CN108753772A - 基于CRISPR/Cas技术敲除CAPNS1基因的人神经母细胞瘤细胞系的构建方法 - Google Patents
基于CRISPR/Cas技术敲除CAPNS1基因的人神经母细胞瘤细胞系的构建方法 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,尤其涉及基于CRISPR/Cas技术敲除CAPNS1基因的人神经母细胞瘤细胞系的构建方法。本发明提供了敲除人神经母细胞瘤细胞CAPNS1基因的方法,质粒转染SK‑N‑SH细胞后,制备单克隆,CruiserTM Enzyme酶切后疑似为阳性克隆,单克隆测序结果显示SK‑N‑SH细胞CAPNS1基因敲除细胞系构建成功;并且,在CAPNS1‑/‑组,CAPNS1蛋白及Calpain1和Calpain2蛋白水平明显降低。结果表明SK‑N‑SH细胞CAPNS1基因敲除细胞系构建成功。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及敲除人神经母细胞瘤细胞 CAPNS1基因的方法。
背景技术
钙蛋白酶(calpain)是一组高度保守的特异性Ca2+依赖性细胞内中性蛋白酶,其活性主要与细胞内Ca2+浓度有关。calpain在机体的生理过程中发挥着重要作用,其不仅与细胞内蛋白质的水解关,还参与细胞自噬、细胞周期调控与凋亡、细胞骨架重构、葡萄糖转运、细胞信号转导等正常的生理过程[3,4]。自在大鼠的脑组织中首次发现一种可溶的Ca2+依赖的中性蛋白酶以来,研究者们越来越关注这种蛋白酶在体内的作用。目前,研究发现在哺乳动物中calpain 存在15种亚型,其中,calpain1(μ-calpain)和calpain2(m-calpain)在体内广泛表达,且研究也较为广泛。Calpain1和calpain2主要由80kD的大亚基和30kD的小亚基(calpain-s1,CAPNS1)组成,CAPNS1是calpain1和calpain2活性所必需的,而且也是细胞迁移,凋亡和存活所必需的。
人神经母细胞瘤细胞(human neuroblastoma cell,HNC)是研究神经系统基因功能和神经毒性机制的良好的体外模型,建立敲除CAPNS1基因的人神经母细胞瘤细胞模型有利于对CAPNS1蛋白的作用的进一步研究,更有利于对神经系统多种疾病机理的研究。
基因敲除是通过DNA序列改变,出现基因功能的“全或无”表象。基因沉默技术是以不改变DNA序列为前提,使基因沉默,即基因不表达或低表达。基因编辑技术是指在基因组水平精确地进行基因编辑,其是通过精确识别靶细胞DNA片段中靶点的核苷酸序列,利用核酸内切酶对DNA靶点序列进行切割,从而完成对靶细胞DNA目的基因片段的敲除、加入等过程。基因敲除构建出的突变型细胞系比基因沉默构建的细胞系稳定。细胞基因沉默的效率并不总是非常高,也不十分稳定,影响实验结果敏感性和可靠性。
继锌指核酸酶(ZFN)和转录激活因子样效应物核酸酶(TALENs)基因编辑技术之后,第三代基因编辑技术CRISPR/Cas技术是目前运用最为广泛的基因技术。CRISPR/Cas是一种来源于原核生物适应性免疫系统,通过人工改良而得。相比ZFN和TALENs,CRISPR/Cas技术具有成本低、可同时进行多位点编辑、效率高等优势。CRISPR/Cas系统分为3种,其中II型最为简单,研究得也较多。该系统主要包含非特异性Cas9核酸酶和sgRNA两部分,其中sgRNA具引导和检测作用;Cas9是一种RNA依赖的DNA内切核酸酶,其利用单一向导 RNA(sgRNA)使双链DNA断裂,起到剪刀作用。
脱靶效应是存在于所有基因组靶向修饰技术中的一道难题,它会对基因组非特性序列进行切割,造成未知突变,增加后期的鉴定工作量。CRISPR/Cas9 系统中,对靶序列的识别主要是依靠一段20bp的短RNA,但是研究表明当存在单个甚至多达5个碱基错配时,切割仍能正常发生。进一步研究发现,在这20bp中只有位于PAM位点前12bp的种子序列对靶位点识别影响较大,即总共只有14bp(PAM中的GG和种子序列)是靶位点识别的关键序列。这在生物体庞大的基因组中很容易出现脱靶位点,从而引入意外突变。除此之外,Cas9蛋白或sgRNA的浓度也对脱靶活性产生影响。
而神经细胞本身较其他细胞的培养较为苛刻,是一种转染质粒较难的细胞系,且转染效率低,而电转对神经细胞的伤害也较大,因此,对于转染条件的摸索较不易。在筛选单克隆时,神经细胞生长培养较难。综上,构建敲除人神经母细胞瘤细胞CAPNS1基因并不容易。
发明内容
有鉴于此,本发明要解决的技术问题在于以CRISPR/Cas技术敲除人神经母细胞瘤细胞CAPNS1基因的方法及细胞系,其能够成功、稳定的敲除掉人神经母细胞瘤细胞中的CAPNS1基因。
本发明提供了人CAPNS1基因第4外显子和/或第5外显子在构建 CAPNS1基因敲除人神经母细胞瘤细胞中的应用。
本发明还提供了靶向CAPNS1基因第4外显子的sgRNA,其核苷酸序列如SEQ IDNO.1和SEQ ID NO.2所示。
靶向CAPNS1基因第5外显子的sgRNA,其核苷酸序列如SEQ ID NO.3 所示。
本发明试验表明,采用CAPNS1基因的第1~3外显子区域的靶位点构建出来的载体在转染细胞后,没有筛选到纯合子。而采用CAPNS1基因的第4~5 外显子的CDS区作为靶点,成功地构建了CAPNS1基因敲除SK-N-SH细胞株。
本发明还提供了如SEQ ID NO.4~9所示的核苷酸序列的6条敲除 CAPNS1基因的靶标寡核苷酸序列。
所述SEQ ID NO.4~9的核苷酸序列中,SEQ ID NO.4~5以SEQ ID NO.1 为靶序列。SEQ ID NO.6~7以SEQ ID NO.2为靶序列。SEQ ID NO.8~9以SEQ ID NO.3为靶序列。
本发明还提供了敲除CAPNS1基因的载体,包括骨架载体和dsDNA片段;所述dsDNA片段由SEQ ID NO.4~5退火形成;或由SEQ ID NO.6~7退火形成;或由SEQ ID NO.8~9退火形成。
本发明中,所述骨架载体为pGK1.1。
所述敲除CAPNS1基因的载体的制备方法为,将dsDNA与线性化的骨架载体T4DNALigase酶连接。
本发明还提供了敲除CAPNS1基因的试剂,包括本发明提供的靶标寡核苷酸序列或本发明提供的敲除载体。本发明还提供了一种敲除人神经母细胞瘤细胞CAPNS1基因的方法,将本发明提供的敲除载体转染入人神经母细胞瘤细胞中,获得敲除CAPNS1基因的人神经母细胞瘤细胞株。
本发明中,所述人神经母细胞瘤细胞为SK-N-SH细胞。
所述SK-N-SH细胞为对数期的SK-N-SH细胞。
本发明中,所述转染采用电转染,转染的电压为1400V。
转染中,所述载体的浓度不低于1μg/μL。
以本发明提供方法构建的敲除CAPNS1基因的人神经母细胞瘤细胞。
本发明提供了敲除人神经母细胞瘤细胞CAPNS1基因的方法,质粒转染 SK-N-SH细胞后,制备单克隆,CruiserTMEnzyme酶切后疑似为阳性克隆,单克隆测序结果显示SK-N-SH细胞CAPNS1基因敲除细胞系构建成功;并且,在CAPNS1-/-组,CAPNS1蛋白及Calpain1和Calpain2蛋白水平明显降低。结果表明SK-N-SH细胞CAPNS1基因敲除细胞系构建成功。
附图说明
图1 Pgk1.1质粒图谱;
图2载体酶切后的电泳检测图;其中泳道1是原质粒对照,M为marker, 2-4为切开的载体
图3菌落PCR检测电泳图;M为Marke菌落PCR检测电泳图k;1、2 为CAPNS1-gRNA1菌落PCR检测电泳图;3、4为CAPNS1-gRNA2菌落PCR 检测电泳图;5、6为CAPNS1-gRNA3菌落PCR检测电泳图
图4 sgRNA测序验证;A、B、C分别代表插入PGK1.1载体的sgRNA1、 sgRNA2、sgRNA3序列测序图;
图5 sgRNA测序比对结果;A、B、C分别表示sgRNA1位点、sgRNA2 位点、sgRNA3位点比对结果;
图6 CAPNS1基因Cruiser验证;
图7 SK-N-SH细胞CAPNS1基因敲除细胞系单克隆测序;
图8 CAPNS1基因敲除SK-N-SH细胞TA克隆测序及比对;
图9 CAPNS1敲除后CAPNS1、calpain1和calpain2蛋白表达情况;
图10不同电压转染SK-N-SH细胞结果;
图11 CAPNS1基因敲除载体转染后的混克隆测序验证。
具体实施方式
本发明提供了以CRISPR/Cas技术敲除人神经母细胞瘤细胞CAPNS1基因的方法及细胞系,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例
1)设计Crispr/cas9敲除靶位点
首先,需要在靶标DNA区域中设计一对20bp左右的oligo DNA,通过以下在线工具设计:
麻省理工学院的CRISPR Design:http://crispr.mit.edu/
选取CAPNS1基因所有转录本公共的CDS区,找出公共CDS区所处的外显子进行靶位点设计,选择第四个和第五个外显子进行靶位点设计。
3个靶位点序列信息:
CAPNS1-gRNA1:AGTTCGACACTGACCGATCAGGG(SEQ ID NO.1)
CAPNS1-gRNA2:TGAACTCCCAGGTGCCTTTGAGG(SEQ ID NO.2)
CAPNS1-gRNA3:CACTTTCATCTGAGTAGCGTCGG(SEQ ID NO.3)
2)引物添加接头
引物合成需在靶序列头部添加额外的碱基,正向引物添加CACC,反向引物添加AAAC,需要特别注意的是靶序列的第一个碱基必须是G,如果你选取的靶序列第一个碱基不是G,可自行在靶序列前加一个G,靶序列引物设计如下:
CAPNS1-1F:CACCGAGTTCGACACTGACCGATCA(SEQ ID NO.4)
CAPNS1-1R:AAACTGATCGGTCAGTGTCGAACTC(SEQ ID NO.5)
CAPNS1-2F:CACCGTGAACTCCCAGGTGCCTTTG(SEQ ID NO.6)
CAPNS1-2R:AAACCAAAGGCACCTGGGAGTTCAC(SEQ ID NO.7)
CAPNS1-3F:CACCGCACTTTCATCTGAGTAGCGT(SEQ ID NO.8)
CAPNS1-3R:AAACACGCTACTCAGATGAAAGTGC(SEQ ID NO.9)
3)Oligo杂交,敲除载体连接反应
将合成后的2条单链oligo DNA稀释成10μM,退火形成dsDNA,再与线性化后的pGK1.1linear vector(cat.no.GP0134,图谱如图1)载体连接,可直接用T4DNA Ligase连接,退火反应体系如下:
将以上体系瞬时离心后,置于PCR仪中95℃孵育3min,孵育后自然冷却20min。取1μl的杂交后的dsDNA进行T4DNA Ligase连接反应,反应体系如下:
将以上体系瞬时离心后,置于PCR仪中16℃孵育30min。
pGK1.1质粒包含了含Cas9核酸酶表达框和gRNA克隆框2个重要原件,其图谱如下,大小为10656bp。VSP primer:CATATGCTTACCGTAACTT
GAAAG序列位于载体的U6启动子区域。下游负链Oligo序列即靶位点的反向互补序列,位于载体的双BbsI酶切位点。(靶位点替换双BbsI酶切位点)。载体经酶切后,获得10kb左右片段,见图2。
4)转化Top10感受态
①从-80℃冰箱中取1管Top10感受态,置冰上融解。
②融解后加入10μl连接产物,轻弹混匀,冰上孵育30min。
③42℃水浴热击60sec,迅速拿出置冰上冷却2~3min。
④向管中加入800μl无抗SOC液体培养基,至摇床(37℃/160rpm) 恢复培养培养45min。
⑤4500rpm离心5min,弃去800μl上清,将沉淀悬在剩余的100μl上清中,均匀涂布于含Kan抗性的的筛选平板上,倒置培养过夜。
5)筛选阳性重组子
第二天使用上游引物VSP primer与下游负链Oligo引物进行菌落PCR筛选,阳性克隆PCR正确大小应为100bp,筛选到的阳性克隆抽质粒进一步测序验证。测序正确的质粒浓缩到1μg/μl浓度以上。
使用上游引物VSP primer与下游负链Oligo引物进行菌落PCR筛选,均能扩增出100bp大小的片段,说明所检测单克隆全为阳性,见图3。测序发现插入序列正确,见图4。SeqMan软件测序比对结果正确,载体构建成功,见图5。
6)电转染靶细胞(cat.no.GP7901)
①取状态良好的对数生长期SK-N-SH细胞悬液台盼蓝计数,确定细胞数及细胞活力(细胞活力>95%)。
②取5×106个细胞于15ml离心管中,离心弃上清(1000rpm/4min)。
③将细胞沉淀悬浮于210μl DPBS中,转移至1.5ml EP管中,加入所需量构建好的敲除质粒5~8μg(质粒浓度要求1μg/μl以上),轻轻混匀。
④将上述细胞质粒混合液用专用电转枪头转移至电击杯中,确定电击杯中溶液无气泡,且液面凸起后,盖上电击杯盖并至于电转仪,设定好电转条件后进行电转。待显示峰图正常后取出细胞液转移至六孔板培养基中(培养基需事先37℃预热且无抗生素)。
结论:1200V、1300V和1400V三个条件,1400V电转效率较高且死细胞较少,建议使用1400V。
药杀浓度结果:Puro 1ug/ml;
单克隆验证结果:能长成单克隆细胞,3cell/孔;图10。
7)pool细胞测序检测敲除效率
①电转72hr后,pool细胞台盼蓝计数。
②在筛选阳性克隆之前,需对pool细胞(混合克隆)的敲除效率进行体内验证。
③一般靶位点附近的序列测序中(图11),阳性样品应在靶点位置及之后的序列中出现套峰,如敲除效率较低时,信号强度往往较低,影响判断;
根据图11的测序结果,可以看出在靶点位置及之后的序列中明显出现套峰,初步认为阳性克隆的存在。
8)单克隆的制备和生长
有限稀释法稀释细胞至10块96孔板中,37℃,CO2培养箱中静置培养;一周后观察单克隆生长情况,约两周后将长起来的单克隆转移至48孔中扩大培养;当细胞长满48孔1/2时,即可取出一部分(102~104),使用Genloci TNA 抽提试剂盒(cat.no.GP0155,GP0156)提取细胞基因组。
9)单克隆基因组DNA的提取
取102~104个细胞于1.5ml EP管中,室温1500rpm离心5min,小心吸掉培养液。
加入150μl PBS重悬细胞,室温1500rpm离心5min,小心弃去上清。
重复步骤2一次。
向离心管中加入适量体积(推荐体积为50~200μl)预先配制的溶液A 与溶液B的混合液,枪头吹打5次,冰上放置10min,使细胞充分裂解。
加入两倍体积的无水乙醇,颠倒混匀,-20℃条件下沉淀20min以上。
4℃,12000rpm,离心20min,弃上清。
加入400~500μl预冷的75%乙醇洗涤沉淀,4℃,12000rpm,离心10 min,小心弃去上清,自然晾干(不超过5min为宜)。
加入适量体积(推荐体积为10-30μl)灭菌的双蒸水溶解沉淀,溶液可直接用于PCR反应,或于-20℃保存。
10)PCR扩增目的片段
引物设计
在敲除靶位点附近设计高特异性的Primers,扩增产物长度为分别为455 bp。Primers引物序列如下:
CAPNS1-seqF3:AAGCCATGGAGACACTATGC
CAPNS1-seqR3:TGGTCCATAGAGGTCATAGG
PCR扩增获得杂交DNA
在灭菌PCR管中配制如下反应体系,使用高特异性的Primers,扩增获得野生型和突变型充分杂交的DNA产物。
PCR反应程序如下:
自然冷却至40℃以下(野生型片段与突变型片段杂交)。
PCR结束后,取2~3μl进行电泳检测,要求目的片段明亮并且单一。
11)CruiserR Enzyme酶切筛选阳性克隆
在灭菌PCR管中配制如下反应体系:
45℃反应20min后立即向上述10μl反应体系内加入2μl 6×Stop Buffer,随后进行琼脂糖电泳检测或置于-20℃保存。
根据扩增引物及理论敲除位点位置,扩增产物约455bp,扩增目的大小明显变小或者可以切出条带的克隆为疑似阳性克隆。从图6可以看出,呈现的条带在400bp和500bp之间,疑似为阳性克隆。
12)测序筛选阳性克隆
对Crusier酶切初步筛选出来的阳性克隆进行测序验证,进一步确认阳性克隆(图7)。TA克隆测序结果比对发现两个亲本都缺失35bp: TCAGGGACCATTTGCAGTAGTGAACTCCCAGGTGC,见图8。CAPNS1基因敲除SK-N-SH细胞株构建成功。
13)TA克隆
对于阳性克隆中,两个等位基因突变情况不一样的阳性克隆,重新做TA 克隆后送测序,跟野生型比对,确定每个等位基因的突变情况。
14)Western blot检测CAPNS1、calpain1和calpain2蛋白表达情况
对稳定CAPNS1基因敲除后的SK-N-SH细胞中的CAPNS1蛋白及活性受 CAPNS1调节的calpain1和calpain2蛋白表达进行western blot分析,结果显示,与对照SK-N-SH细胞(CAPNS1+/+)相比,稳定CAPNS1基因敲除后的 SK-N-SH细胞(CAPNS1-/-)中CAPNS1、calpain1和calpain2的蛋白表达明显降低。见图9。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 南华大学
<120> 基于CRISPR/Cas技术敲除CAPNS1基因的人神经母细胞瘤细胞系的构建方法
<130> MP1803791
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
agttcgacac tgaccgatca ggg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgaactccca ggtgcctttg agg 23
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cactttcatc tgagtagcgt cgg 23
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
caccgagttc gacactgacc gatca 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aaactgatcg gtcagtgtcg aactc 25
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caccgtgaac tcccaggtgc ctttg 25
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaaccaaagg cacctgggag ttcac 25
<210> 8
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caccgcactt tcatctgagt agcgt 25
<210> 9
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aaacacgcta ctcagatgaa agtgc 25
Claims (10)
1.人CAPNS1基因CDS区第4外显子和/或第5外显子在构建CAPNS1基因敲除的人神经母细胞瘤细胞中的应用。
2.靶向CAPNS1基因第4外显子的sgRNA,其核苷酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
3.靶向CAPNS1基因第5外显子的sgRNA,其核苷酸序列如SEQ ID NO.3所示。
4.如SEQ ID NO.4~9所示的核苷酸序列的6条敲除CAPNS1基因的靶标寡核苷酸序列。
5.敲除CAPNS1基因的载体,其特征在于,包括骨架载体和dsDNA片段;所述dsDNA片段由SEQ ID NO.4~5退火形成;或由SEQ ID NO.6~7退火形成;或由SEQ ID NO.8~9退火形成。
6.敲除CAPNS1基因的试剂,其特征在于,包括权利要求4所述的靶标寡核苷酸序列或权利要求5所述的载体。
7.一种敲除人神经母细胞瘤细胞CAPNS1基因的方法,其特征在于,将权利要求5所述的载体转染入人神经母细胞瘤细胞中,获得敲除CAPNS1基因的人神经母细胞瘤细胞系。
8.根据权利要求7所述的方法,其特征在于,所述人神经母细胞瘤细胞为SK-N-SH细胞。
9.根据权利要求7所述的方法,其特征在于,所述转染采用电转染,转染的电压为1400V。
10.权利要求7~9任一项所述方法构建的敲除CAPNS1基因的人神经母细胞瘤细胞。
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