CN108913691A - Card3 gene knockout is carried out using CRISPR-Cas system in epidermal stem cells - Google Patents

Card3 gene knockout is carried out using CRISPR-Cas system in epidermal stem cells Download PDF

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CN108913691A
CN108913691A CN201810780112.1A CN201810780112A CN108913691A CN 108913691 A CN108913691 A CN 108913691A CN 201810780112 A CN201810780112 A CN 201810780112A CN 108913691 A CN108913691 A CN 108913691A
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stem cells
card3
epidermal stem
glu
lys
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CN108913691B (en
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杨骏
朱成光
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Shandong Huayu Biotechnology Co., Ltd
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Luoyang Xuan Zhi Biological Technology Co Ltd
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Abstract

Card3 gene editing is carried out for epidermal stem cells using CRISPR-cas system the present invention provides a kind of, more particularly to a kind of foundation of epidermal stem cells cell line for constructing Card3 gene knockout.Two special gRNA are wherein constructed and obtained, the gene editing efficiency that CRISPR/Cas9 in epidermal stem cells is directed to Card3 can be significantly improved.Epidermal stem cells Card3 provided by the invention, which knocks out plasmid, has preferable genetic stability and higher target practice efficiency.

Description

Card3 gene knockout is carried out using CRISPR-Cas system in epidermal stem cells
Technical field
The present invention provide it is a kind of using CRISPR-cas system for epidermal stem cells carry out CARD3 gene editing, especially It is to be related to a kind of foundation of epidermal stem cells cell line for constructing CARD3 gene knockout.
Background technique
Epidermal stem cells (Epidermal stem cells, EpiSCS) have self duplication and multi-lineage potential Stem cell, its normal proliferative and differentiation are to maintain skin and its appendicle (sweat gland hair, sebaceous glands) structure and function complete The basic demand of property.In physiological conditions, epidermal stem cells are divided into a stem cell and one by Asymmetric division mode Transit amplifying cells (transit amplifying cellsTA cell), TA cell is using being divided into silk after multiple division Postmitotic cell (Post-mitotic cells) and terminally differentiated cells (terminally-differentiated Cells), to supplement the needs that epidermal cell is constantly updated.Research shows that epidermal stem cells can not only be trained Long Term Passages in vitro Support, and keep its Proliferation, Differentiation potential (Dunnwald et al, Exp Dermatol, 2001,10：45-54.Papini et Al.stem cells, 2003,21：481-494), moreover, also being shown under the conditions of certain environment similar to embryonic stem cell Differentiation potential [Liang et al, stem cells, 2002：20：21-31].Therefore, the epidermal stem cells of purifying are obtained not Only seed cell can be provided to be built with the artificial skin of physiological function, and can be used for gene therapy and the life of transgenic animals It produces.
Human pluripotent stem cell (Humanpluripotent stem cells, hPSCs) and genome editing technique combine The cell model established provides a unique experiment porch for disease research.Utilize this platform system, researcher Specific gene mutation even influence of the chromosomal structural variation to mankind's various kinds of cell type and tissue organ function can be studied And its detailed molecular mechanism, and " personalization " disease model for carrying different genetic mutations can be established for large-scale medicine sieve Choosing.The foundation of the model system has benefited from genome editing technique, especially CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins9,CRISPR/ Cas9) the rapid development of technology.
Card3 (Caspase activat1n and recruitment domain 3) is also referred to as RIP2, receptor phase Interaction albumen 2 (receptor_interacting protein 2), belongs to RIP family member, has in Various Tissues Distribution, is a protein serine/threonine, the media field between kinase domain and the domain CARD, initially in 1998 by 3 It is found when a independent research group's screening RIP and CARD homologous protein, Card3 is distributed in cytoplasm, wide in tissue expression General, mRNA high is expressed in the tissue such as spleen, peripheral white blood cells, placenta, testis and heart.Card3 is in inherent immunity, adaptation Property immune and inflammatory reaction in play an important role, and participate in a variety of diseases and occur.Bacterial infection and virus are worked as in many research discoveries When, Card3 can mediate the activation of NF- κ Β and cause inflammatory reaction, and especially in N0D/RIP2 signal pathway, Card3 is as it Downstream elements cause host's immune response appropriate.One of Card3 is noteworthy characterized by its expression in transcriptional level with cell spy Different, time dependent mode is adjusted.The expression of Card3 increases the stimulation for reacting on cell-specific, and the peptide including bacterium is poly- Sugar and lipopolysaccharides.
CN103877576A discloses a kind of Caspase activation and to raise (Card3) gene of binding domain 3 athero- in coronary artery Function and application in hardenability heart disease.The present invention is small with Card3 knock out mice and heartspecific Card3 transgenosis Mouse is object, by blocking mouse heart ramus descendens anterior arteriae coronariae sinistrae that myocardial infarction model is caused to be studied, the results showed that with The comparison of WT mouse, the degree of Card3 knock out mice cardiac infarction ratio, myocardial hypertrophy and fibrosis are obviously suppressed, the heart Function is clearly better；And Card3 transgenic mice is opposite with Card3 knock out mice phenotype.Show that Card3 gene can promote Into and aggravate the occurrence and development of coronary atherosclerotic heart disease, it is coronal dynamic that Card3 can be used as drug targets screening treatment The cardiopathic drug of pulse atherosclerosis, the inhibitor of Card3 can be used for preparing treatment coronary atherosclerotic heart disease Drug.
Versatility based on epidermal stem cells, in order to study the epidermal stem cells for knocking out CARD3 gene in disease treatment The function of aspect, the epidermal stem cells cell line for establishing knockout CARD3 gene become particularly important.
Summary of the invention
The object of the present invention is to provide a kind of epidermal stem cells for knocking out CARD3 gene, and effectively overcoming the prior art makes The technological deficiency of heredity cannot be stablized by carrying out interference with siRNA.
To achieve the above object, the present invention provides a kind of target of CRISPR-cas system, according to the gene sequence of CARD3 Column, the specific sgRNA of design are as follows：
CARD3-sgRNA1：5'to 3'agtagcaaatctgcaccaga
CARD3-sgRNA2：5'to 3'ataatgtatagtgtgtcaca.
To achieve the above object, the present invention provides a kind of raising CRISPR-cas system and knocks out in epidermal stem cells The method of CARD3 gene, including synergistic protein is introduced into host cell, the synergistic protein ESCS-higher 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, a kind of system carrying out gene editing using CRISPR/Cas9 in epidermal stem cells is provided, Be characterized in that the system comprises：(1) for expressing SEQ ID NO：The plasmid of ESCS-higher described in 1；(2) sgRNA has been The plasmid of expression PX330 through being inserted into (it can express sgRNA and cas9).
Further, sgRNA the and cas9 expression vector is also possible to other expression vectors commonly used in the art.
The method that the present invention provides a kind of to use CRISPR/Cas9 gene editing CARD3 in epidermal stem cells, this hair It is bright to have the following advantages that：
It being capable of specificity in epidermal stem cells the present invention provides obtaining by gRNA design and optimization nearly 100 times The 2 Non-specific gRNA knocked out for CARD3 gene, there is stronger knockout effect, and the present invention is in epidermis In stem cell, construct the cell line of CARD3 gene knockout, screen and optimize and obtain optimal sgRNA, knock out it is high-efficient, Passage is stablized.
Detailed description of the invention
Fig. 1 Western-blot detects CARD3 protein expression result figure；4 be epidermal stem cells blank control；3 be not lead Enter the effect of the gRNA1 of synergistic protein；1 is the effect for importing the gRNA1 of synergistic protein；2 be the gRNA2 for importing synergistic protein Effect.
Below by embodiment, technical scheme of the present invention will be described in further detail.
Specific embodiment
Further illustrate that the present invention improves the technical solution of the method for genome editorial efficiency below by specific embodiment.
The building of embodiment 1, CRISPR expression vector
The design of gRNA
According to the gene order of target gene, by applicant's early period, optimization design is specifically screened and is obtained from tens gRNA The form of specific sgRNA is as follows：
CARD3-sgRNA1：5'to 3'agtagcaaatctgcaccaga
CARD3-sgRNA2：5'to 3'ataatgtatagtgtgtcaca
According to above-mentioned gRNA, positive oligonucleotide sequence is obtained plus CACC at its end 5', at the end 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 end, as follows：It is positive：5'- CACCNNNNNNNNNNNNNNNNNNNN is reversed：NNNNNNNNNNNNNNNNNNNNCAAA-5 ', denaturation, annealing system are：2μl Positive 2 μ l reverse oligonucleotide chain (50 μM) of oligonucleotide chain (50 μM), 46 μ l l*NEB buffer presses following journey in PCR instrument Sort run：90 DEG C, 4min；72 DEG C, 10min；37 DEG C, 22min；25 DEG C, 25min.
Double chain oligonucleotide chain after annealing contains the cohesive end of BbsI, directly and by the pX330- of BbsI digestion U6-Chimeric_BB-CBh-hSpCas9 (hereinafter referred to as PX330) carrier is attached, and can obtain PX330-gRNA-Cas9 weight Group plasmid.
Digestion system：39.3 μ l, 10*FD buffer of water, 52 37 DEG C of water-bath 2h of μ l, PX3303.7 μ l (2 μ g) of μ l, BbsI Plasmid after digestion is directly recycled with plastic recovery kit.
Linked system：0.5 μ l of annealed product, PX330 plasmid 2 μ 1,5*ligation the buffer2 μ l 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 cell is coated on the LB plate 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 plasmid.
Embodiment 2 clones synergistic protein ESCS-higher and carrier construction
Synergistic protein ESCS-higher gene is cloned, by full genome synthetic method, obtains SEQ ID NO：Described in 1 Gene order using the sequence as template be respectively 5'-atgatatactttattagaat-3' according to upstream and downstream primer sequence, 5'-tcaagggatttccatttctc-3', primer and full-length genome are synthesized by Shanghai Sheng Gong Co., Ltd.PCR reaction amplification ESCS-higher gene target gene fragment, 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 is sequenced by Shanghai Sheng Gong Co., Ltd, by sequencing, in conjunction with SEQ ID NO： 1 exact matching.Then, the target gene of PCR amplification is connected on empty carrier slow virus carrier pHIV-CS-CDF-CG-PRE, Recombined lentivirus vector is identified by the methods of PCR amplification, digestion, sequencing.It is constructed successfully in conjunction with proof recombined lentivirus vector. Then by the recombined lentivirus vector plasmid with helper plasmid together coinfection epidermal stem cells (ESCs) (according to CN1253558C Middle method of claim 1 is separately cultured acquisition), the epidermal stem that can express ESCS-higher gene is packaged by recombination Cell.By PCR screening and identification, the stem cell of stable transfection is used for subsequent gene editor application.
Applied analysis of 3 CRISPR/Cas9 of embodiment in epidermal stem cells
SgRNA expression plasmid prepared by embodiment 1, well known Cas9 expression plasmid cotransfection epidermal stem cells.Using The transfection epidermal stem cells rotaring redyeing system and reagent that build are made Lipofectamine by lipofectionTM2000 (invitrogen company), transfection detailed step is referring to transfection specification.With the stem cell of the synergy gene of untransfected embodiment 2 As positive control.
4 Western-blot of embodiment detects CARD3 protein expression situation
1. total protein extraction
Cultivate cell cracking
(1) by epidermal stem cells attached cell, culture solution is removed, is washed one time with PBS, suspension cell is collected by centrifugation, PBS It washes one time.
(2) usually every 106 cells can add 0.1ml RIPA buffer, and lysate and cell come into full contact with.
(3) it places on ice several minutes, is gently blown and beaten with pipette tips, crack cell sufficiently, then gently inclination culture dish makes to split Solution product flow to bottle ware one side or one jiao, it is then transferred to 1.5ml centrifuge tube, acutely oscillation 30 seconds.
DEG C centrifugation of (4) 12,000 × g, 45 minutes, takes supernatant, can carry out subsequent electrophoresis, Western or immunoprecipitation Operation.
Tissue block cracking
(1) tissue cuts into tiny fragment.1ml RIPA lysate is added in every 100 milligrams of tissues.Use glass homogenizer Homogenate homogenate 20 times manually up and down.
(2) homogenate is transferred to 1.5ml centrifuge tube.
DEG C centrifugation of (3) 12,000 × g, 45 minutes, takes supernatant, can carry out subsequent electrophoresis, Western or immunoprecipitation Operation.
2. determination of protein concentration (BCA surveys protein concentration)
The preparation of working solution
(1) before measuring, according to BCA Reagent A:BCA Reagent B=100:Work is configured to after 1 ratio mixing When making liquid, such as preparing the working solution of 30ml, the BCA Reagent B of 0.3ml is added in the BCA Reagent A of 30ml Afterwards, the working solution sufficiently after oscillation mixture system can be saved three days at 4 DEG C and be used.
(2) calculation method of work liquid measure needed for is as follows：
Required working solution total volume (ml)=[(8 parts of BSA standard solution or 7 parts+test sample number) × parallel samples number (n)+1] × 1 working solution volume needed for sample
Example) Standard Operating Procedure【1ml reaction system】When test sample number is 12, Duplicate Samples (n=2)：
[(8+12) × 2+1] × 1ml=41ml
Example) Standard Operating Procedure【200 μ l reaction systems】, test sample number be 20, Duplicate Samples (n=2) when：
[(8+20) × 2+1] × 0.2ml=11.4ml
Example) low concentration protein example measurement operating process【1ml reaction system】, test sample number be 12, it is parallel When sample (n=2)：[(7+12) × 2+1] × 0.5ml=19.5ml
3. Standard Operating Procedure (the quantification range of low concentration protein sample：0~200 μ g/ml)
【0.2ml reaction system is measured using microwell plate】
1) preparation of BSA standard solution.
(1) preparation of 0.2mg/ml BSA standard solution：Take 120 μ l BSA Standard Solution (2mg/ Ml), it is sufficiently mixed after 1,080 μ l dilution being added.
(2) according to different concentration dilution BSA standard solutions, the dilution of BSA standard solution and test sample can make With deionized water, 0.9%NaCl or PBS.
2) preparation of BSA standard curve
(1) the BSA standard solution after taking 100 μ l to dilute respectively is added in microwell plate, each concentration take 2 it is parallel Sample.
(2) it after 100ul working solution being added, mixes immediately.
It reacts after sixty minutes, is cooled to room temperature in (3) 37 DEG C of water baths.
(4) using the absorbance value at spectrophotometric determination 562nm.When measurement, using 1mL cuvette, with water school zero. Detection finishes all samples as far as possible in 20 minutes.
(5) absorbance value of each concentration BSA standard solution subtracts the average value of Blank value, draws BSA standard solution Standard curve.
3) measurement of test sample
When test sample measures, it is proposed that be measured simultaneously with BSA standard solution.
(1) 100 μ l test samples are taken to be added in microwell plate respectively, each sample takes 2 Duplicate Samples to be measured.
(being measured after dilution process dilution test sample identical with BSA standard solution if necessary, also may be selected)
(2) it after 100 μ l working solutions being added, mixes immediately.
It reacts after sixty minutes, is cooled to room temperature in (3) 37 DEG C of water-baths.
(4) microplate reader wavelength is set at 562nm and is measured.With water school zero.It detects and finishes in 20 minutes as far as possible All samples.
(5) absorbance value of each sample solution subtracts the average value of Blank value, calculates test sample according to standard curve Protein concentration.
4.SDS-PAGE electrophoresis
(1) clamping in folder is put into after glass plate alignment.Then vertical card prepares encapsulating on the top of the shelf.
(2) 10% separation gel is prepared, shaking up immediately after addition TEMED can encapsulating.
(3) when having a fringence between Dang Shui and glue, illustrate that glue has coagulated.Equal 3min make glue sufficiently solidify to fall again It removes photoresist and upper water and is blotted water with blotting paper.
(4) 4% concentration glue is prepared, shaking up immediately after addition TEMED can encapsulating.It is right that remaining space is filled into concentration glue Comb is inserted into concentration glue afterwards.
(5) it is rinsed with water and glue is concentrated, put it into electrophoresis tank.(small glass-board surface is inside, big glass-board surface to Outside.If only running one piece of glue, that slot another side will pad one piece of plastic plate and have facing out on one side for word.)
(6) it takes out loading sample and 5 × SDS sample-loading buffer presses 4：The mixing of 1 ratio, boiling 5min in boiling water after mixing makes egg Leucismus.
(7) plus after enough electrophoresis liquids equal protein loading is pressed.
(8) electrophoresis, conversion voltage was gone to offset plate bottom to bromjophenol blue and was not run just to 120V after 80V runs concentration glue Out.
(9) opening clip keeps black one side holding horizontal, is successively padding foam-rubber cushion, filter paper, glue, pvdf membrane (warp above Methanol activation), filter paper, foam-rubber cushion；Change electrophoresis liquid into transfer liquid simultaneously.
(10) electric current is adjusted to constant current 200mA, shifted about 1 hour.
(11) film is taken out, and carries out front and back sides label, cleans 1 minute in TBST, is then closed with confining liquid.
(12) corresponding primary antibody is diluted to certain concentration (1 with confining liquid:500), the dilution final concentration of internal reference primary antibody It is 1：3000, it then incubates 1.5 hours or 4 DEG C and is incubated overnight.
(13) it is cleaned 3 times, every time 5 minutes with TBST.
(14) secondary antibody is diluted to certain concentration (1 with confining liquid:3000) it, then incubates 1.5 hours.
(15) it is cleaned 4 times, every time 5 minutes with TBST.
5. chemiluminescence is developed, fixing
(1) two kinds of reagents of A and B are in vitro mixed in equal volume, is then added in the front of pvdf membrane, incubate general 2 points Clock.
(2) enter darkroom, one layer of preservative film of pvdf membrane upper cover wipes extra luminous agent.Film is pressed on preservative film, The different time for exposure is selected according to luminous intensity.
(3) film is put into developer solution, after there is band, is immediately placed in fixing solution, flowing water dries after developing photographic film.
(4) film is scanned, then analyzes purpose item with UVP gel images processing system Labworks4.6 software The gray value of band.
(5) CARD3 protein expression in epidermal stem cells after transfection is detected by Western-blot, as a result such as Shown in Fig. 1, epidermal stem cells blank control, protein expression is not affected；The gRNA1 for not importing synergistic protein can be knocked out Target gene, protein expression can be preferably suppressed, and inhibiting rate reaches 82.6%；And import the gRNA1 tool of synergistic protein The gene knockout effect albumen inhibiting rate that increases significantly reaches 99.2%；Import the protein expression suppression of the gRNA2 of synergistic protein Rate processed reaches 99.0%, and effect is equally extremely significant.With fabulous application prospect and application value.
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, those skilled in the art should understand that, it can be to the present invention Technical solution be modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention.
Sequence table
<110>Luoyang Xuan Zhi Biotechnology Co., Ltd
<120>Card3 gene knockout is carried out using CRISPR-Cas system in epidermal stem cells
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2280
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
atgatatact ttattagaat aatcatgggc cagactggga agaaatctga gaagggacca 60
gtttgttggc ggaagcgtgt aaaatcagag tacatgcgac tgagacagct caagaggttc 120
agacgagctg atgaagtaaa gagtatgttt agttccaatc gtcagaaaat tttggaaaga 180
acggaaatct taaaccaaga atggaaacag cgaaggatac agcctgtgca catcctgact 240
tctgtgagct cattgcgcgg gactagggag tgttcggtga ccagtgactt ggattttcca 300
acacaagtca tcccattaaa gactctgaat gcagttgctt cagtacccat aatgtattct 360
tggtctcccc tacagcagaa ttttatggtg gaagatgaaa ctgttttaca taacattcct 420
tatatgggag atgaagtttt agatcaggat ggtactttca ttgaagaact aataaaaaat 480
tatgatggga aagtacacgg ggatagagaa tgtgggttta taaatgatga aatttttgtg 540
gagttggtga atgcccttgg tcaatataat gatgatgacg atgatgatga tggagacgat 600
cctgaagaaa gagaagaaaa gcagaaagat ctggaggatc accgagatga taaagaaagc 660
cgcccacctc ggaaatttcc ttctgataaa atttttgaag ccatttcctc aatgtttcca 720
gataagggca cagcagaaga actaaaggaa aaatataaag aactcaccga acagcagctc 780
ccaggcgcac ttcctcctga atgtaccccc aacatagatg gaccaaatgc taaatctgtt 840
cagagagagc aaagcttaca ctcctttcat acgcttttct gtaggcgatg ttttaaatat 900
gactgcttcc tacatcgtaa gtgcaattat tcttttcatg caacacccaa cacttataag 960
cggaagaaca cagaaacagc tctagacaac aaaccttgtg gaccacagtg ttaccagcat 1020
ttggagggag caaaggagtt tgctgctgct ctcaccgctg agcggataaa gaccccacca 1080
aaacgtccag gaggccgcag aagaggacgg cttcccaata acagtagcag gcccagcacc 1140
cccaccatta atgtgctgga atcaaaggat acagacagtg atagggaagc agggactgaa 1200
acggggggag agaacaatga taaagaagaa gaagagaaga aagatgaaac ttcgagctcc 1260
tctgaagcaa attctcggtg tcaaacacca ataaagatga agccaaatat tgaacctcct 1320
gagaatgtgg agtggagtgg tgctgaagcc tcaatgttta gagtcctcat tggcacttac 1380
tatgacaatt tctgtgccat tgctaggtta attgggacca aaacatgtag acaggtgtat 1440
gagtttagag tcaaagaatc tagcatcata gctccagctc ccgctgagga tgtggatact 1500
cctccaagga aaaagaagag gaaacaccgg ttgtgggctg cacactgcag aaagatacag 1560
ctgaaaaagg acggctcctc taaccatgtt tacaactatc aaccctgtga tcatccacgg 1620
cagccttgtg acagttcgtg cccttgtgtg atagcacaaa atttttgtga aaagttttgt 1680
caatgtagtt cagagtgtca aaaccgcttt ccgggatgcc gctgcaaagc acagtgcaac 1740
accaagcagt gcccgtgcta cctggctgtc cgagagtgtg accctgacct ctgtcttact 1800
tgtggagccg ctgaccattg ggacagtaaa aatgtgtcct gcaagaactg cagtattcag 1860
cggggctcca aaaagcatct attgctggca ccatctgacg tggcaggctg ggggattttt 1920
atcaaagatc ctgtgcagaa aaatgaattc atctcagaat actgtggaga gattatttct 1980
caagatgaag ctgacagaag agggaaagtg tatgataaat acatgtgcag ctttctgttc 2040
aacttgaaca atgattttgt ggtggatgca acccgcaagg gtaacaaaat tcgttttgca 2100
aatcattcgg taaatccaaa ctgctatgca aaagttatga tggttaacgg tgatcacagg 2160
ataggtattt ttgccaagag agccatccag actggcgaag agctgttttt tgattacaga 2220
tacagccagg ctgatgccct gaagtatgtc ggcatcgaaa gagaaatgga aatcccttga 2280
<210> 2
<211> 759
<212> PRT
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
Met Ile Tyr Phe Ile Arg Ile Ile Met Gly Gln Thr Gly Lys Lys Ser
1 5 10 15
Glu Lys Gly Pro Val Cys Trp Arg Lys Arg Val Lys Ser Glu Tyr Met
20 25 30
Arg Leu Arg Gln Leu Lys Arg Phe Arg Arg Ala Asp Glu Val Lys Ser
35 40 45
Met Phe Ser Ser Asn Arg Gln Lys Ile Leu Glu Arg Thr Glu Ile Leu
50 55 60
Asn Gln Glu Trp Lys Gln Arg Arg Ile Gln Pro Val His Ile Leu Thr
65 70 75 80
Ser Val Ser Ser Leu Arg Gly Thr Arg Glu Cys Ser Val Thr Ser Asp
85 90 95
Leu Asp Phe Pro Thr Gln Val Ile Pro Leu Lys Thr Leu Asn Ala Val
100 105 110
Ala Ser Val Pro Ile Met Tyr Ser Trp Ser Pro Leu Gln Gln Asn Phe
115 120 125
Met Val Glu Asp Glu Thr Val Leu His Asn Ile Pro Tyr Met Gly Asp
130 135 140
Glu Val Leu Asp Gln Asp Gly Thr Phe Ile Glu Glu Leu Ile Lys Asn
145 150 155 160
Tyr Asp Gly Lys Val His Gly Asp Arg Glu Cys Gly Phe Ile Asn Asp
165 170 175
Glu Ile Phe Val Glu Leu Val Asn Ala Leu Gly Gln Tyr Asn Asp Asp
180 185 190
Asp Asp Asp Asp Asp Gly Asp Asp Pro Glu Glu Arg Glu Glu Lys Gln
195 200 205
Lys Asp Leu Glu Asp His Arg Asp Asp Lys Glu Ser Arg Pro Pro Arg
210 215 220
Lys Phe Pro Ser Asp Lys Ile Phe Glu Ala Ile Ser Ser Met Phe Pro
225 230 235 240
Asp Lys Gly Thr Ala Glu Glu Leu Lys Glu Lys Tyr Lys Glu Leu Thr
245 250 255
Glu Gln Gln Leu Pro Gly Ala Leu Pro Pro Glu Cys Thr Pro Asn Ile
260 265 270
Asp Gly Pro Asn Ala Lys Ser Val Gln Arg Glu Gln Ser Leu His Ser
275 280 285
Phe His Thr Leu Phe Cys Arg Arg Cys Phe Lys Tyr Asp Cys Phe Leu
290 295 300
His Arg Lys Cys Asn Tyr Ser Phe His Ala Thr Pro Asn Thr Tyr Lys
305 310 315 320
Arg Lys Asn Thr Glu Thr Ala Leu Asp Asn Lys Pro Cys Gly Pro Gln
325 330 335
Cys Tyr Gln His Leu Glu Gly Ala Lys Glu Phe Ala Ala Ala Leu Thr
340 345 350
Ala Glu Arg Ile Lys Thr Pro Pro Lys Arg Pro Gly Gly Arg Arg Arg
355 360 365
Gly Arg Leu Pro Asn Asn Ser Ser Arg Pro Ser Thr Pro Thr Ile Asn
370 375 380
Val Leu Glu Ser Lys Asp Thr Asp Ser Asp Arg Glu Ala Gly Thr Glu
385 390 395 400
Thr Gly Gly Glu Asn Asn Asp Lys Glu Glu Glu Glu Lys Lys Asp Glu
405 410 415
Thr Ser Ser Ser Ser Glu Ala Asn Ser Arg Cys Gln Thr Pro Ile Lys
420 425 430
Met Lys Pro Asn Ile Glu Pro Pro Glu Asn Val Glu Trp Ser Gly Ala
435 440 445
Glu Ala Ser Met Phe Arg Val Leu Ile Gly Thr Tyr Tyr Asp Asn Phe
450 455 460
Cys Ala Ile Ala Arg Leu Ile Gly Thr Lys Thr Cys Arg Gln Val Tyr
465 470 475 480
Glu Phe Arg Val Lys Glu Ser Ser Ile Ile Ala Pro Ala Pro Ala Glu
485 490 495
Asp Val Asp Thr Pro Pro Arg Lys Lys Lys Arg Lys His Arg Leu Trp
500 505 510
Ala Ala His Cys Arg Lys Ile Gln Leu Lys Lys Asp Gly Ser Ser Asn
515 520 525
His Val Tyr Asn Tyr Gln Pro Cys Asp His Pro Arg Gln Pro Cys Asp
530 535 540
Ser Ser Cys Pro Cys Val Ile Ala Gln Asn Phe Cys Glu Lys Phe Cys
545 550 555 560
Gln Cys Ser Ser Glu Cys Gln Asn Arg Phe Pro Gly Cys Arg Cys Lys
565 570 575
Ala Gln Cys Asn Thr Lys Gln Cys Pro Cys Tyr Leu Ala Val Arg Glu
580 585 590
Cys Asp Pro Asp Leu Cys Leu Thr Cys Gly Ala Ala Asp His Trp Asp
595 600 605
Ser Lys Asn Val Ser Cys Lys Asn Cys Ser Ile Gln Arg Gly Ser Lys
610 615 620
Lys His Leu Leu Leu Ala Pro Ser Asp Val Ala Gly Trp Gly Ile Phe
625 630 635 640
Ile Lys Asp Pro Val Gln Lys Asn Glu Phe Ile Ser Glu Tyr Cys Gly
645 650 655
Glu Ile Ile Ser Gln Asp Glu Ala Asp Arg Arg Gly Lys Val Tyr Asp
660 665 670
Lys Tyr Met Cys Ser Phe Leu Phe Asn Leu Asn Asn Asp Phe Val Val
675 680 685
Asp Ala Thr Arg Lys Gly Asn Lys Ile Arg Phe Ala Asn His Ser Val
690 695 700
Asn Pro Asn Cys Tyr Ala Lys Val Met Met Val Asn Gly Asp His Arg
705 710 715 720
Ile Gly Ile Phe Ala Lys Arg Ala Ile Gln Thr Gly Glu Glu Leu Phe
725 730 735
Phe Asp Tyr Arg Tyr Ser Gln Ala Asp Ala Leu Lys Tyr Val Gly Ile
740 745 750
Glu Arg Glu Met Glu Ile Pro
755
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
agtagcaaat ctgcaccaga 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
ataatgtata gtgtgtcaca 20

Claims (8)

1. a kind of sgRNA, is used for gene editing.
2. a kind of sgRNA, sequence such as SEQ ID NO：3 or 4 is any shown.
3. CRISPR-cas system method that Card3 gene is knocked out in vitro epidermal stem cells is improved a kind of, including to place Synergistic protein is introduced in chief cell, the synergistic protein ESCS-higher is by SEQ ID NO:Nucleotide sequence shown in 1 is compiled The protein of code；The sgRNA, sequence such as SEQ ID NO：3 or 4 is any shown.
4. method as claimed in claim 3, 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.
5. a kind of system for carrying out gene editing using CRISPR/Cas9 in epidermal stem cells, it is characterised in that the system Including：(1) for expressing SEQ ID NO：The plasmid of ESCS-higher described in 1；(2) expression that sgRNA is already inserted into The plasmid of PX330 can express sgRNA and cas9.
6. system as claimed in claim 5, it is characterised in that：(1) plasmid can be imported into advance in gene editing cell, After screening obtains positive cell, then it is transferred to the plasmid of (2).
7. purposes of the system of claim 5 in the reagent that preparation is used for epidermal stem cells gene editing.
8. purposes of the gRNA as claimed in claim 2 in preparation in epidermal stem cells and in the reagent of progress Card3 knockout.
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