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

The method that GINS2 gene knockouts are carried out to mescenchymal stem cell using CRISPR-CAS systems Download PDF

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CN107619829B
CN107619829B CN201710955489.1A CN201710955489A CN107619829B CN 107619829 B CN107619829 B CN 107619829B CN 201710955489 A CN201710955489 A CN 201710955489A CN 107619829 B CN107619829 B CN 107619829B
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gins2
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CN107619829A (en
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杨骏
刘语方
张立平
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Nanjing Pinggang Biotechnology Co ltd
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Nanjing Ping Gang Biotechnology Co Ltd
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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 systems Method
Technical field
Present invention offer is a kind of to carry out GINS2 gene editings using CRISPR-cas systems for mescenchymal stem cell, special It 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 multidirectional The adult stem cell of differentiation potential, this stem cell can develop into os osseum, cartilage, fat and other kinds of cell.Between fill Matter stem cell can receive transplanting, and they can grow into what type of cell and depend on its position injected.For example, by The 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.Mainly It 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 endothelium The 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 protective Still there is multi-lineage potential.Whether self or allogenic mescenchymal stem cell generally will not all cause host's Immune response.Due to this immunological characteristic that mescenchymal stem cell has, makes it in autoimmune disease and various replace Generation 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, with The 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 mesenchyma The stage of cell core technical research.The development of stem-cell research work, including the high match cell of country are reinforced energetically in China More authoritative research institutions including genetic engineering Co., Ltd, cell products National Engineering Research Centre and each place umbilical cord Into 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 heart Cranial vascular disease, hepatic sclerosis, bone and muscle degenerative disease, brain and neurologic defict, senile dementia and lupus erythematosus and hard The Therapy study of the autoimmune diseases such as skin disease, the partial clinical test result having been achieved with are encouraging.Research so far Show that the mescenchymal stem cell in umbilical cord source is not only able to the ideal substitute as mesenchymal stem cell, and has The application potential of bigger.Umbilical cord mesenchymal stem cells express the peculiar molecular marker of a variety of embryonic stem cells, have differentiation potential Greatly, 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 is 1196bp, the protein that coding relative molecular mass is 21000.GINS is a kind of replicative helicase, before being moved to replication fork Open DNA double chain.Studies have shown that GINS family members play a role in the generation of cancer, as GINS family members are invading It 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 as In terms of determining that centerbody replicates quantity and the pathogenetic different phase of disease, GINS has played certain function, especially with dye The 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 GINS2 The siRNA sequence of gene, rna interference vector and RNA interfere slow virus, further have detected the heavy of GINS2 genes The silent influence of efficiency, GINS2-siRNA slow virus to tumor cell proliferation ability and level of apoptosis, as a result display use the side RNAi The proliferation of tumour cell can effectively be inhibited under method after the expression of mediator GINS2 genes and growth and promote its apoptosis, show GINS2 Gene 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 genes The effective means of tumor development.But in this study, it is interfered using SiRNA, the method has knockout not thorough Bottom, the defect for the knockout heredity that cannot stablize.In (Integration of Genomic, Biologic, and Chemical Approaches to Target p53 Loss and Gain-of-Function in Triple Negative BreastCancer 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 set Meter, 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 cancer Function 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 art The 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 GINS2 The 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 protein CREnhancer1.0 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 cloned CREnhancer1.0 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 knockouts Method, including editor's positive cell will be obtained in the gene editing system introducing mescenchymal stem cell, it then breeds, harvest The stem cell.
Specific mescenchymal stem cell is human marrow mesenchymal stem cell (hMSCs) PC015, and purchase is biological from Shanghai Ai Yan Science and Technology Ltd..
The present invention provides a kind of mescenchymal stem cell cell system, methods of structure GINS2 gene knockouts, have following excellent Point:The present invention constructs the cell line of GINS2 gene knockouts in mescenchymal stem cell, screen and optimize obtain it is best SgRNA 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 specific The 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 strand In addition AAAC obtains reverse oligonucleotide sequence, it is respectively synthesized forward and reverse oligonucleotide sequence, then by the sequence of synthesis Denaturation, 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:2μl Positive 2 μ l reverse oligonucleotides chain (50 μM) of oligonucleotide chain (50 μM), 46 μ ll*NEBbuffer press following procedure in PCR instrument Operation: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 digestions U6-Chimeric_BB-CBh-hSpCas9 (hereinafter referred to as PX330) (SEQ ID NO.3) carrier is attached, can obtain PX330-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, BbsI Plasmid 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 convert JM109 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 extraction Kit 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 institute The gene order stated is respectively 5'- according to upstream and downstream primer sequence using the sequence as template ATGCAGGAGAACCTGGCCCCCTG-3', 5'-CAGGCAGCTCACGCTCCTCTCG-3', primer and full-length genome are by Shanghai Sheng Gong Co., Ltds synthesize.PCR reaction amplification CREnhancer1.0 gene target gene fragments, amplification reaction system are as follows:95 DEG 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 out Sequencing, by sequencing, in conjunction with SEQ ID NO:1 exactly matches.Then, the target gene of PCR amplification is connected to empty carrier On 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 coinfection Human marrow mesenchymal stem cell (hMSCs) PC015, the people's bone that can express CREnhancer1.0 genes is packaged by recombination Marrow 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 expanded The sites HindIII, 3 '-PCR primers introduce the sites I-SceI and EcoRI.PCR product (BSD) is inserted into EGFP plasmid (EGFP cores The sequence that nucleotide sequence is known in the art, such as shown in sequence 1 in CN105647968A and sequence 2) in CMV drivings and The sites HindIII and EcoRI between the code areas EGFP generate the plasmid pBGN, BSD- of the fusion containing BSD-fsEGFP FsEGFP fusion nucleotides sequences be classified as in CN105647968A sequence 3 and sequence 4 shown in).The fusion is by CMV Driving 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 untransfected The stem cell of the synergy gene of embodiment 2 as positive control, meanwhile, by the parallel transfectional cell of GFP expression plasmids routinely To 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 sun Property cell frequencies and transfection efficiency ratio represent, the results are shown in Figure 3.We have found that the synergistic protein of untransfected embodiment 2 Stem cell in GFP positive cell frequencies be significantly lower than transfected embodiment 2 synergistic protein cell.Wherein at 25 In sgRNA, only GINS2-sgRNA7 and GINS2-sgRNA23 have preferable gene editing effect.It is prepared using empty carrier Negative 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-sgRNA23 Stem cell) and bone marrow interstital stem cell GINS2-sgRNA7 stablize secondary culture, after culture 40 instead of, by being directed to GINS2PCR is sequenced, it is found that the gene is still mutant inactive, maintain the effect that gene is knocked.This absolutely proves, this The 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 ginseng It 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 invention Technical 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 gttccaggcc 1020
atcctgaacc acgagaacgg caacctggag aagagctgcc tggccaagct gagggtggac 1080
ggcgccgcct tcctgcagat ccccgccgag ggcgtgcccg cctacatcag cctgcacagg 1140
ctgctgagga agatgctgag gggcagcggc ctgcccgccg tgagcaggga gaaccccgtg 1200
gccagcgaca gctacgaggc cgccgtgctg agcctggcca ccggcctggc ccacagcggc 1260
accgactgca gccagctggc ccagggcctg ggcacctgga cctgcagcag cagcctgtac 1320
cccagcaagc accccatcac cgtggagatc aacggcatca accccggcaa caacgactgc 1380
tgggtgagca ccggcctgta cctgctggag ggccagaacg ccgaggtgag cctgagcgag 1440
gccgccgcca gcgccggcct gagggtgcag atcggctgcc acaccgacga cctgaccaag 1500
gccaggaagc tgagcagggc ccccatggtg acccaccagt gctggatgga caggaccgag 1560
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 gctgcgacgc 1020
tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1080
accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1140
ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1200
ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1260
aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1320
tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1380
acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1440
acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1500
agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1560
agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1620
tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1680
tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1740
aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1800
gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1860
ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1920
tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1980
gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2040
tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2100
gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2160
agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2220
agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2280
tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2340
acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2400
agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2460
gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2520
gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2580
tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2640
ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2700
ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2760
tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2820
gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2880
agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2940
ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3000
aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3060
aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3120
atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3180
acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3240
tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3300
acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3360
agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3420
ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3480
aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3540
ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3600
acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3660
gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3720
aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3780
agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3840
accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3900
gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3960
gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4020
agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4080
gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4140
ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4200
agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4260
atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4320
acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4380
aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4440
gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4500
ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4560
caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4620
aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4680
tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4740
actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4800
tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4860
ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4920
agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4980
agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5040
aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5100
tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5160
acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5220
ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5280
aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5340
agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5400
ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5460
tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 5520
aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5580
tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5640
ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5700
tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5760
gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5820
cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5880
gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 5940
tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 6000
gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 6060
ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 6120
cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 6180
gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 6240
gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6300
ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 6360
tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6420
atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 6480
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 6540
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 6600
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 6660
gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 6720
cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 6780
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6840
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 6900
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 6960
gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 7020
atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 7080
caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 7140
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 7200
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 7260
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 7320
gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 7380
acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7440
atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 7500
ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 7560
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 7620
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 7680
tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7740
taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 7800
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 7860
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 7920
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 7980
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 8040
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 8100
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 8160
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 8220
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 8280
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8340
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 8400
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 8460
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 system It 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:6 Or 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。
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