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CN108913691B - 表皮干细胞中采用CRISPR-Cas系统进行Card3基因敲除 - Google Patents

表皮干细胞中采用CRISPR-Cas系统进行Card3基因敲除 Download PDF

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CN108913691B
CN108913691B CN201810780112.1A CN201810780112A CN108913691B CN 108913691 B CN108913691 B CN 108913691B CN 201810780112 A CN201810780112 A CN 201810780112A CN 108913691 B CN108913691 B CN 108913691B
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杨骏
朱成光
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Shandong Huayu Biotechnology Co., Ltd
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Abstract

本发明提供了一种采用CRISPR‑cas系统针对表皮干细胞进行Card3基因编辑，特别是涉及一种构建Card3基因敲除的表皮干细胞细胞系的建立。其中构建并获得了二个特异的gRNA，能够显著提高表皮干细胞内CRISPR/Cas9针对Card3的基因编辑效率。本发明提供的表皮干细胞Card3敲除质粒具有较好的遗传稳定性以及较高的打靶效率。

Description

表皮干细胞中采用CRISPR-Cas系统进行Card3基因敲除
技术领域
本发明提供一种采用CRISPR-cas系统针对表皮干细胞进行CARD3基因编辑，特别是涉及一种构建CARD3基因敲除的表皮干细胞细胞系的建立。
背景技术
表皮干细胞(Epidermal stem cells，EpiSCS)是具有自我增殖和多向分化潜能的干细胞，它的正常增殖和分化是维持皮肤及其附属器(汗腺毛发、皮脂腺)结构和功能完整性的基本要求。在生理条件下，表皮干细胞通过不对称分裂方式分化为一个干细胞和一个短暂扩充细胞(transit amplifying cellsTA细胞)，TA细胞再经过多次分裂后分化为有丝分裂后细胞(Post-mitotic cells)及终末分化细胞(terminally-differentiatedcells)，以补充表皮细胞不断更新的需要。研究表明表皮干细胞不仅能在体外长期传代培养，并保持其增殖分化潜能(Dunnwald et al，Exp Dermatol，2001，10：45-54.Papini etal.stem cells，2003，21：481-494)，而且，在一定环境条件下还表现出与胚胎干细胞相似的分化潜能[Liang et al，stem cells，2002：20：21-31]。因此，获得纯化的表皮干细胞不仅可为构建有生理功能的人工皮肤提供种子细胞，而且可用于基因治疗和转基因动物的生产。
人类多能干细胞(Humanpluripotent stem cells,hPSCs)和基因组编辑技术结合所建立的细胞模型，为疾病研究提供了一个独特的实验平台。利用这个平台体系，研究人员可以研究特定基因突变甚至染色体结构变异对人类多种细胞类型和组织器官功能的影响及其详细的分子机制，并可建立携带不同遗传突变的“个性化”疾病模型用于大规模药物筛选。该模型体系的建立得益于基因组编辑技术，尤其是CRISPR/Cas9(Clustered regularlyinterspaced short palindromic repeats/CRISPR-associated proteins9,CRISPR/Cas9)技术的飞速发展。
Card3(Caspase activat1n and recruitment domain 3)也被称作RIP2，受体相互作用蛋白2(receptor_interacting protein 2)，属于RIP家族成员，在多种组织中均有分布，是一个丝/苏氨酸蛋白激酶，介于激酶域和CARD域之间的媒介领域，最初在1998年由3个独立的研究小组筛选RIP和CARD同源蛋白时发现的，Card3分布于胞质内，在组织表达广泛，其mRNA高表达于脾、外周血白细胞、胎盘、睾丸和心脏等组织中。Card3在固有免疫、适应性免疫和炎症反应中起重要作用，并参与多种疾病发生。许多研究发现当感染细菌及病毒时，Card3可介导NF-κΒ的激活并引起炎症反应，尤其在N0D/RIP2信号途径中，Card3作为其下游因子引起宿主适当的免疫反应。Card3的一个显著特征是其表达在转录水平以细胞特异的、时间依赖的方式被调控。Card3的表达增加反应于细胞特异的刺激，包括细菌的肽聚糖和脂多糖。
CN103877576A公开一种Caspase激活及募集结合域3(Card3)基因在冠状动脉粥样硬化性心脏病中的功能和应用。本发明以Card3基因敲除小鼠和心脏特异性Card3转基因小鼠为对象，通过阻断小鼠心脏左冠状动脉前降支造成心肌梗死模型进行研究，结果表明与WT小鼠对比，Card3基因敲除小鼠心脏梗死比例、心肌肥厚和纤维化的程度明显被抑制，心功能明显好转；而Card3转基因小鼠与Card3基因敲除小鼠表型相反。表明Card3基因能够促进并加重冠状动脉粥样硬化性心脏病的发生发展，Card3可作为药物靶标筛选治疗冠状动脉粥样硬化性心脏病的药物，Card3的抑制剂可用于制备治疗冠状动脉粥样硬化性心脏病的药物。
基于表皮干细胞的多能性，为了研究敲除CARD3基因的表皮干细胞在心脏病治疗方面的功能，建立敲除CARD3基因的表皮干细胞细胞系变得尤为重要。
发明内容
本发明的目的是提供一种敲除CARD3基因的表皮干细胞，有效克服了现有技术使用siRNA进行干扰不能稳定遗传的技术缺陷。
为实现上述目的，本发明提供一种CRISPR-cas系统的靶标，根据CARD3的基因序列，设计的具体的sgRNA如下：
CARD3-sgRNA1：5’to 3’agtagcaaatctgcaccaga
CARD3-sgRNA2：5’to 3’ataatgtatagtgtgtcaca。
为实现上述目的，本发明提供一种提高CRISPR-cas系统在表皮干细胞中敲除CARD3基因的方法，包括向宿主细胞中引入增效蛋白，所述增效蛋白ESCS-higher是由SEQID NO:1所示的核苷酸序列编码的蛋白质。
进一步地，所述增效蛋白包含a)或b)：
a)SEQ ID NO:1所示的核苷酸序列编码的蛋白质的多核苷酸序列；
b)SEQ ID NO:2所示的氨基酸序列。
更进一步地，提供一种在表皮干细胞中采用CRISPR/Cas9进行基因编辑的系统，其特征在于所述系统包括：(1)用于表达SEQ ID NO：1所述的ESCS-higher的质粒；(2)sgRNA已经插入的表达PX330的质粒(其可以表达sgRNA和cas9)。
更进一步地，所述sgRNA和cas9表达载体也可以是本领域常用的其他表达载体。
本发明提供了一种在表皮干细胞中采用CRISPR/Cas9基因编辑CARD3的方法，本发明具有以下优点：
本发明提供了通过近百次的gRNA设计与优化，获得了在表皮干细胞中能够特异性的针对CARD3基因进行敲除的2个特意性的gRNA，具有较强的敲除效果，而且本发明在表皮干细胞中，构建了CARD3基因敲除的细胞系，筛选并优化获得了最佳的sgRNA，敲除效率高，传代稳定。
附图说明
图1 Western-blot检测CARD3蛋白表达结果图；4为表皮干细胞空白对照；3为未导入增效蛋白的gRNA1的效果；1为导入增效蛋白的gRNA1的效果；2为导入增效蛋白的gRNA2的效果。
下面通过实施例，对本发明的技术方案做进一步的详细描述。
具体实施方式
下面通过具体实施例进一步说明本发明提高基因组编辑效率的方法的技术方案。
实施例1、CRISPR表达载体的构建
gRNA的设计
根据靶基因的基因序列，通过申请人前期从几十个gRNA中优化设计具体筛选得到具体的sgRNA的形式如下：
CARD3-sgRNA1：5’to 3’agtagcaaatctgcaccaga
CARD3-sgRNA2：5’to 3’ataatgtatagtgtgtcaca
根据上述的gRNA，在其5'端加上CACC得到正向寡核苷酸序列，在其互补链的5'端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后将合成的序列变性、退火，得到具有BbsI粘性末端的双链DNA片段，如下：正向：5’-CACCNNNNNNNNNNNNNNNNNNNN反向：NNNNNNNNNNNNNNNNNNNNCAAA-5’，变性、退火体系为：2μl正向寡核苷酸链(50μM)2μl反向寡核苷酸链(50μM)46μl l*NEB buffer在PCR仪中按以下程序运行：90℃，4min；72℃，10min；37℃，22min；25℃，25min。
退火后的双链寡核苷酸链含有BbsI的粘性末端，直接与被BbsI酶切过的pX330-U6-Chimeric_BB-CBh-hSpCas9(以下简称PX330)载体进行连接，可以得PX330-gRNA-Cas9重组质粒。
酶切体系：水39.3μl，10*FD buffer 5μl，BbsI 2μl，PX3303.7μl(2μg)37℃水浴2h酶切后的质粒直接用胶回收试剂盒回收。
连接体系：退火产物0.5μl，线性化的PX330质粒2μ1，5*ligation buffer2μl，T4DNA Ligase(3units/μ1)，1μl，水4.5μl，16℃水浴2h将上述步骤得到的连接产物转化JM109感受态细胞，涂布于Amp+的LB平板，挑取阳性克隆接菌，37℃摇床摇菌过夜，质粒抽提试剂盒提取质粒并进行测序鉴定，得到PX330-gRNA质粒。
实施例2克隆增效蛋白ESCS-higher及构建载体
克隆增效蛋白ESCS-higher基因，通过全基因合成的方法，获得SEQ ID NO：1所述的基因序列，以该序列为模板，根据上下游引物序列分别为5'-atgatatactttattagaat-3'，5'-tcaagggatttccatttctc-3'，引物和全基因组由上海生工有限公司合成。PCR反应扩增ESCS-higher基因目的基因片段，扩增反应体系如下:95℃、40s，57℃、1min，72℃、1min，72℃、10min，循环35次，PCR产物由上海生工有限公司进行测序，通过测序，结合与SEQ ID NO：1完全匹配。随后，将PCR扩增的目的基因连接在空载体慢病毒载体pHIV-CS-CDF-CG-PRE上，通过PCR扩增、酶切、测序等方法鉴定重组慢病毒载体。结合证明重组慢病毒载体构建成功。随后将该重组慢病毒载体质粒同辅助质粒一起共感染表皮干细胞(ESCs)(按照CN1253558C中权利要求1的方法分离培养获得)，通过重组而包装成能表达ESCS-higher基因的表皮干细胞。通过PCR筛选鉴定，将稳定转染的干细胞用于后续基因编辑应用。
实施例3 CRISPR/Cas9在表皮干细胞中的应用分析
将实施例1制备的sgRNA表达质粒，公知的Cas9表达质粒共转染表皮干细胞。采用脂质体转染法将构建好的转染表皮干细胞转染体系及试剂使LipofectamineTM2000(invitrogen公司)，转染详细步骤参照转染说明书。以未转染实施例2的增效基因的干细胞作为阳性对照。
实施例4 Western-blot检测CARD3蛋白表达情况
1.总蛋白提取
培养细胞裂解
(1)将表皮干细胞贴壁细胞，去除培养液，用PBS洗一遍，悬浮细胞，离心收集，PBS洗一遍。
(2)通常每106个细胞可加0.1ml RIPA buffer，裂解液和细胞充分接触。
(3)冰上放置数分钟，用枪头轻轻吹打，使细胞充分裂解，再轻轻倾斜培养皿使裂解产物流向瓶皿的一边或一角，然后将之转移到1.5ml离心管，剧烈振荡30秒。
(4)12,000×g，4℃离心5分钟，取上清，即可进行后续的电泳、Western或免疫沉淀操作。
组织块裂解
(1)组织剪切成细小的碎片。每100毫克组织加入1ml RIPA裂解液。用玻璃匀浆器匀浆上下手动匀浆20次。
(2)将匀浆物转移到1.5ml离心管。
(3)12,000×g，4℃离心5分钟，取上清，即可进行后续的电泳、Western或免疫沉淀操作。
2.蛋白浓度测定(BCA测蛋白浓度)
工作液的配制
(1)测定前，按照BCA Reagent A:BCA Reagent B＝100:1的比例混合后配制成工作液，例如配制30ml的工作液时，在30ml的BCA Reagent A中添加0.3ml的BCA Reagent B后，充分振荡混配制后的工作液可在4℃保存三天使用。
(2)所需工作液量的计算方法如下：
所需工作液总体积(ml)＝[(BSA标准溶液8份或7份+检测样品数)×平行样本数(n)+1]×1个样品所需的工作液体积
例)标准操作流程【1ml反应体系】检测样品数为12个、平行样(n＝2)时：
[(8+12)×2+1]×1ml＝41ml
例)标准操作流程【200μl反应体系】、检测样品数为20个、平行样(n＝2)时：
[(8+20)×2+1]×0.2ml＝11.4ml
例)低浓度蛋白质样品测定的操作流程【1ml反应体系】、检测样品数为12个、平行样(n＝2)时：[(7+12)×2+1]×0.5ml＝19.5ml
3.低浓度蛋白样品的标准操作流程(定量范围：0～200μg/ml)
【0.2ml反应体系.使用微孔板测定】
1)BSA标准品溶液的配制。
(1)0.2mg/ml BSA标准品溶液的制备：取120μl BSA Standard Solution(2mg/ml)，加入1,080μl稀释液后充分混合。
(2)按照不同的浓度稀释BSA标准品溶液，BSA标准品溶液和检测样品的稀释可使用去离子水、0.9％NaCl或PBS。
2)BSA标准曲线的制备
(1)分别取100μl稀释后的BSA标准品溶液加入到微孔板中，每个浓度取2个平行样。
(2)加入100ul工作液后，立即混匀。
(3)37℃水浴槽中反应60分钟后，冷却至室温。
(4)使用分光光度计测定562nm处的吸光度值。测定时，使用1mL比色皿，用水校零。尽可能在20分钟内检测完毕所有样品。
(5)各浓度BSA标准品溶液的吸光度值减去Blank值的平均值，绘制BSA标准品溶液的标准曲线。
3)检测样品的测定
检测样品测定时，建议同BSA标准品溶液同时进行测定。
(1)分别取100μl检测样品加入到微孔板中，每个样品取2个平行样进行测定。
(如果必要，也可选择与BSA标准品溶液相同的稀释方法稀释检测样品后测定)
(2)加入100μl工作液后，立即混匀。
(3)37℃水浴中反应60分钟后，冷却至室温。
(4)酶标仪波长设定在562nm处进行测定。用水校零。尽可能在20分钟内检测完毕所有样品。
(5)各样品溶液的吸光度值减去Blank值的平均值，根据标准曲线计算出检测样品的蛋白浓度。
4.SDS－PAGE电泳
(1)玻璃板对齐后放入夹中卡紧。然后垂直卡在架子上准备灌胶。
(2)配制10％分离胶，加入TEMED后立即摇匀即可灌胶。
(3)当水和胶之间有一条折射线时，说明胶已凝了。再等3min使胶充分凝固就可倒去胶上层水并用吸水纸将水吸干。
(4)配制4％的浓缩胶，加入TEMED后立即摇匀即可灌胶。将剩余空间灌满浓缩胶然后将梳子插入浓缩胶中。
(5)用水冲洗一下浓缩胶，将其放入电泳槽中。(小玻璃板面向内，大玻璃板面向外。若只跑一块胶，那槽另一边要垫一块塑料板且有字的一面面向外。)
(6)取出上样样品与5×SDS上样缓冲液按4：1比例混合，混匀后沸水中煮5min使蛋白变性。
(7)加足够的电泳液后按等量蛋白上样。
(8)电泳，80V跑过浓缩胶后转换电压至120V，待溴酚兰跑到胶板底部刚好没有跑出即可。
(9)将夹子打开使黑的一面保持水平，在上面依次垫海绵垫、滤纸、胶、PVDF膜(经甲醇活化)、滤纸、海绵垫；同时将电泳液换成转移液。
(10)将电流调整到恒流200mA，转移约1小时。
(11)取出膜，并做好正反面标记，在TBST中清洗1分钟，然后用封闭液封闭。
(12)用封闭液将对应的一抗稀释成一定的浓度(1:500)，内参一抗的稀释终浓度为1：3000,然后温育1.5小时或4℃孵育过夜。
(13)用TBST清洗3次，每次5分钟。
(14)用封闭液将二抗稀释成一定的浓度(1:3000)，然后温育1.5小时。
(15)用TBST清洗4次，每次5分钟。
5.化学发光，显影，定影
(1)将A和B两种试剂在试管内等体积混合，然后加在PVDF膜的正面，温育大概2分钟。
(2)进入暗室，PVDF膜上盖一层保鲜膜，擦去多余的发光剂。将胶片压在保鲜膜上，依照发光的强度选择不同的曝光时间。
(3)将胶片放入显影液中，出现条带后，立即放入定影液中,流水冲洗胶片后晾干。
(4)对胶片进行扫描，然后用UVP凝胶图象处理系统Labworks4.6软件分析目的条带的灰度值。
(5)通过Western-blot对转染后表皮干细胞中CARD3蛋白质表达进行检测，结果如图1所示，表皮干细胞空白对照，蛋白表达没有受到影响；未导入增效蛋白的gRNA1能够敲除目的基因，蛋白表达能够较好的受到抑制，抑制率达到82.6％；而导入增效蛋白的gRNA1具有显著的提高基因敲除效果蛋白抑制率达到99.2％；导入增效蛋白的gRNA2的蛋白表达抑制率达到99.0％，效果同样极其显著。具有极好的应用前景和应用价值。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。
序列表
<110> 洛阳轩智生物科技有限公司
<120> 表皮干细胞中采用CRISPR-Cas系统进行Card3基因敲除
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2280
<212> DNA
<213> 人工序列(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> 人工序列(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> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
agtagcaaat ctgcaccaga 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
ataatgtata gtgtgtcaca 20

Claims (3)

1.一种用于表皮干细胞Card3基因编辑的CRISPR-CAS系统，其特征在于：系统的组成包括：（1）用于表达SEQ ID NO：1所示的ESCS-higher的质粒；（2)表达SEQ ID NO：3或4所示sgRNA和Cas9的PX330质粒。
2.如权利要求1所述的系统，其特征在于：（1）的质粒提前导入到基因编辑细胞中，筛选得到阳性细胞后，再转入（2）的质粒。
3.权利要求1的系统在制备用于表皮干细胞Card3基因编辑的试剂中的用途。
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