CN106434663A - CRISPR/Cas9靶向敲除人ezrin基因增强子关键区的方法及其特异性gRNA - Google Patents

CRISPR/Cas9靶向敲除人ezrin基因增强子关键区的方法及其特异性gRNA Download PDF

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CN106434663A
CN106434663A CN201610889009.1A CN201610889009A CN106434663A CN 106434663 A CN106434663 A CN 106434663A CN 201610889009 A CN201610889009 A CN 201610889009A CN 106434663 A CN106434663 A CN 106434663A
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高书颖
张青峰
郭晓龙
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Abstract

本发明属于分子生物学领域，具体涉及CRISPR/Cas9靶向敲除人ezrin基因增强子关键区的方法及其特异性gRNA，本发明根据CRISPR/Cas9的设计原则，在人ezrin基因增强子关键区的上下游设计两个靶位点，合成相应的寡核苷酸序列，并连接至载体pX459上构建重组质粒，将其转染人食管癌细胞株，能够特异性敲除人ezrin基因增强子关键区。本发明对研究以人ezrin基因增强子为靶点的肿瘤临床治疗具有重要意义。

Description

CRISPR/Cas9靶向敲除人ezrin基因增强子关键区的方法及其 特异性gRNA
技术领域
本发明属于分子生物学领域，具体涉及的是CRISPR/Cas9靶向敲除人食管癌细胞ezrin基因增强子关键区的方法以及用于靶向的人ezrin基因增强子关键区的gRNA。
背景技术
CRISPR/Cas(clustered regularly interspaced short palindromic repeats-associated)是很多细菌和大部分古生菌的天然免疫系统，通过对入侵的病毒和核酸进行特异性的识别，利用Cas蛋白进行切割，从而达到对自身的免疫。CRISPR/Cas9系统借鉴细菌的防御策略，由gRNA(guide RNA)寻找特定的DNA序列，然后利用Cas9核酸内切酶对靶DNA进行切割，造成双链断裂，在没有模板的情况下，发生非同源末端连接，造成DNA缺失突变(Shalem O,Sanjana NE,Hartenian E,et al.Genome-scale CRISPR-Cas9 knockoutscreening in human cells.Science,2014,343(6166):84-87.)。
肿瘤相关基因ezrin在食管癌、鼻咽癌、肺癌、胰腺癌等多种肿瘤中存在异常表达现象，其表达上调与肿瘤细胞的移动侵袭相关，抑制ezrin基因的过表达可有效阻止食管癌等肿瘤细胞的侵袭移动(Yang L,Guo T,Jiang S,et al.Expression of ezrin,HGF andc-met and its clinicopathological significance in the benign and malignantlesions of the gallbladder.Hepatogastroenterology,2012,59(118):1769-1775.)。我们既往采用双荧光素酶报告基因检测系统研究发现，在人ezrin基因编码区的上游存在启动子和增强子区(Gao SY,Li EM,Cui L,et al.Sp1 and AP-1 regulate expression ofthe human gene VIL2 in esophageal carcinoma cells.J Biol Chem,2009,284(12):7995-8004.)，增强子关键区(如图1)很可能是决定ezrin基因在食管癌等肿瘤细胞中过高表达的关键因素，有望成为控制ezrin基因表达的有效靶点(张青峰,卫金岐,张芳婷,等.几种肿瘤细胞中ezrin基因增强子区转录调控特性的研究.中国细胞生物学学报,2014,36(5):610-616.)。人ezrin基因增强子关键区为非转录区域，不能用传统的RNA干扰的方法进行研究。而采用CRISPR/Cas9系统，在人ezrin基因增强子关键区的上游和下游分别设计筛选1个特异性gRNA靶位点，对2个靶位点同时进行双链断裂，有望实现人ezrin增强子关键区的靶向敲除。靶向敲除人ezrin基因增强子关键区，对于研究人ezrin基因增强子在Ezrin蛋白过表达中的调控作用，以及研究人ezrin基因增强子与肿瘤细胞生物学行为之间的关系具有重要意义。
发明内容
本发明的目的在于通过设计、构建和检测，提供靶向人ezrin基因增强子关键区的gRNA及其靶位点序列，并用其实现ezrin基因增强子关键区的靶向敲除。
为实现上述目的，本发明以CRISPR/Cas9系统原理和gRNA设计原则为基础，利用软件设计2个gRNA，分别靶向人ezrin基因增强子关键区的上游和下游，合成gRNA相对应的正向和反向互补寡核苷酸链，退火后形成双链，连接至载体pX459构建重组质粒；将重组质粒转染食管癌EC109细胞中进行靶位点敲除验证。本发明提供的gRNA能够实现人ezrin基因增强子关键区的特异性敲除，对于研究人ezrin基因增强子在Ezrin蛋白过表达中的调控作用，以及研究人ezrin基因增强子与肿瘤细胞生物学行为之间的关系具有重要意义。
本发明申请的技术方案如下：
1、靶向人ezrin基因增强子关键区的gRNA设计，gRNA对应的寡核苷酸链的合成，携带gRNA寡核苷酸链的CRISPR/Cas9重组质粒构建。
2、在肿瘤细胞模型中分析鉴定本发明gRNA指导的CRISPR/Cas9系统对于靶向敲除人ezrin基因增强子关键区的特异性。
一种CRISPR/Cas9靶向敲除人ezrin基因增强子关键区中用于特异性靶向人ezrin基因增强子关键区的gRNA：
1)、所述gRNA在人ezrin基因的靶序列符合5′-N(20)-NGG-3′或者5′-CCN-N(20)-3′序列的排列规则；
2)、所述gRNA在人ezrin基因的靶序列是唯一的；
3)、所述gRNA在人ezrin基因的靶序列位于人ezrin基因增强子区。
上述CRISPR/Cas9靶向敲除人ezrin基因增强子关键区中用于特异性靶向人ezrin基因增强子关键区的gRNA，其对应的DNA序列如序列表SEQ ID NO.1-2任意一条序列所示。
一种CRISPR/Cas9靶向敲除人ezrin基因增强子关键区的方法，该方法用于非诊断或治疗目的，步骤如下：
1)、如上述CRISPR/Cas9靶向敲除人ezrin基因增强子关键区中用于特异性靶向人ezrin基因增强子关键区的gRNA，在gRNA序列SEQ ID NO.1的互补链的5′端加上CACCG，合成得到正向寡核苷酸，在所述的SEQ ID NO.1序列的5′端加上AAAC，3′端加上C，合成得到反向寡核苷酸链。在gRNA序列SEQ ID NO.2的5′端加上CACC，合成得到正向寡核苷酸，在所述的SEQ ID NO.2所述序列的互补链的5′端加上AAAC，合成得到反向寡核苷酸链。将合成的一对互补正、反寡核苷酸链退火，形成双链gRNA寡核苷酸链；
2)、将载体pX459经酶BbsⅠ切反应线性化，与上述双链gRNA寡核苷酸链连接，连接产物转化大肠杆菌DH5α感受态细胞，在氨苄青霉素抗性平板上筛选阳性克隆，提取质粒进行测序鉴定，构建重组质粒pX459-sgRNA1和pX459-sgRNA2；
3)、采用LipofectamineTM 2000转染试剂将质粒pX459-sgRNA1和pX459-sgRNA2共转染至人食管癌EC109细胞，提取细胞基因组DNA进行PCR扩增，扩增产物连接至载体pMD18-T，测序鉴定所构建的重组质粒分别在预定位点对基因组DNA进行的切割，实现人ezrin基因增强子关键区的靶向敲除。
本发明根据CRISPR/Cas9的设计原则，在人ezrin基因增强子关键区的上下游设计两个靶位点，合成相应的寡核苷酸序列，并连接至载体pX459上构建重组质粒，将其转染人食管癌细胞株，能够特异性敲除人ezrin基因增强子关键区。本发明对研究以人ezrin基因增强子为靶点的肿瘤临床治疗具有重要意义。
附图说明
图1为人ezrin基因增强子关键区结构示意图；
图2为重组质粒测序鉴定图；
图3为转染细胞基因突变的PCR鉴定图；
图4为亚克隆测序的CLUSTAL序列比对分析图；
图5为亚克隆测序图。
具体实施方式
下面结合具体实施例和附图进一步阐述本发明。
实施例1 靶向人ezrin基因增强子关键区的gRNA设计以及载体构建
1、靶向人ezrin基因增强子关键区的gRNA设计及寡核苷酸链的合成
从Genebank中查找人ezrin基因序列(http://www.ncbi.nlm.nih.gov/gene/ 7430)，利用在线软件http://www.e-crisp.org/E-CRISP/设计gRNA靶位点，分别位于人ezrin基因增强子关键区(–1297/–1186)的上游和下游。在本发明中将gRNA对应的DNA序列又称为gRNA序列，是gRNA在目标基因上的靶位点。gRNA对应的寡核苷酸链(Oligo DNA)按照5′-G(N)20NGG-3′的PAM结构(protospacer adjacent motif)为设计原则，选择分值较高的序列，如果序列的正向寡核苷酸链(Forward oligo)5′端第一个碱基不是G，则在5′端添加一个G，相应地在反向寡核苷酸链(Reverse oligo)的3′端添加一个C。同时在每对互补序列的正向寡核苷酸链的5′端添加CACC，反向寡核苷酸链的5′端添加AAAC，使其退火后形成的末端与pSpCas9(BB)-2A-Puro质粒(Addgene plasmid ID：48139，以下简称pX459)经BbsⅠ酶切后形成的黏性末端互补。本发明设计筛选的gRNA1靶向人ezrin基因增强子关键区的上游(–1319/–1300)，gRNA2靶向人ezrin基因增强子关键区的下游(–1192/–1173)。在gRNA1序列SEQ ID NO.1的互补链的5′端加上CACCG，合成得到正向寡核苷酸SEQ ID NO.3，在SEQ IDNO.1序列的5′端加上AAAC，3′端加上C，合成得到反向寡核苷酸链SEQ ID NO.4。在gRNA2对应的DNA序列SEQ ID NO.2的5′端加上CACC，合成得到正向寡核苷酸SEQ ID NO.5，在SEQ IDNO.2所述序列的互补链的5′端加上AAAC，合成得到反向寡核苷酸链SEQ ID NO.6。
2、靶向人ezrin基因增强子关键区的CRISPR/Cas9重组质粒构建
将上述寡核苷酸链稀释至终浓度为100μM，进行退火反应。反应体系如下：两条互补Oligo DNA各0.5μl，2μl Annealing Buffer(10×)，17μl ddH2O。将以上体系瞬时离心后，置于65℃水浴中孵育10min，随后取出，室温下缓慢冷却1～2h。退火后形成的双链如下：
gRNA1 Forward oligo：5′-CACCGCTCCCCTCGCAGATGCAAGT-3′
Reverse oligo：3′-CGAGGGGAGCGTCTACGTTCACAAA-5′
gRNA2 Forward oligo：5′-CACCGGTCCCGGGACCCGCCCCGC-3′
Reverse oligo：3′-CCAGGGCCCTGGGCGGGGCGCAAA-5′
将载体pX459进行Bbs I(NEB，Code No.R0539S)酶切反应，纯化回收目的片段，与上述gRNA1和gRNA2杂交双链DNA分别连接，构建重组质粒pX459-sgRNA1和pX459-sgRNA2。连接反应体系如下：2μl杂交双链DNA，2μl pX459酶切片段，1μl T4 DNA ligation buffer，1μl T4 DNA Ligase(TAKARA，Code No.2011B)，4μl ddH2O。将以上体系瞬时离心后，置于16℃水浴中孵育2h。连接产物转化大肠杆菌DH5α感受态细胞，在氨苄青霉素抗性平板上筛选阳性克隆，提取质粒进行测序鉴定。测序鉴定引物序列见SEQ ID NO.7。
测序结果如图2所示，图中A：重组质粒pX459-sgRNA1，实线框为gRNA1序列；B：重组质粒pX459-sgRNA2，虚线框为gRNA2序列。测序结果表明，分别位于人ezrin基因增强子关键区上游和下游的gRNA1和gRNA2序列在载体pX459上的连接位置和方向完全正确，重组质粒pX459-sgRNA1和pX459-sgRNA2构建成功。
实施例2 细胞培养及CRISPR/Cas9重组质粒转染
人食管癌EC109细胞在含10％灭活胎牛血清的DMEM培养基中贴壁生长，用含0.25％胰蛋白酶和0.02％EDTA的消化液消化细胞，进行传代培养。将细胞接种于96孔细胞培养板，每孔100μl。24h后细胞汇合率达50％～60％时即可用于转染。采用脂质体法共转染质粒pX459-sgRNA1和pX459-sgRNA2，转染步骤参照LipofectamineTM 2000转染试剂说明进行。
实施例3 人食管癌细胞ezrin增强子关键区靶向敲除检测
重组质粒pX459-sgRNA1和pX459-sgRNA2共转染食管癌EC109细胞48h后，不经抗性筛选直接收取细胞。以细胞基因组DNA为模板，PCR扩增ezrin基因增强子DNA序列。PCR引物位于拟敲除序列的上、下游，引物序列见SEQ ID NO.8和SEQ ID NO.9。
gRNA1和gRNA2序列分别位于ezrin增强子关键区的两侧，预计非突变基因组DNA扩增片段长766bp(ezrin基因-1543/-778序列)；突变基因组DNA删除147bp(ezrin基因-1319/-1173序列)，扩增片段长约619bp。PCR产物琼脂糖凝胶电泳检测结果见附图3，图中，M：DL 1 000 DNA marker；1：对照EC109细胞；2：共转染质粒pX459-sgRNA1和pX459-sgRNA2的EC109细胞。由于质粒转染细胞后未经药物抗性筛选，存在ezrin基因增强子关键区未被敲除的细胞，因此在对照组和转染组均能检测到与预计766bp相符的目的条带，为ezrin基因增强子关键区非缺失突变片段大小。而转染组还检测到小于700bp的微弱条带，有可能是CRISPR/Cas9系统在EC109细胞基因组上导致了定点突变。
将共转染重组质粒的EC109细胞基因组PCR产物与pMD18-T(TAKARA，CodeNo.6011)连接，转化大肠杆菌DH5α感受态细胞，在氨苄青霉素抗性平板上筛选阳性克隆。随机取20个克隆进行测序鉴定。测序引物序列SEQ ID NO.10。
在测序的20个克隆中，有2个克隆(编号为CL-3和CL-20)在人ezrin增强子关键区发生了碱基的缺失，20个克隆的测序比对分析结果见图4。克隆CL-3、CL-10、CL-19和CL-20测序结果见图5，图中，A：克隆CL-10；B：克隆CL-3；C：克隆CL-19；D：克隆CL-20。深色区域为人ezrin基因增强子关键区gRNA靶位点两侧序列，实线框为gRNA1靶位点，虚线框为gRNA2靶位点，结果显示，CRISPR/Cas9重组质粒pX459-sgRNA1和pX459-sgRNA2分别在预定位点对基因组DNA进行的切割，实现人ezrin基因增强子关键区的靶向敲除。
SEQUENCE LISTING
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caccggtccc gggacccgcc ccgc 24
<210> 6
<211> 24
<212> DNA
<213> 人工序列
<400> 6
aaacgcgggg cgggtcccgg gacc 24
<210> 7
<211> 22
<212> DNA
<213> 人工序列
<400> 7
ccaagtagga aagtcccata ag 22
<210> 8
<211> 21
<212> DNA
<213> 人工序列
<400> 8
cacaaacgtg ccacttaacc a 21
<210> 9
<211> 21
<212> DNA
<213> 人工序列
<400> 9
aaccgtcaag cctttgagaa a 21
<210> 10
<211> 24
<212> DNA
<213> 人工序列
<400> 10
cgccagggtt ttcccagtca cgac 24

Claims (3)

1.一种CRISPR/Cas9靶向敲除人ezrin基因增强子关键区中用于特异性靶向人ezrin基因增强子关键区的gRNA，其特征为：
1)、所述gRNA在人ezrin基因的靶序列符合5′-N(20)-NGG-3′或者5′-CCN-N(20)-3′序列的排列规则；
2)、所述gRNA在人ezrin基因的靶序列是唯一的；
3)、所述gRNA在人ezrin基因的靶序列位于人ezrin基因增强子区。
2.如权利要求1所述的CRISPR/Cas9靶向敲除人ezrin基因增强子关键区中用于特异性靶向人ezrin基因增强子关键区的gRNA，其特征为：其对应的DNA序列如序列表SEQ IDNO.1-2任意一条序列所示。
3.一种CRISPR/Cas9靶向敲除人ezrin基因增强子关键区的方法，该方法用于非诊断或治疗目的，其特征为如下步骤：
1)、如权利要求1-2任意一项所述的gRNA，在gRNA序列SEQ ID NO.1的互补链的5′端加上CACCG，合成得到正向寡核苷酸，在所述的SEQ ID NO.1序列的5′端加上AAAC，3′端加上C，合成得到反向寡核苷酸链。在gRNA序列SEQ ID NO.2的5′端加上CACC，合成得到正向寡核苷酸，在所述的SEQ ID NO.2所述序列的互补链的5′端加上AAAC，合成得到反向寡核苷酸链。将合成的一对互补正、反寡核苷酸链退火，形成双链gRNA寡核苷酸链；
2)、将载体pX459经酶BbsⅠ切反应线性化，与上述双链gRNA寡核苷酸链连接，连接产物转化大肠杆菌DH5α感受态细胞，在氨苄青霉素抗性平板上筛选阳性克隆，提取质粒进行测序鉴定，构建重组质粒pX459-sgRNA1和pX459-sgRNA2；
3)、采用LipofectamineTM2000转染试剂将质粒pX459-sgRNA1和pX459-sgRNA2共转染至人食管癌EC109细胞，提取细胞基因组DNA进行PCR扩增，扩增产物连接至载体pMD18-T，测序鉴定所构建的重组质粒分别在预定位点对基因组DNA进行的切割，实现人ezrin基因增强子关键区的靶向敲除。
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