gaaagagata aggcgagaaa atcttggggg accgtagaga atatcaaaga actaaaagaa 2820

ggctatttat ccatagtaat tcatcaaatt tcaaaactca tggtcgaaaa taatgcgatc 2880

gttgtattgg aggacttgaa tataggattt aagagggggc gtcaaaaagt agaaaggcag 2940

gtttatcaaa aatttgagaa aatgttaatt gataaactga atttccttgt attcaaagaa 3000

aataaaccaa cggagccggg aggagtgttg aaagcttatc aattaacgga tgagtttcaa 3060

agtttcgaaa aattaagtaa gcagactgga tttctttttt atgtgccctc ctggaatact 3120

agtaagatag atccaagaac gggatttatc gattttttac acccggcata tgaaaatata 3180

gaaaaagcta aacaatggat caataaattt gattcgattc ggtttaattc taaaatggac 3240

tggtttgaat ttactgctga cactagaaaa ttcagcgaaa atttaatgtt gggtaaaaat 3300

cgggtttggg ttatttgtac aacgaatgta gaaagatatt ttacgtctaa aactgcaaat 3360

tcatccattc aatataattc cattcaaatc acggaaaaat taaaagagct atttgtcgat 3420

attccctttt cgaatgggca agatctgaaa cctgaaatat tgaggaagaa tgatgcagta 3480

ttttttaaaa gcctattgtt ttatataaaa accactcttt ctcttaggca gaataatgga 3540

aaaaaggggg aggaggaaaa agattttata ctctctccag tagtggattc caaaggacgg 3600

ttttttaact ctttggaagc aagtgacgat gagccgaaag atgctgatgc caacggtgct 3660

tatcatatcg ctctgaaggg tcttatgaac cttctggttc tgaacgaaac taaagaagaa 3720

aatctgagta ggccgaaatg gaaaatcaaa aacaaagatt ggctggagtt tgtgtgggaa 3780

agaaatcggt aa 3792

<210> 2

<211> 21

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 2

taatttctac taagtgtaga t 21

<210> 3

<211> 20

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 3

taatttctac tgttgtagat 20

<210> 4

<211> 27

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 4

tttaaataaa gatttgtcct tggagaa 27

<210> 5

<211> 27

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 5

tttggaaagg ggtttggggg ggctgtt 27

<210> 6

<211> 27

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 6

tttacgcggc cccgccctct cctcgcg 27

<210> 7

<211> 27

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 7

tttgaggcag cgctgcgtcc tgctgcg 27

<210> 8

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 8

tttctgtact cctggtcaca tgggcg 26

<210> 9

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 9

tttaacgcag accgcagcta catggt 26

<210> 10

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 10

tttaatgatg atttcagccg cgctgt 26

<210> 11

<211> 27

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 11

tttcggcggt gaaattatcg atgagcg 27

<210> 12

<211> 27

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 12

tttatccagg ctgtgaaaca acgcgtg 27

<210> 13

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 13

tttgaagact ttgctcaaaa aaatgc 26

<210> 14

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 14

tttaagaaat tcgccgggat tgatgt 26

<210> 15

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 15

tttccggcgc gggtgccgca gcaatc 26

<210> 16

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 16

tttattcgct gtaaaaggaa accagg 26

<210> 17

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 17

tttccgctga aagaattaaa taatcc 26

<210> 18

<211> 25

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 18

tttacgcagc gcggagttcg gtttt 25

<210> 19

<211> 26

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 19

tttcgaagtg acttcctaca tcggtg 26

Claims (9)

1. a kind of LiCas12a gene for CRISPR/Cas12a system optimization, SEQ ID No:1 in sequence such as sequence table It is shown.

2. the recombinant vector for constructing simple substance grain or double-mass model CRISPR/LiCas12a system requires described for carrying right 1 The recombinant vector phCas or pCas of the LiCas12a gene of screening, the LiCas12a gene are coding LiCas12a albumen Gene.

3. a kind of CRISPR/LiCas12a gene editing system, including described in gene described in claim 1 or claim 2 Recombinant vector, and cannot detect undershooting-effect in genomic level.

4. carrier phCas, the crRNA sequence comprising the driving expression of U6 promoter, sequence such as SEQ ID No:2 institute in sequence table Show；Carrier pCas contains the crRNA sequence of pJ23119 promoter driving expression, SEQ ID No in sequence such as sequence table: Shown in 3.

5. simple substance grain or double-mass model CRISPR/LiCas12a system, single pUC pUC include carrying described in claim 2 or 4 The recombinant vector phCas of the LiCas12a gene of screening；Double-mass model system includes carrying screening described in claim 2 or 4 Recombinant vector pCas and the pcrRNA plasmid of LiCas12a gene.

6. simple substance grain as claimed in claim 5 or double-mass model CRISPR/LiCas12a system or as claimed in claim 3 one Kind CRISPR/LiCas12a gene editing system, it is comprising two or more restriction enzyme positions that target sequence, which is connected into site, The multiple cloning sites of point, while containing selectable marker gene on phCas the and pCas plasmid, and pCas includes that temperature is sensitive Property replicon repA101ts.

7. a kind of CRISPR/LiCas12a gene editing system, including such as simple substance grain described in claim 5 or 6 or double-mass model CRISPR/LiCas12a system, the application in mammalian cell and bacillus coli gene editor.

8. the application of simple substance grain CRISPR/LiCas12a system as claimed in claim 7 in mammalian cells, feature Be, by carrying out the gene editing of CRISPR/Cas12a mediation in mammalian cells, specifically include gene knockout and Point mutation genetic manipulation, template sequence used in point mutation is single stranded DNA or double-stranded DNA.

9. application of the double-mass model CRISPR/LiCas12a system as claimed in claim 7 in bacillus coli gene editor, It is characterized in that, gene editing, tool are realized in the homologous recombination operation by carrying out CRISPR/LiCas12a auxiliary in Escherichia coli Body includes knockout, insertion and the point mutation genetic manipulation of gene, and used template sequence is single stranded DNA or double-stranded DNA.