CN107760684A - The method for carrying out RBM17 gene knockouts to mescenchymal stem cell using CRISPR cas systems - Google Patents
The method for carrying out RBM17 gene knockouts to mescenchymal stem cell using CRISPR cas systems Download PDFInfo
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Abstract
RBM17 gene editings are carried out for mescenchymal stem cell using CRISPR cas systems the invention provides one kind, more particularly to a kind of foundation of the mescenchymal stem cell cell line of structure RBM17 gene knockouts.New synergistic protein CREnhancer1.0 is which used, intracellular CRISPR/Cas9 gene editings efficiency can be significantly improved.Mesenchymal stem cells MSCs RBM17 provided by the invention, which knocks out plasmid, has preferable genetic stability.
Description
Technical field
The present invention provides one kind and carries out RBM17 gene editings for mescenchymal stem cell using CRISPR-cas systems, special
It is not to be related to a kind of foundation of the mescenchymal stem cell cell line of structure RBM17 gene knockouts.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC), it 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 being injected into.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, comes 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, because marrow is its main source, therefore
It is referred to as mesenchymal stem cells MSCs.Mescenchymal stem cell belongs to non-terminally differentiated cells, its existing interstitial cell, there is endothelium again
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 autologous or allogenic mescenchymal stem cell, typically all without causing host's
Immune response.Due to this immunological characteristic that mescenchymal stem cell possesses, make it in autoimmune disease and various replace
Generation treatment etc. has wide potential applicability in clinical practice.The 26S Proteasome Structure and Function of histoorgan can be rebuild by autotransplantation,
And immunological rejection can be avoided.
The clinical research of mescenchymal stem cell is carried out in many countries, and the U.S. have approved 60 remainder clinical tests, with
The increasingly mature of mescenchymal stem cell and its correlation technique, China also have approved multinomial clinical test, has entered into mesenchyma and has done
The stage of cell core technical research.The development of stem-cell research work, including the high match cell of country are strengthened energetically in China
More authoritative research institutions and each place umbilical cord including genetic engineering Co., Ltd, cell products National Engineering Research Centre
Into clinic is guided investigative technique by blood bank.It is used for the Therapy study for treating more than ten kind refractory diseases for mescenchymal stem cell,
Except for promoting to recover hematopoiesis, being improved with candidate stem cell co-transplantation beyond leukaemia and refractory anemia etc., being 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 result of the test having been achieved with are encouraging.Research so far
Show, the mescenchymal stem cell in umbilical cord source is not only able to the ideal substitute as mesenchymal stem cells MSCs, and has
Bigger application potential.Umbilical cord mesenchymal stem cells express the peculiar molecular marker of a variety of embryonic stem cells, have differentiation potential
Greatly, multiplication 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 cell as most potential applicability in clinical practice.
RNA binding motif albumens 17 (RNA binding motif protein, RBM), also known as selective mRNA montages
The factor 45 (splicing factor 45, SPF45) encodes a kind of rna binding protein, and the albumen participates in RNA spliceosome complexs
Composition, and played a role in the second catalytic step of mRNA montages.RBM17 regulation Codocyte death receptors FAS's
The alternative splicing of Pre-mRNA, exon 6 of the induction comprising membrane spaning domain jump, and exon 6 jump can produce solvable
Property dominant Fas albumen (Corsini L, et al.2007.U2AF-homology motif interactions are
required for alternative splicing regulation by SPF45.Nat.Struct.Mol.Biol.14:
620-629) .RBM17 includes a unstructured N- terminal domains, a G- patch motif, participates in protein-protein
Between and the interaction between protein and nucleic acid, and a mRNA montage necessary to C-terminal RNA identification motif
(RNA recognition motif, RRM) (Lallena MJ, Chalmers KJ, Llamazares S, Lamond Al,
Valcarcel J.2002.Splicing regulation at the second catalytic step by Sex-
lethal involves 3'splice site recognition by SPF45.Cell 109:285-296).In the mankind
In, RBM17 has a low expression in the ductal epithelium of normal breast, liver, prostate and pancreas, but it is in many human cancers
In have overexpression, such as：Carcinoma of urinary bladder, breast cancer, colon cancer, lung cancer, oophoroma, cancer of pancreas, and prostate cancer etc.；Grind simultaneously
Study carefully proof, RBM17 overexpression (Sampath J, Long PR, Shepard RL, et relevant with the resistance to the action of a drug of many cancers
al.Human SPF45,a splicing factor,has limited expression in normal tissues,is
overexpressed in many tumors and can confer multidrug resistant phenotype to
cells.Am J Pathol2003；163:1781-90).
CN105803053A discloses purposes of the people RBM17 genes in oncotherapy, diagnosing tumor and medicine preparation.This
Invention also further constructs people's RBM17 genes small molecules interference RNA, people's RBM17 gene RNAs construct, people RBM17
Gene disturbs slow virus and discloses their purposes.SiRNA provided by the invention or the nucleic acid comprising the siRNA sequence
Construct, slow virus are capable of the expression of specificity suppression people's RBM17 genes, especially slow virus, can efficiently infect target cell,
Expeditiously suppress the expression of RBM17 genes in target cell, and then suppress the growth of tumour cell, promote apoptosis of tumor cells,
It is significant in oncotherapy.But it is low using siRNA progress jamming effectiveness, interference effect is not permanent, unstable, because
Improved space be present in this.
Versatility based on mescenchymal stem cell, controlled to study the mescenchymal stem cell of knockout RBM17 genes in cancer
Function in terms for the treatment of, establishing the mescenchymal stem cell cell line of knockout RBM17 genes becomes particularly important.
The content of the invention
It is an object of the invention to provide a kind of mescenchymal stem cell of knockout RBM17 genes, prior art is effectively overcome
The technological deficiency of heredity can not 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 RBM17 gene sequence
Row, the specific selectable target site of design are following (dashed part represents PAM motifs)：
RBM17-sgRNA1:5’to 3’gtctcagcttcaggtgaagaagg
RBM17-sgRNA2:5’to 3’actccaccgcatgtagcagctgg
RBM17-sgRNA3:5’to 3’aagacagacatgaagcaagtggg
RBM17-sgRNA4:5’to 3’ttccgggagggccagggtctggg
RBM17-sgRNA5:5’to 3’actagagcacgagtcatctccgg
RBM17-sgRNA6:5’to 3’ggacttggtttggagacataagg
RBM17-sgRNA7:5’to 3’atgtttgtgcttacaagtaccgg
According to these target sites, it is as follows to design specific sgRNA：
RBM17-sgRNA1:5’to 3’gtctcagcttcaggtgaaga
RBM17-sgRNA2:5’to 3’actccaccgcatgtagcagc
RBM17-sgRNA3:5’to 3’aagacagacatgaagcaagt
RBM17-sgRNA4:5’to 3’ttccgggagggccagggtct
RBM17-sgRNA5:5’to 3’actagagcacgagtcatctc
RBM17-sgRNA6:5’to 3’ggacttggtttggagacata
RBM17-sgRNA7:5’to 3’atgtttgtgcttacaagtac
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 marks are cloned
CREnhancer1.0 Lentivirals, slow virus, modified stem cell are packed with GP2-293T cells.
Further, there is provided a kind of system for carrying out gene editing using CRISPR/Cas9 in mescenchymal stem cell,
It is characterized in that the system includes：(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 line of structure RBM17 gene knockouts
Method, including editor's positive cell will be obtained in the gene editing system introducing mescenchymal stem cell, then breed, harvest
The stem cell.
Specific mescenchymal stem cell is human marrow mesenchymal stem cell (hMSCs) PC015, is bought from Shanghai Ai Yan biologies
Science and Technology Ltd..
The invention provides a kind of mescenchymal stem cell cell system, method of structure RBM17 gene knockouts, has following excellent
Point：The present invention in mescenchymal stem cell, construct the cell line of RBM17 gene knockouts, screen and optimize obtain it is optimal
SgRNA, knocks out efficiency high, and passage is stable.
Below by drawings and examples, technical scheme is described in further detail.
Brief description of the drawings
Fig. 1 PX330 plasmid figures；
SgRNA insertion points figure in Fig. 2 PX330；
7 sgRNA genetic marker efficiency schematic diagrames of Fig. 3；
Embodiment
Further illustrate that the present invention improves the technical scheme of the method for genome editorial efficiency below by specific embodiment.
The structure of embodiment 1, CRISPR expression vectors
GRNA design
According to the gene order of target gene, by the unique Optimization Design of applicant, specific screening obtains specific
SgRNA form is as follows：
RBM17-sgRNA1:5’to 3’gtctcagcttcaggtgaaga
RBM17-sgRNA2:5’to 3’actccaccgcatgtagcagc
RBM17-sgRNA3:5’to 3’aagacagacatgaagcaagt
RBM17-sgRNA4:5’to 3’ttccgggagggccagggtct
RBM17-sgRNA5:5’to 3’actagagcacgagtcatctc
RBM17-sgRNA6:5’to 3’ggacttggtttggagacata
RBM17-sgRNA7:5’to 3’atgtttgtgcttacaagtac
According to above-mentioned gRNA, positive oligonucleotide sequence is obtained plus CACC at its 5' end, at the 5' ends of its complementary strand
Reverse oligonucleotide sequence is obtained plus AAAC, 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’-CACCNNNNNNNNNNNNNNNN
NNNN is reverse：NNNNNNNNNNNNNNNNNNNNCAAA-5 ', denaturation, annealing system are：2 μ l forward directions oligonucleotide chains (50 μM)
The μ l l*NEB buffer of 2 μ l reverse oligonucleotides chain (50 μM) 46 are run in PCR instrument by following procedure：90 DEG C, 4min；70
DEG C, 10min；37 DEG C, 20min；25 DEG C, 20min.
Double chain oligonucleotide chain after annealing contains BbsI cohesive end, 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 obtained
PX330-gRNA-Cas9 recombinant plasmids.
Digestion system：39.3 μ l, 10*FD buffer of water 5,2 37 DEG C of water-baths of μ l, PX3303.7 μ l (2 μ g) of μ l, BbsI
Plasmid after 2h digestions is directly reclaimed with glue reclaim kit.
Linked system：The μ l of annealed product 0.5, the μ l of PX330 plasmids 2 μ 1,5*ligation buffer 2 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 obtains above-mentioned steps convert
JM109 competent cells, Amp+ LB flat boards are coated on, picking positive colony connects bacterium, and 37 DEG C of shaking tables shake bacterium and stayed 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, the method synthesized by full genome, obtain SEQ ID NO：1 institute
The gene order stated, using the sequence as template, it is respectively according to upstream and downstream primer sequence
5'-ATGCAGGAGAACCTGGCCCCCTG-3', 5'-CAGGCAGCTCACGCTCCTCTCG -3', primer and complete
Genome is synthesized by Shanghai Sheng Gong Co., Ltds.PCR reaction amplification CREnhancer1.0 gene target gene fragments, amplification are anti-
Answer system as follows:95 DEG C, 40s, 57 DEG C, 1min, 72 DEG C, 1min, 72 DEG C, 10min, circulate 35 times, PCR primer is given birth to by Shanghai
Work Co., Ltd is sequenced, by sequencing, with reference to SEQ ID NO：1 matching completely.Then, purpose base PCR expanded
Because being connected on empty carrier slow virus carrier pHIV-CS-CDF-CG-PRE, expanded by PCR, digestion, sequencing the methods of identify
Recombined lentivirus vector.Successfully constructed with reference to proof recombined lentivirus vector.Then by the recombined lentivirus vector plasmid with auxiliary
Plasmid coinfection human marrow mesenchymal stem cell (hMSCs) PC015 together is helped, can be expressed by restructuring to be packaged into
The human marrow-interstitial stem cell of CREnhancer1.0 genes.By PCR Screening and Identifications, after the stem cell of stable transfection is used for
Continuous gene editing application.
Applications of the CRISPR/Cas9 of embodiment 3 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, BSD genes known to amplification, 5 '-PCR primer bands
HindIII sites, 3 '-PCR primer introduce I-SceI and EcoRI sites.By PCR primer (BSD) insertion EGFP plasmids (EGFP
The sequence that nucleotide sequence is known in the art, such as shown in sequence 1 and sequence 2 in CN105647968A) in CMV driving
HindIII and EcoRI sites between EGFP code areas, the plasmid pBGN of the fusion containing BSD-fsEGFP is generated,
BSD-fsEGFP fusion nucleotides sequences are classified as shown in sequence 3 and sequence 4 in CN105647968A).The fusion
Son or PGK driving son drivings are driven by CMV, but EGFP is inactive due to frameshit, therefore claim fsEGFP.
5 '-PCR primer is
CTCAAGCTTAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAG,
3 '-PCR primer is
AGAATTCCAGTAGGGATAACAGGGTAATGCCAGGTCCGCCCTCCCACACA TAACCAGAG。
(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 determine transfection efficiency, the CRISPR/Cas9 gene editing relative efficiencies obtained are corrected using transfection efficiency.
(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 of specific sgRNA mediations are calculated.This relative efficiency is by GFP sun
Property cell frequencies and transfection efficiency ratio represent, as a result as shown in Figure 3.We have found that the synergistic protein of untransfected embodiment 2
Stem cell in GFP positive cells frequency be significantly lower than transfected embodiment 2 synergistic protein cell.
| Positive frequency | sgRNA1 | sgRNA2 | sgRNA3 | sgRNA4 | sgRNA5 | sgRNA6 | sgRNA7 |
| Import synergistic protein | 0.6 | 0.23 | 0.89 | 0.3 | 0.5 | 0.4 | 0.29 |
| Synergistic protein is not imported | 0.24 | 0.03 | 0.31 | 0.05 | 0.1 | 0.09 | 0.04 |
Wherein in 7 sgRNA, only RBM17-sgRNA3 has preferable gene editing effect, and this is also applicant
The obtained sgRNA with preferable edit effect is just screened in the work for having paid hardships.The negative control prepared using empty carrier
There is no GFP positive cells, wherein P values are respectively less than 0.01, have statistical significance.
Bone marrow interstital stem cell RBM17-sgRNA3 (is obtained after being edited using RBM17-sgRNA3 progress CRISPR
Stem cell) stablize Secondary Culture, after culture 38 instead of, by being sequenced for RBM17PCR, it is found that the gene is still
Mutant inactive, maintain the effect that gene is knocked.This absolutely proves that the system that knocks out of the invention has preferably stabilization
Property.
It should be noted last that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although ginseng
The present invention is described in detail according to preferred embodiment, it will be understood by those within the art that, can be to the present invention
Technical scheme modify or equivalent substitution, without departing from the spirit and scope of technical solution of the present invention.
Sequence table
<110>Luoyang Xuan Zhi bio tech ltd
<120>The method for carrying out RBM17 gene knockouts to mescenchymal stem cell using CRISPR-CAS systems
<160> 12
<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
gtctcagctt caggtgaaga 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
actccaccgc atgtagcagc 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
aagacagaca tgaagcaagt 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
ttccgggagg gccagggtct 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
actagagcac gagtcatctc 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 11
ggacttggtt tggagacata 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 12
atgtttgtgc ttacaagtac 20
Claims (6)
1. a kind of sgRNA, it is used for gene editing.
2. a kind of sgRNA, its sequence such as SEQ ID NO：Shown in 8.
A kind of 3. CRISPR-CAS systems for stem cell cytogene editor, it is characterised in that：The composition bag of system
Include：（1）For expressing SEQ ID NO：The plasmid of CREnhancer1.0 genes described in 1；（2) it is used to express SEQ ID NO：8
Shown sgRNA PX330 plasmid.
4. system as claimed in claim 3, it is characterised in that：（1）Plasmid can imported into advance in gene editing cell,
After screening obtains positive cell, then it is transferred to（2）Plasmid.
5. purposes of the system of claim 3 in the reagent for mesenchymal stem cells MSCs gene editing is prepared.
6. purposes as claimed in claim 5, wherein mesenchymal stem cells MSCs are human marrow mesenchymal stem cell (hMSCs)
PC015。
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