CN107619829B - The method that GINS2 gene knockouts are carried out to mescenchymal stem cell using CRISPR-CAS systems - Google Patents

Abstract

GINS2 gene editings are carried out for mescenchymal stem cell using CRISPR cas9 systems the present invention provides a kind of, more particularly to a kind of foundation of the mescenchymal stem cell cell line of structure GINS2 gene knockouts.Which use new synergistic protein CREnhancer1.0, can significantly improve intracellular CRISPR/Cas9 gene editings efficiency.Mesenchymal stem cell GINS2 provided by the invention, which knocks out plasmid, has preferable genetic stability.

Description

GINS2 gene knockouts are carried out to mescenchymal stem cell using CRISPR-CAS systemsMethod

Technical field

Present invention offer is a kind of to carry out GINS2 gene editings using CRISPR-cas systems for mescenchymal stem cell, specialIt is not to be related to a kind of foundation of the mescenchymal stem cell cell line of structure GINS2 gene knockouts.

Background technology

Mescenchymal stem cell (mesenchymal stem cells, MSC) is a kind of with the of self-replication capacity and multidirectionalThe adult stem cell of differentiation potential, this stem cell can develop into os osseum, cartilage, fat and other kinds of cell.Between fillMatter stem cell can receive transplanting, and they can grow into what type of cell and depend on its position injected.For example, byThe mescenchymal stem cell of injection heart can form new tissue of health etc..

Mescenchymal stem cell (MSCs) is a kind of multipotential stem cell, is derived from the mesoderm and ectoderm of mesoderm growing early stage.MainlyIt is present in connective tissue and organ interstitial, it is the abundantest with content in myeloid tissue, since marrow is its main source,It is referred to as mesenchymal stem cell.Mescenchymal stem cell belongs to non-terminally differentiated cells, its existing interstitial cell, and has endotheliumThe feature of cell and epithelial cell.Mescenchymal stem cell is in vitro under specific inductive condition, can be divided into fat, cartilage, bone,The Various Tissues cell such as muscle, tendon, nerve, liver, cardiac muscle, beta Cell of islet and endothelium, after continuous passage culture and freezen protectiveStill there is multi-lineage potential.Whether self or allogenic mescenchymal stem cell generally will not all cause host'sImmune response.Due to this immunological characteristic that mescenchymal stem cell has, makes it in autoimmune disease and various replaceGeneration treatment etc. has wide potential applicability in clinical practice.The structure and function of histoorgan can be rebuild by autotransplantation,And it can avoid immunological rejection.

The clinical research of mescenchymal stem cell is carried out in many countries, and the U.S. has approved 60 remainder clinical tests, withThe increasingly mature of mescenchymal stem cell and its relevant technologies, China also has approved multinomial clinical test, and it is dry to have entered into mesenchymaThe stage of cell core technical research.The development of stem-cell research work, including the high match cell of country are reinforced energetically in ChinaMore authoritative research institutions including genetic engineering Co., Ltd, cell products National Engineering Research Centre and each place umbilical cordInto clinic is guided investigative technique by blood bank.It is used to treat the Therapy study of more than ten kind refractory diseases for mescenchymal stem cell,Restore hematopoiesis in addition to being used for promoting, is improved other than leukaemia and refractory anemia etc. with candidate stem cell co-transplantation, be additionally operable to the heartCranial vascular disease, hepatic sclerosis, bone and muscle degenerative disease, brain and neurologic defict, senile dementia and lupus erythematosus and hardThe Therapy study of the autoimmune diseases such as skin disease, the partial clinical test result having been achieved with are encouraging.Research so farShow that the mescenchymal stem cell in umbilical cord source is not only able to the ideal substitute as mesenchymal stem cell, and hasThe application potential of bigger.Umbilical cord mesenchymal stem cells express the peculiar molecular marker of a variety of embryonic stem cells, have differentiation potentialGreatly, proliferative capacity is strong, immunogenicity is low, the limitation of convenient material drawing, amoral ethics problem, is easy to the features such as preparation of industrialization,It is therefore possible to the multipotential stem cells as most potential applicability in clinical practice.

GINS2 is one of DNA replication dna complex GINS family members, is located on human chromosome 16q24, mRNA length is1196bp, the protein that coding relative molecular mass is 21000.GINS is a kind of replicative helicase, before being moved to replication forkOpen DNA double chain.Studies have shown that GINS family members play a role in the generation of cancer, as GINS family members are invadingIt is overexpressed in attacking property melanoma, also there is document to show that DNA replication dna GAP-associated protein GAP plays the role of in different cells different, such asIn terms of determining that centerbody replicates quantity and the pathogenetic different phase of disease, GINS has played certain function, especially with dyeThe separation of colour solid is closely related.

And it is seldom in the report of tumour related field about GINS2 at present.In CN106620703 A, for people GINS2The siRNA sequence of gene, rna interference vector and RNA interfere slow virus, further have detected the heavy of GINS2 genesThe silent influence of efficiency, GINS2-siRNA slow virus to tumor cell proliferation ability and level of apoptosis, as a result display use the side RNAiThe proliferation of tumour cell can effectively be inhibited under method after the expression of mediator GINS2 genes and growth and promote its apoptosis, show GINS2Gene is proto-oncogene, can be used as the target spot of oncotherapy, can be used as inhibition by the expression of RNAi mode silence GINS2 genesThe effective means of tumor development.But in this study, it is interfered using SiRNA, the method has knockout not thoroughBottom, the defect for the knockout heredity that cannot stablize.In (Integration of Genomic, Biologic, and ChemicalApproaches to Target p53 Loss and Gain-of-Function in Triple NegativeBreastCancer in), although referring to CRISPR/Cas can be used for MCM2, GINS2, C19orf43, ELOVL2, ARL4D,DNM3OS, FGFR2, IFIT2, MPPED2, B2M, ERRFI1, GLUL CASP4, CPED1, SPTLC2, CMTM6, CFH, CARS2,SUMF1, but an only conception, there is no implement.CRISPR modifications are carried out for different genes to be not simply easy to setMeter, it needs to overcome numerous obstacles, has greatly experiment difficult.

Versatility based on mescenchymal stem cell, the mescenchymal stem cell in order to study knockout GINS2 genes are controlled in cancerFunction in terms for the treatment of, establishing the mescenchymal stem cell cell line of knockout GINS2 genes becomes particularly important.

Invention content

The object of the present invention is to provide a kind of mescenchymal stem cells knocking out GINS2 genes, effectively overcome the prior artThe technological deficiency of heredity cannot be stablized by carrying out interference using siRNA.

To achieve the above object, the present invention provides a kind of target of CRISPR-cas systems, according to the gene sequence of GINS2The specific selectable target site of row, design is following (dashed part indicates PAM motifs)：

GINS2-sgRNA1:5’to 3’gcgtctcctccgggacgctgagg

GINS2-sgRNA2:5’to 3’ccgaggagaccgtgaggctctgg

GINS2-sgRNA3:5’to 3’attcctcgccgagaaggagctgg

GINS2-sgRNA4:5’to 3’gtcgcctgctccctccagagtgg

GINS2-sgRNA5:5’to 3’cctgctccctccagagtggatgg

GINS2-sgRNA6:5’to 3’gtggatggatgtagaaaagttgg

GINS2-sgRNA7:5’to 3’aatgcccagcccttactacatgg

GINS2-sgRNA8:5’to 3’tcccgaaggcagacgaaatccgg

GINS2-sgRNA9:5’to 3’ggcagacgaaatccggaccctgg

GINS2-sgRNA10:5’to 3’tgacagctttgtgagacagcagg

GINS2-sgRNA11:5’to 3’cagctttgtgagacagcaggagg

GINS2-sgRNA12:5’to 3’gctggataacttgaccttgatgg

GINS2-sgRNA13:5’to 3’ttgatggagatcaacaccagcgg

GINS2-sgRNA14:5’to 3’ccgcacgaacctccagcctctgg

GINS2-sgRNA15:5’to 3’tctggagagtactcagtctcagg

GINS2-sgRNA16:5’to 3’gtctcaggacttctagagaaagg

GINS2-sgRNA17:5’to 3’gatgcatgaaaaatgtgtgatgg

GINS2-sgRNA18:5’to 3’gaaaaatgtgtgatggtgcaagg

GINS2-sgRNA19:5’to 3’atggtgcaaggaatggattcagg

GINS2-sgRNA20:5’to 3’gtccttaaaacttagctccctgg

GINS2-sgRNA21:5’to 3’tccttaaaacttagctccctggg

GINS2-sgRNA22:5’to 3’tctccctagcagagccacttggg

GINS2-sgRNA23:5’to 3’tgcatggaagccatcacacttgg

GINS2-sgRNA24:5’to 3’gcaggtgttcagtgactggtagg

GINS2-sgRNA25:5’to 3’ctggtaggtgtagatacagcagg。

According to these target sites, it is as follows to design specific sgRNA：

GINS2-sgRNA1:5’to 3’gcgtctcctccgggacgctg

GINS2-sgRNA2:5’to 3’ccgaggagaccgtgaggctc

GINS2-sgRNA3:5’to 3’attcctcgccgagaaggagc

GINS2-sgRNA4:5’to 3’gtcgcctgctccctccagag

GINS2-sgRNA5:5’to 3’cctgctccctccagagtgga

GINS2-sgRNA6:5’to 3’gtggatggatgtagaaaagt

GINS2-sgRNA7:5’to 3’aatgcccagcccttactaca

GINS2-sgRNA8:5’to 3’tcccgaaggcagacgaaatc

GINS2-sgRNA9:5’to 3’ggcagacgaaatccggaccc

GINS2-sgRNA10:5’to 3’tgacagctttgtgagacagc

GINS2-sgRNA11:5’to 3’cagctttgtgagacagcagg

GINS2-sgRNA12:5’to 3’gctggataacttgaccttga

GINS2-sgRNA13:5’to 3’ttgatggagatcaacaccag

GINS2-sgRNA14:5’to 3’ccgcacgaacctccagcctc

GINS2-sgRNA15:5’to 3’tctggagagtactcagtctc

GINS2-sgRNA16:5’to 3’gtctcaggacttctagagaa

GINS2-sgRNA17:5’to 3’gatgcatgaaaaatgtgtga

GINS2-sgRNA18:5’to 3’gaaaaatgtgtgatggtgca

GINS2-sgRNA19:5’to 3’atggtgcaaggaatggattc

GINS2-sgRNA20:5’to 3’gtccttaaaacttagctccc

GINS2-sgRNA21:5’to 3’tccttaaaacttagctccct

GINS2-sgRNA22:5’to 3’tctccctagcagagccactt

GINS2-sgRNA23:5’to 3’tgcatggaagccatcacact

GINS2-sgRNA24:5’to 3’gcaggtgttcagtgactggt

GINS2-sgRNA25:5’to 3’ctggtaggtgtagatacagc

In order to improve gene editing efficiency, including synergistic protein is introduced into mescenchymal stem cell, the synergistic proteinCREnhancer1.0 is by SEQ ID NO:Nucleotide sequence coded protein shown in 1.

Further, the synergistic protein is comprising a) or b)：

a)SEQ ID NO:The polynucleotide sequence of nucleotide sequence coded protein shown in 1；

b)SEQ ID NO:Amino acid sequence shown in 2.

Further, synergistic protein CREnhancer1.0 genes, the synergistic protein of structure EGFP labels are clonedCREnhancer1.0 Lentivirals pack slow virus, modified stem cell with GP2-293T cells.

Further, a kind of system carrying out gene editing using CRISPR/Cas9 in mescenchymal stem cell is provided,It is characterized in that the system comprises：(1) it is used to express SEQ ID NO：The plasmid of CREnhancer1.0 genes described in 1；(2) plasmid for the expression PX330 that sgRNA is already inserted into (it can express sgRNA and cas9).

Further, sgRNA the and cas9 expression vectors can also be other expression vectors commonly used in the art.

To achieve the above object, the present invention also provides a kind of mescenchymal stem cell cell lines of structure GINS2 gene knockoutsMethod, including editor's positive cell will be obtained in the gene editing system introducing mescenchymal stem cell, it then breeds, harvestThe stem cell.

Specific mescenchymal stem cell is human marrow mesenchymal stem cell (hMSCs) PC015, and purchase is biological from Shanghai Ai YanScience and Technology Ltd..

The present invention provides a kind of mescenchymal stem cell cell system, methods of structure GINS2 gene knockouts, have following excellentPoint：The present invention constructs the cell line of GINS2 gene knockouts in mescenchymal stem cell, screen and optimize obtain it is bestSgRNA knocks out efficient, passage stabilization.

Below by drawings and examples, technical scheme of the present invention will be described in further detail.

Description of the drawings

Fig. 1 PX330 plasmid figures；

SgRNA insertion points figure in Fig. 2 PX330；

25 sgRNA genetic marker efficiency schematic diagrames of Fig. 3；

Specific implementation mode

The technical solution for the method for improving genome editorial efficiency is further illustrated the present invention below by specific embodiment.

The structure of embodiment 1, CRISPR expression vectors

The design of gRNA

According to the gene order of target gene, by the unique optimum design method of applicant, specific screening obtains specificThe form of sgRNA is as follows：

GINS2-sgRNA1:5’to 3’gcgtctcctccgggacgctg

GINS2-sgRNA2:5’to 3’ccgaggagaccgtgaggctc

GINS2-sgRNA3:5’to 3’attcctcgccgagaaggagc

GINS2-sgRNA4:5’to 3’gtcgcctgctccctccagag

GINS2-sgRNA5:5’to 3’cctgctccctccagagtgga

GINS2-sgRNA6:5’to 3’gtggatggatgtagaaaagt

GINS2-sgRNA7:5’to 3’aatgcccagcccttactaca

GINS2-sgRNA8:5’to 3’tcccgaaggcagacgaaatc

GINS2-sgRNA9:5’to 3’ggcagacgaaatccggaccc

GINS2-sgRNA10:5’to 3’tgacagctttgtgagacagc

GINS2-sgRNA11:5’to 3’cagctttgtgagacagcagg

GINS2-sgRNA12:5’to 3’gctggataacttgaccttga

GINS2-sgRNA13:5’to 3’ttgatggagatcaacaccag

GINS2-sgRNA14:5’to 3’ccgcacgaacctccagcctc

GINS2-sgRNA15:5’to 3’tctggagagtactcagtctc

GINS2-sgRNA16:5’to 3’gtctcaggacttctagagaa

GINS2-sgRNA17:5’to 3’gatgcatgaaaaatgtgtga

GINS2-sgRNA18:5’to 3’gaaaaatgtgtgatggtgca

GINS2-sgRNA19:5’to 3’atggtgcaaggaatggattc

GINS2-sgRNA20:5’to 3’gtccttaaaacttagctccc

GINS2-sgRNA21:5’to 3’tccttaaaacttagctccct

GINS2-sgRNA22:5’to 3’tctccctagcagagccactt

GINS2-sgRNA23:5’to 3’tgcatggaagccatcacact

GINS2-sgRNA24:5’to 3’gcaggtgttcagtgactggt

GINS2-sgRNA25:5’to 3’ctggtaggtgtagatacagc

According to above-mentioned gRNA, positive oligonucleotide sequence is obtained plus CACC at its end 5', at the ends 5' of its complementary strandIn addition AAAC obtains reverse oligonucleotide sequence, it is respectively synthesized forward and reverse oligonucleotide sequence, then by the sequence of synthesisDenaturation, annealing, obtain the double chain DNA fragment with BbsI cohesive ends, as follows：It is positive：5’-CACCNNNNNNNNNNNNNNNNNNNN is reversed：NNNNNNNNNNNNNNNNNNNNCAAA-5 ', denaturation, annealing system are：2μlPositive 2 μ l reverse oligonucleotides chain (50 μM) of oligonucleotide chain (50 μM), 46 μ ll*NEBbuffer press following procedure in PCR instrumentOperation：90 DEG C, 4min；70 DEG C, 10min；37 DEG C, 20min；25 DEG C, 20min.

Double chain oligonucleotide chain after annealing contains the cohesive end of BbsI, directly with by the pX330- of BbsI digestionsU6-Chimeric_BB-CBh-hSpCas9 (hereinafter referred to as PX330) (SEQ ID NO.3) carrier is attached, can obtainPX330-gRNA-Cas9 recombinant plasmids.

Digestion system：39.3 μ l, 10*FD buffer of water, 52 3.7 37 DEG C of water-baths of μ l (2 μ g) of μ l, PX330 of μ l, BbsIPlasmid after 2h digestions is directly recycled with plastic recovery kit.

Linked system：0.5 μ l of annealed product, the PX330 plasmids 2 μ l of 2 μ 1,5*ligation buffer of linearisation,T4DNA Ligase (3units/ μ 1), 1 μ l, the connection product that water 4.5 μ l, 16 DEG C of water-bath 2h obtain above-mentioned steps convertJM109 competent cells are coated on the LB tablets of Amp+, and picking positive colony connects bacterium, and 37 DEG C of shaking tables shake bacterium and stay overnight, plasmid extractionKit extracts plasmid and carries out sequencing identification, obtains PX330-gRNA plasmids.

Embodiment 2 clones synergistic protein CREnhancer1.0 and carrier construction

Synergistic protein CREnhancer1.0 genes are cloned, by full genome synthetic method, obtain SEQ ID NO：1 instituteThe gene order stated is respectively 5'- according to upstream and downstream primer sequence using the sequence as templateATGCAGGAGAACCTGGCCCCCTG-3', 5'-CAGGCAGCTCACGCTCCTCTCG-3', primer and full-length genome are by ShanghaiSheng Gong Co., Ltds synthesize.PCR reaction amplification CREnhancer1.0 gene target gene fragments, amplification reaction system are as follows:95DEG C, 40s, 57 DEG C, 1min, 72 DEG C, 1min, 72 DEG C, 10min, recycle 35 times, PCR product by Shanghai Sheng Gong Co., Ltds carry outSequencing, by sequencing, in conjunction with SEQ ID NO：1 exactly matches.Then, the target gene of PCR amplification is connected to empty carrierOn slow virus carrier pHIV-CS-CDF-CG-PRE, recombined lentivirus vector is identified by the methods of PCR amplification, digestion, sequencing.It is built successfully in conjunction with proof recombined lentivirus vector.Then by the recombined lentivirus vector plasmid with helper plasmid together coinfectionHuman marrow mesenchymal stem cell (hMSCs) PC015, the people's bone that can express CREnhancer1.0 genes is packaged by recombinationMarrow interstital stem cell.By PCR screening and identifications, the stem cell of stable transfection is applied for subsequent gene editor.

Applications of 3 CRISPR/Cas9 of embodiment in bone marrow interstital stem cell

CRISPR/Cas9 based on the pBGN plasmids of fusion containing BSD-fsEGFP edits carrier

(1) BSD-fsEGFP fusions：Using Standard PCR, well known BSD genes, 5 '-PCR primer bands are expandedThe sites HindIII, 3 '-PCR primers introduce the sites I-SceI and EcoRI.PCR product (BSD) is inserted into EGFP plasmid (EGFP coresThe sequence that nucleotide sequence is known in the art, such as shown in sequence 1 in CN105647968A and sequence 2) in CMV drivings andThe sites HindIII and EcoRI between the code areas EGFP generate the plasmid pBGN, BSD- of the fusion containing BSD-fsEGFPFsEGFP fusion nucleotides sequences be classified as in CN105647968A sequence 3 and sequence 4 shown in).The fusion is by CMVDriving or PGK driving son drivings, but EGFP is inactive due to frameshit, claims fsEGFP.

5 '-PCR primers are

CTCAAGCTTAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAG,

3 '-PCR primers are

AGAATTCCAGTAGGGATAACAGGGTAATGCCAGGTCCGCCCTCCCACACATAACCAGAG。

(2) plasmid pBGN, expression plasmid cotransfection bone marrow interstital stem cell prepared by embodiment 1 will be tested.With untransfectedThe stem cell of the synergy gene of embodiment 2 as positive control, meanwhile, by the parallel transfectional cell of GFP expression plasmids routinelyTo measure transfection efficiency, the CRISPR/Cas9 gene editing relative efficiencies of transfection efficiency correction acquisition are utilized.

(4) after transfecting 2-3 days, measured by flow cytometry GFP is utilized⁺The frequency of cell.

(5) the CRISPR/Cas9 gene editing relative efficiencies that specific sgRNA is mediated are calculated.This relative efficiency is by GFP sunProperty cell frequencies and transfection efficiency ratio represent, the results are shown in Figure 3.We have found that the synergistic protein of untransfected embodiment 2Stem cell in GFP positive cell frequencies be significantly lower than transfected embodiment 2 synergistic protein cell.Wherein at 25In sgRNA, only GINS2-sgRNA7 and GINS2-sgRNA23 have preferable gene editing effect.It is prepared using empty carrierNegative control there is no GFP positive cells, wherein P values to be respectively less than 0.01, have statistical significance.

(GINS2-sgRNA23 is used obtained after CRISPR is edited bone marrow interstital stem cell GINS2-sgRNA23Stem cell) and bone marrow interstital stem cell GINS2-sgRNA7 stablize secondary culture, after culture 40 instead of, by being directed toGINS2PCR is sequenced, it is found that the gene is still mutant inactive, maintain the effect that gene is knocked.This absolutely proves, thisThe system that knocks out of invention has preferable stability.

It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although ginsengIt is described the invention in detail according to preferred embodiment, it will be understood by those of ordinary skill in the art that, it can be to the present inventionTechnical solution be modified or replaced equivalently, without departing from the spirit of the technical scheme of the invention and range.

Sequence table

<110>The Luoyang bio tech ltd Xuan Zhi

<120>The method that GINS2 gene knockouts are carried out to mescenchymal stem cell using CRISPR-CAS systems

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1578

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

atgcaggaga acctggcccc ctggggcgag ctggccaccg acaacatcat cctgaccgtg 60

cccaccacca acctgcaggc cctgaaggac cccgagcccg tgctgaggct gtgggacgag 120

atgatgcagg ccgtggccag gctggccgcc gagcccttcc ccttcaggag gcccgagagg 180

atcgtggccg acgtgcagat cagcgccggc tggatgcaca gcggctaccc catcatgtgc 240

cacctggaga gcgtgaagga gatcatcaac gagatggaca tgaggagcag gggcgtgtgg 300

ggccccatcc acgagctggg ccacaaccag cagaggcacg gctgggagtt ccccccccac 360

accaccgagg ccacctgcaa cctgtggagc gtgtacgtgc acgagaccgt gctgggcatc 420

cccagggccc aggcccacga ggccctgagc ccccccgaga gggagaggag gatcaaggcc 480

cacctgggca agggcgcccc cctgtgcgac tggaacgtgt ggaccgccct ggagacctac 540

ctgcaggtgc tgagcaggaa cagcggcagg aggggcgtgg acggcaggct ggtgcacacc 600

tgcatcaagg ccggcgccgt gaggtggctg gccaggggcc agaccggcaa ggtgggcgtg 660

aacaccaacc tgaaggacct gtgccccctg ctgagcgagc acggcctgca gtgcagcctg 720

gagccccacc tgaacagcga cctgtgcgtg tactgctgca aggcctacag cgacaaggag 780

gccaagcagc tgcaggagtt cgtggccgag ggcggcggcc tgctgatcgg cggccaggcc 840

tggtggtggg ccagccagaa ccccggccac tgccccctgg ccggcttccc cggcaacatc 900

atcctgaact gcttcggcct gagcatcctg ccccagaccc tgaaggccgg ctgcttcccc 960

gtgcccaccc ccgagatgag gagctaccac ttcaggaagg ccctgagcca gttccaggcc1020

atcctgaacc acgagaacgg caacctggag aagagctgcc tggccaagct gagggtggac1080

ggcgccgcct tcctgcagat ccccgccgag ggcgtgcccg cctacatcag cctgcacagg1140

ctgctgagga agatgctgag gggcagcggc ctgcccgccg tgagcaggga gaaccccgtg1200

gccagcgaca gctacgaggc cgccgtgctg agcctggcca ccggcctggc ccacagcggc1260

accgactgca gccagctggc ccagggcctg ggcacctgga cctgcagcag cagcctgtac1320

cccagcaagc accccatcac cgtggagatc aacggcatca accccggcaa caacgactgc1380

tgggtgagca ccggcctgta cctgctggag ggccagaacg ccgaggtgag cctgagcgag1440

gccgccgcca gcgccggcct gagggtgcag atcggctgcc acaccgacga cctgaccaag1500

gccaggaagc tgagcagggc ccccatggtg acccaccagt gctggatgga caggaccgag1560

aggagcgtga gctgcctg 1578

<210> 2

<211> 526

<212> PRT

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

Met Gln Glu Asn Leu Ala Pro Trp Gly Glu Leu Ala Thr Asp Asn Ile

1 5 10 15

Ile Leu Thr Val Pro Thr Thr Asn Leu Gln Ala Leu Lys Asp Pro Glu

20 25 30

Pro Val Leu Arg Leu Trp Asp Glu Met Met Gln Ala Val Ala Arg Leu

35 40 45

Ala Ala Glu Pro Phe Pro Phe Arg Arg Pro Glu Arg Ile Val Ala Asp

50 55 60

Val Gln Ile Ser Ala Gly Trp Met His Ser Gly Tyr Pro Ile Met Cys

65 70 75 80

His Leu Glu Ser Val Lys Glu Ile Ile Asn Glu Met Asp Met Arg Ser

85 90 95

Arg Gly Val Trp Gly Pro Ile His Glu Leu Gly His Asn Gln Gln Arg

100 105 110

His Gly Trp Glu Phe Pro Pro His Thr Thr Glu Ala Thr Cys Asn Leu

115 120 125

Trp Ser Val Tyr Val His Glu Thr Val Leu Gly Ile Pro Arg Ala Gln

130 135 140

Ala His Glu Ala Leu Ser Pro Pro Glu Arg Glu Arg Arg Ile Lys Ala

145 150 155 160

His Leu Gly Lys Gly Ala Pro Leu Cys Asp Trp Asn Val Trp Thr Ala

165 170 175

Leu Glu Thr Tyr Leu Gln Val Leu Ser Arg Asn Ser Gly Arg Arg Gly

180 185 190

Val Asp Gly Arg Leu Val His Thr Cys Ile Lys Ala Gly Ala Val Arg

195 200 205

Trp Leu Ala Arg Gly Gln Thr Gly Lys Val Gly Val Asn Thr Asn Leu

210 215 220

Lys Asp Leu Cys Pro Leu Leu Ser Glu His Gly Leu Gln Cys Ser Leu

225 230 235 240

Glu Pro His Leu Asn Ser Asp Leu Cys Val Tyr Cys Cys Lys Ala Tyr

245 250 255

Ser Asp Lys Glu Ala Lys Gln Leu Gln Glu Phe Val Ala Glu Gly Gly

260 265 270

Gly Leu Leu Ile Gly Gly Gln Ala Trp Trp Trp Ala Ser Gln Asn Pro

275 280 285

Gly His Cys Pro Leu Ala Gly Phe Pro Gly Asn Ile Ile Leu Asn Cys

290 295 300

Phe Gly Leu Ser Ile Leu Pro Gln Thr Leu Lys Ala Gly Cys Phe Pro

305 310 315 320

Val Pro Thr Pro Glu Met Arg Ser Tyr His Phe Arg Lys Ala Leu Ser

325 330 335

Gln Phe Gln Ala Ile Leu Asn His Glu Asn Gly Asn Leu Glu Lys Ser

340 345 350

Cys Leu Ala Lys Leu Arg Val Asp Gly Ala Ala Phe Leu Gln Ile Pro

355 360 365

Ala Glu Gly Val Pro Ala Tyr Ile Ser Leu His Arg Leu Leu Arg Lys

370 375 380

Met Leu Arg Gly Ser Gly Leu Pro Ala Val Ser Arg Glu Asn Pro Val

385 390 395 400

Ala Ser Asp Ser Tyr Glu Ala Ala Val Leu Ser Leu Ala Thr Gly Leu

405 410 415

Ala His Ser Gly Thr Asp Cys Ser Gln Leu Ala Gln Gly Leu Gly Thr

420 425 430

Trp Thr Cys Ser Ser Ser Leu Tyr Pro Ser Lys His Pro Ile Thr Val

435 440 445

Glu Ile Asn Gly Ile Asn Pro Gly Asn Asn Asp Cys Trp Val Ser Thr

450 455 460

Gly Leu Tyr Leu Leu Glu Gly Gln Asn Ala Glu Val Ser Leu Ser Glu

465 470 475 480

Ala Ala Ala Ser Ala Gly Leu Arg Val Gln Ile Gly Cys His Thr Asp

485 490 495

Asp Leu Thr Lys Ala Arg Lys Leu Ser Arg Ala Pro Met Val Thr His

500 505 510

Gln Cys Trp Met Asp Arg Thr Glu Arg Ser Val Ser Cys Leu

515 520 525

<210> 3

<211> 8506

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360

agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420

aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480

ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720

ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780

ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840

ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 900

gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 960

ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc1020

tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg1080

accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag1140

ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac1200

ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata1260

aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga1320

tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt1380

acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt1440

acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga1500

agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga1560

agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga1620

tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct1680

tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg1740

aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca1800

gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc1860

ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt1920

tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg1980

gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc2040

tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga2100

gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc2160

agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc2220

agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca2280

tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat2340

acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg2400

agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg2460

gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg2520

gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct2580

tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc2640

ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga2700

ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga2760

tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg2820

gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg2880

agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga2940

ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga3000

aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga3060

aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag3120

atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg3180

acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac3240

tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg3300

acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga3360

agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt3420

ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta3480

aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg3540

ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg3600

acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca3660

gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg3720

aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc3780

agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg3840

accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga3900

gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg3960

gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc4020

agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga4080

gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc4140

ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg4200

agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg4260

atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc4320

acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg4380

aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga4440

gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact4500

ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga4560

caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga4620

aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct4680

tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg4740

actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg4800

tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg4860

ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca4920

agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg4980

agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg5040

aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc5100

tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact5160

acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg5220

ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc5280

aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca5340

agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg5400

ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc5460

tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt5520

aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt5580

tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc5640

ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg5700

tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga5760

gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct5820

cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt5880

gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca5940

tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc6000

gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc6060

ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc6120

cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc6180

gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg6240

gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact6300

ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt6360

tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa6420

atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag6480

ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc6540

ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt6600

tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag6660

gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg6720

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga6780

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat6840

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca6900

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc6960

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca7020

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg7080

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca7140

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata7200

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag7260

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg7320

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca7380

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta7440

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct7500

ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca7560

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag7620

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat7680

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt7740

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa7800

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga7860

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg7920

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc7980

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag8040

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc8100

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg8160

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac8220

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga8280

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt8340

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag8400

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg8460

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt 8506

<210> 4

<211> 23

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 4

atgcaggaga acctggcccc ctg 23

<210> 5

<211> 22

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

caggcagctc acgctcctct cg 22

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

aatgcccagc ccttactaca 20

<210> 7

<211> 20

<212> DNA

<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 7

tgcatggaag ccatcacact 20

Claims (4)

1. a kind of CRISPR-CAS systems for stem cell cytogene editor, it is characterised in that：The composition packet of systemIt includes：(1) it is used to express SEQ ID NO：The plasmid of CREnhancer1.0 genes described in 1；(2) it is used to express SEQ ID NO：6Or 7 it is any shown in sgRNA PX330 plasmid.

2. the system as claimed in claim 1, it is characterised in that：(1) plasmid can in advance be imported into gene editing cell,After screening obtains positive cell, then it is transferred to the plasmid of (2).

3. purposes of the system of claim 1 in preparing the reagent for mesenchymal stem cell gene editing.

4. purposes as claimed in claim 3, wherein mesenchymal stem cell are human marrow mesenchymal stem cell (hMSCs)PC015。