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

Abstract

The present invention provides a GINS2 gene editing method for mesenchymal stem cells using a CRISPR-cas9 system, and in particular relates to the establishment of a GINS2 gene knockout mesenchymal stem cell line. Among them, the new enhancer protein CREnhancer1.0 is used, which can significantly improve the efficiency of CRISPR/Cas9 gene editing in cells. The bone marrow mesenchymal stem cell GINS2 knockout plasmid provided by the invention has better genetic stability.

Description

Knockout of GINS2 gene in mesenchymal stem cells using CRISPR-Cas system method

technical field

The present invention provides a GINS2 gene editing method for mesenchymal stem cells using a CRISPR-cas system, and in particular relates to the establishment of a GINS2 gene knockout mesenchymal stem cell line.

Background technique

Mesenchymal stem cells (MSCs) are adult stem cells with self-replication ability and multi-directional differentiation potential, which can develop into bone, cartilage, fat and other types of cells. Mesenchymal stem cells can be transplanted, and the type of cells they grow into depends on where they are injected. For example, mesenchymal stem cells injected into the heart can form healthy new tissue, among other things.

Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from the mesoderm and ectoderm in early development. Mainly exist in connective tissue and interstitium of organs, and the content is most abundant in bone marrow tissue. Since bone marrow is its main source, they are collectively called bone marrow mesenchymal stem cells. Mesenchymal stem cells are non-terminal differentiated cells, which have the characteristics of mesenchymal cells, endothelial cells and epithelial cells. Under specific induction conditions in vitro, mesenchymal stem cells can differentiate into fat, cartilage, bone, muscle, tendon, nerve, liver, myocardium, pancreatic β cells, and endothelial cells. Potential for multidirectional differentiation. Whether autologous or allogeneic mesenchymal stem cells generally do not cause the host's immune response. Due to the immunological characteristics of mesenchymal stem cells, it has broad clinical application prospects in autoimmune diseases and various alternative treatments. The structure and function of tissues and organs can be reconstructed through autologous transplantation, and immune rejection can be avoided.

Clinical research on mesenchymal stem cells has been carried out in many countries. The United States has approved more than 60 clinical trials. With the maturation of mesenchymal stem cells and related technologies, my country has also approved a number of clinical trials. The stage for the research and development of the core technology of mesenchymal stem cells. my country has vigorously strengthened the development of stem cell research work, and many authoritative research institutions including the National Oncell Cell Gene Engineering Co., Ltd., the National Engineering Research Center for Cell Products, and local cord blood banks have introduced the research technology to the clinic. For the research on the use of mesenchymal stem cells in the treatment of more than ten kinds of refractory diseases, in addition to promoting the recovery of hematopoiesis, co-transplantation with hematopoietic stem cells to improve leukemia and refractory anemia, etc., it is also used for cardiovascular and cerebrovascular diseases, liver Some clinical trial results have been encouraging for research on the treatment of sclerosis, bone and muscle degenerative diseases, brain and spinal cord nerve injuries, Alzheimer's, and autoimmune diseases such as lupus erythematosus and scleroderma. Studies so far have shown that umbilical cord-derived mesenchymal stem cells can not only become an ideal substitute for bone marrow mesenchymal stem cells, but also have greater application potential. Umbilical cord mesenchymal stem cells express a variety of specific molecular markers of embryonic stem cells, and have the characteristics of high differentiation potential, strong proliferation ability, low immunogenicity, convenient material extraction, no restrictions on moral and ethical issues, and easy industrial production. Pluripotent stem cells with clinical application prospects.

GINS2 is a member of the DNA replication complex GINS family, located on human chromosome 16q24, its mRNA length is 1196bp, and it encodes a protein with a relative molecular mass of 21000. GINS is a replicative helicase that unwinds the DNA double strand before moving to the replication fork. Studies have shown that GINS family members play a role in the occurrence of cancer, for example, GINS family members are overexpressed in invasive melanoma, and there are also literatures showing that DNA replication-related proteins have different roles in different cells, such as in determining the centrosome GINS has played a certain role in terms of the number of copies and different stages of disease occurrence, especially closely related to the segregation of chromosomes.

However, there are few reports about GINS2 in tumor-related fields. In CN106620703 A, aiming at the small molecule interference RNA sequence of human GINS2 gene, RNA interference carrier and RNA interference lentivirus, the silencing efficiency of GINS2 gene and the effect of GINS2-siRNA lentivirus on tumor cell proliferation ability and apoptosis level were further detected , the results showed that down-regulating the expression of human GINS2 gene by RNAi method can effectively inhibit the proliferation and growth of tumor cells and promote their apoptosis, indicating that GINS2 gene is a proto-oncogene and can be used as a target for tumor therapy. GINS2 gene can be silenced by RNAi The expression of can be used as an effective means to inhibit tumor development. However, in this study, siRNA was used for interference, and the method has the defects of incomplete knockout and unstable genetic knockout. In (Integration of Genomic, Biologic, and Chemical Approaches to Target p53 Loss and Gain-of-Function in Triple Negative Breast Cancer), although it is mentioned that 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 it is only an idea and has not been implemented. CRISPR modification for different genes is not simple and easy to design, many obstacles need to be overcome, and there are great experimental difficulties.

Based on the pluripotency of mesenchymal stem cells, in order to study the function of GINS2 gene knockout mesenchymal stem cells in cancer therapy, it is particularly important to establish GINS2 gene knockout mesenchymal stem cell lines.

Contents of the invention

The purpose of the present invention is to provide a GINS2 gene knockout mesenchymal stem cell, which effectively overcomes the technical defect of using siRNA to interfere with stable inheritance in the prior art.

In order to achieve the above object, the present invention provides a target of the CRISPR-cas system. According to the gene sequence of GINS2, the specific selectable target sites designed are as follows (the underlined part indicates the PAM motif):

GINS2-sgRNA1: 5'to 3'gcgtctcctccgggacgctg agg

GINS2-sgRNA2: 5'to 3'ccgaggagaccgtgaggctctgg

GINS2-sgRNA3: 5'to 3'attcctcgccgagaaggagc tgg

GINS2-sgRNA4: 5'to 3'gtcgcctgctccctccagagtgg

GINS2-sgRNA5: 5'to 3'cctgctccctccagagtgga tgg

GINS2-sgRNA6: 5'to 3'gtggatggatgtagaaaagttgg

GINS2-sgRNA7: 5'to 3'aatgcccagcccttactaca tgg

GINS2-sgRNA8: 5'to 3'tcccgaaggcagacgaaatc cgg

GINS2-sgRNA9: 5'to 3'ggcagacgaaatccggaccctgg

GINS2-sgRNA10: 5'to 3'tgacagctttgtgagacagc agg

GINS2-sgRNA11: 5'to 3' cagctttgtgagacagcagg agg

GINS2-sgRNA12: 5'to 3'gctggataacttgaccttga tgg

GINS2-sgRNA13: 5'to 3'ttgatggagatcaacaccag -cgg

GINS2-sgRNA14:5'to 3'ccgcacgaacctccagcctctgg

GINS2-sgRNA15: 5'to 3'tctggagagtactcagtctc agg

GINS2-sgRNA16: 5'to 3'gtctcaggacttctagagaa agg

GINS2-sgRNA17: 5'to 3'gatgcatgaaaaatgtgtga tgg

GINS2-sgRNA18: 5'to 3'gaaaaatgtgtgatggtgca agg

GINS2-sgRNA19: 5'to 3'atggtgcaaggaatggattc agg

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'gcaggtgttcagtgactggt agg

GINS2-sgRNA25:5'to 3'ctggtaggtgtagatacagc agg .

According to these target sites, design specific sgRNA 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

In order to improve gene editing efficiency, it includes introducing a potentiating protein into the mesenchymal stem cells, and the synergizing protein CREnhancer1.0 is a protein encoded by the nucleotide sequence shown in SEQ ID NO:1.

Further, the synergistic protein comprises a) or b):

a) the polynucleotide sequence of the protein encoded by the nucleotide sequence shown in SEQ ID NO:1;

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

Further, clone the synergistic protein CREnhancer1.0 gene, construct the EGFP-labeled synergistic protein CREnhancer1.0 lentiviral expression vector, use GP2-293T cells to package the lentivirus, and modify the stem cells.

Furthermore, a system for gene editing using CRISPR/Cas9 in mesenchymal stem cells is provided, characterized in that the system includes: (1) for expressing the CREnhancer1.0 gene described in SEQ ID NO: 1 Plasmid; (2) PX330-expressing plasmid (which can express sgRNA and cas9) into which sgRNA has been inserted.

Furthermore, the sgRNA and cas9 expression vectors may also be other expression vectors commonly used in the art.

To achieve the above purpose, the present invention also provides a method for constructing a GINS2 gene knockout mesenchymal stem cell line, comprising introducing the gene editing system into the mesenchymal stem cells to obtain edited positive cells, and then propagating and harvesting the stem cell.

The specific mesenchymal stem cells are human bone marrow mesenchymal stem cells (hMSCs) PC015, purchased from Shanghai Ayan Biotechnology Co., Ltd.

The invention provides a method for constructing a GINS2 gene knockout mesenchymal stem cell line, which has the following advantages: the present invention constructs a GINS2 gene knockout cell line in the mesenchymal stem cells, screens and optimizes to obtain the best sgRNA with high knockout efficiency and stable passage.

The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Description of drawings

Figure 1 PX330 plasmid map;

Figure 2 sgRNA insertion site map in PX330;

Figure 3 Schematic diagram of 25 sgRNA gene labeling efficiencies;

Detailed ways

The technical scheme of the method for improving genome editing efficiency of the present invention will be further illustrated below through specific examples.

Embodiment 1, construction of CRISPR expression vector

gRNA design

According to the gene sequence of the target gene, through the applicant's unique optimization design method, the specific form of sgRNA obtained through specific screening 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 the above gRNA, add CACC to its 5' end to obtain the forward oligonucleotide sequence, add AAAC to the 5' end of its complementary strand to obtain the reverse oligonucleotide sequence, and synthesize forward and reverse oligonucleotides respectively Nucleotide sequence, and then denature and anneal the synthesized sequence to obtain a double-stranded DNA fragment with BbsI sticky ends, as follows: Forward: 5'-CACCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAAA-5', denaturation, annealing system: 2μl forward To the oligonucleotide chain (50 μM) 2 μl reverse oligonucleotide chain (50 μM) 46 μll*NEBbuffer in the PCR machine according to the following procedures: 90 ° C, 4 min; 70 ° C, 10 min; 37 ° C, 20 min; 25 ° C, 20min.

The annealed double-stranded oligonucleotide chain contains the cohesive end of BbsI, and is directly connected to the pX330-U6-Chimeric_BB-CBh-hSpCas9 (hereinafter referred to as PX330) (SEQ ID NO.3) vector cut by BbsI, which can be The PX330-gRNA-Cas9 recombinant plasmid was obtained.

Enzyme digestion system: 39.3 μl of water, 5 μl of 10*FD buffer, 2 μl of BbsI, 3.7 μl (2 μg) of PX330 in a water bath at 37°C for 2 hours. The digested plasmid was directly recovered with a gel recovery kit.

Ligation system: 0.5 μl of annealed product, 2 μl of linearized PX330 plasmid, 2 μl of 5*ligation buffer, 1 μl of T4 DNA Ligase (3units/μ1), 4.5 μl of water, 2 hours in a water bath at 16°C, and transform the ligation product obtained in the above steps into JM109 competent The cells were spread on the Amp+ LB plate, the positive clones were picked and inoculated, and the bacteria were shaken overnight at 37°C, and the plasmid was extracted by the plasmid extraction kit and sequenced to obtain the PX330-gRNA plasmid.

Example 2 Cloning of enhancer protein CREnhancer1.0 and constructing vector

The CREnhancer1.0 gene was cloned, and the gene sequence described in SEQ ID NO: 1 was obtained by the method of whole gene synthesis. Using this sequence as a template, according to the sequences of the upstream and downstream primers, they were 5'-ATGCAGGAGAACCTGGCCCCCTG-3', 5 '-CAGGCAGCTCACGCTCCTCTCG-3', primers and whole genome were synthesized by Shanghai Sangong Co., Ltd. The target gene fragment of CREnhancer1.0 gene was amplified by PCR reaction. The amplification reaction system was as follows: 95°C, 40s, 57°C, 1min, 72°C, 1min, 72°C, 10min, cycled 35 times, and the PCR product was produced by Shanghai Shenggong Co., Ltd. Sequencing was performed, and the binding was a complete match to SEQ ID NO:1 by sequencing. Subsequently, the target gene amplified by PCR was connected to the empty vector lentiviral vector pHIV-CS-CDF-CG-PRE, and the recombinant lentiviral vector was identified by methods such as PCR amplification, enzyme digestion, and sequencing. The combination proved that the recombinant lentiviral vector was constructed successfully. Subsequently, the recombinant lentiviral vector plasmid and the helper plasmid were co-infected into human bone marrow mesenchymal stem cells (hMSCs) PC015, and packaged into human bone marrow mesenchymal stem cells capable of expressing CREnhancer1.0 gene through recombination. Identified by PCR screening, the stably transfected stem cells were used for subsequent gene editing applications.

Example 3 Application of CRISPR/Cas9 in bone marrow mesenchymal stem cells

CRISPR/Cas9 editing vector based on pBGN plasmid containing BSD-fsEGFP fusion gene

(1) BSD-fsEGFP fusion gene: use conventional PCR to amplify the known BSD gene, 5'-PCR primers with HindIII sites, and 3'-PCR primers to introduce I-SceI and EcoRI sites. Insert the PCR product (BSD) into the HindIII and EcoRI positions between the CMV driver and the EGFP coding region in the EGFP plasmid (the EGFP nucleotide sequence is a sequence known in the art, such as shown in sequence 1 and sequence 2 in CN105647968A) Point, generate the plasmid pBGN containing the BSD-fsEGFP fusion gene, the nucleotide sequence of the BSD-fsEGFP fusion gene is as shown in sequence 3 and sequence 4 in CN105647968A). The fusion gene is driven by CMV driver or PGK driver, but EGFP is inactive due to frameshift, so it is called fsEGFP.

5'-PCR primers are

CTCAAGCTTAACTAAACCATGGCCAAGCCCTTTGTCTCAAGAAG,

3'-PCR primers are

AGAATTCCAGTAGGGATAACAGGGTAATGCCAGGTCCGCCCTCCCACACATAACCAGAG.

(2) The test plasmid pBGN and the expression plasmid prepared in Example 1 were co-transfected into bone marrow mesenchymal stem cells. Stem cells that were not transfected with the synergistic gene of Example 2 were used as a positive control. At the same time, cells were transfected in parallel with conventional GFP expression plasmids to determine the transfection efficiency, and the relative efficiency of CRISPR/Cas9 gene editing obtained was corrected using the transfection efficiency .

(4) 2-3 days after transfection, the frequency of GFP ⁺ cells was measured by flow cytometry.

(5) Calculate the relative efficiency of specific sgRNA-mediated CRISPR/Cas9 gene editing. This relative efficiency is represented by the ratio of the frequency of GFP-positive cells to the transfection efficiency, and the results are shown in FIG. 3 . We found that the frequency of GFP-positive cells in stem cells not transfected with the potentiating protein of Example 2 was significantly lower than that of cells transfected with the potentiating protein of Example 2. Among the 25 sgRNAs, only GINS2-sgRNA7 and GINS2-sgRNA23 had good gene editing effects. The negative control prepared with empty vector had no GFP-positive cells, and the P values were all less than 0.01, which was statistically significant.

Bone marrow mesenchymal stem cells GINS2-sgRNA23 (that is, stem cells obtained after CRISPR editing with GINS2-sgRNA23) and bone marrow mesenchymal stem cells GINS2-sgRNA7 were stably subcultured. After 40 generations of culture, the gene was found to be still It is inactivated by mutation and maintains the effect of gene knockout. This fully demonstrates that the knockout system of the present invention has better stability.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The scheme shall be modified or equivalently replaced without departing from the spirit and scope of the technical scheme of the present invention.

sequence listing

<110> Luoyang Xuanzhi Biotechnology Co., Ltd.

<120> A method for knocking out GINS2 gene in mesenchymal stem cells using CRISPR-CAS system

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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 gctggggagtt 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 accccaccagt gctggatgga caggaccgag1560

aggagcgtga gctgcctg 1578

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<211> 526

<212> PRT

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

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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

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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

aggaccga 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 atcgtgtgggg 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 aggggggagga 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 gagtccacg 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 catggggat 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 ttgattaaa 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 gtagttacc 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 cctactaca 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 CRISPR-CAS system for bone marrow stem cell gene editing, characterized in that: the composition of the system comprises: (1) a plasmid for expressing the CREnhancer1.0 gene described in SEQ ID NO: 1; (2) A plasmid for PX330 expressing either sgRNA shown in SEQ ID NO: 6 or 7. 2. The system according to claim 1, characterized in that: the plasmid of (1) can be introduced into gene-edited cells in advance, and after positive cells are screened, the plasmid of (2) can be transferred. 3. The use of the system of claim 1 in the preparation of reagents for gene editing of bone marrow mesenchymal stem cells. 4. The use according to claim 3, wherein the bone marrow mesenchymal stem cells are human bone marrow mesenchymal stem cells (hMSCs) PC015.