CN107523567A - 一种敲除人ezrin基因增强子的食管癌细胞株的构建方法 - Google Patents

一种敲除人ezrin基因增强子的食管癌细胞株的构建方法 Download PDF

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CN107523567A
CN107523567A CN201710957308.9A CN201710957308A CN107523567A CN 107523567 A CN107523567 A CN 107523567A CN 201710957308 A CN201710957308 A CN 201710957308A CN 107523567 A CN107523567 A CN 107523567A
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高书颖
张青峰
李文娜
莫镇涛
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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基因增强子的方法、用于靶向人ezrin基因增强子的特异性gRNA以及一种敲除人ezrin基因增强子的食管癌细胞株。本发明提供了用于敲除人ezrin基因增强子的特异性gRNA，所述gRNA的靶DNA序列为SEQ ID NO.1‑4所示序列中的任意一条序列。本发明提供了一种敲除人食管癌细胞ezrin基因增强子的方法，为利用CRISPR/Cas系统在人食管癌细胞中对ezrin基因进行改造。本发明还提供了一种敲除人ezrin基因增强子的食管癌细胞株，为研究ezrin基因表达与肿瘤侵袭移动的相关性提供了有效的平台。

Description

一种敲除人ezrin基因增强子的食管癌细胞株的构建方法
技术领域
本发明属于分子生物学领域，具体涉及CRISPR/Cas9靶向敲除人ezrin基因增强子的方法、用于靶向人ezrin基因增强子的特异性gRNA以及一种敲除人ezrin基因增强子的食管癌细胞株。
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（Lau，2017）。
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，2012）。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基因编码区上游的启动子和增强子区（Gao，2009），有望成为控制ezrin基因表达的有效靶点（张青峰，2014）。采用CRISPR/Cas9系统，在人ezrin基因增强子区的上游和下游分别设计筛选特异性gRNA靶位点，对靶位点同时进行双链断裂，有望实现人ezrin增强子的靶向敲除，获得敲除人ezrin基因增强子的细胞株，这对于研究人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。
参考文献：
1.Lau V, Davie JR. The discovery and development of the CRISPR system inapplications in genome manipulation. Biochem Cell Biol, 2017, 95(2): 203-210.
2.Yang L, Guo T, Jiang S, et al. Expression of ezrin, HGF and c-met andits clinicopathological significance in the benign and malignant lesions ofthe gallbladder. Hepatogastroenterology, 2012, 59(118): 1769-1775.
3.Gao SY, Li EM, Cui L, et al. Sp1 and AP-1 regulate expression of thehuman gene VIL2 in esophageal carcinoma cells. J Biol Chem, 2009, 284(12):7995-8004.
4.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.
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基因增强子的gRNA及其靶位点序列，并用其实现目标序列的靶向敲除，获得敲除人ezrin基因增强子的食管癌细胞株。
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4个gRNA，靶向人ezrin基因增强子上游和下游的gRNA各2个。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CRISPR/Cas9重组质粒；将重组质粒转染至人食管癌细胞Eca-109中进行gRNA活性验证；筛选、鉴定靶向敲除目的片段的细胞株。本发明提供的gRNA能够实现人ezrin基因增强子的特异性敲除，本发明获得敲除人ezrin基因增强子的食管癌细胞株对于研究人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基因增强子的特异性，验证gRNA活性。
3、CRISPR/Cas9重组质粒转染人食管癌细胞，利用嘌呤霉素筛选单细胞克隆。测序鉴定突变等位基因的基因型，筛选纯合突变单克隆细胞系, 获得敲除人ezrin基因增强子的食管癌细胞株。
一种在CRISPR/Cas9靶向敲除人ezrin基因增强子中用于特异性靶向人ezrin基因增强子的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基因增强子的上游或下游。
上述其对应的DNA序列如序列表SEQ ID NO.1-4任意一条序列所示。
上述方法用于非诊断或治疗目的，其包括如下步骤：
（1）权利要求1-2任意一项所述的gRNA，在gRNA序列SEQ ID NO.1和SEQ ID NO.2的互补链的5′端加上CACCG，合成得到正向寡核苷酸链；在SEQ ID NO.1和SEQ ID NO.2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.3和SEQ ID NO.4的5′端加上CACCG，合成得到正向寡核苷酸链；在SEQ ID NO.3和SEQ ID NO.4的互补链的5′端加上AAAC，3′端加上C，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BbsⅠ酶切反应线性化，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CRISPR/Cas9重组质粒pX459-sgRNA-L1、pX459-sgRNA-L2、pX459-sgRNA-R1和pX459-sgRNA-R2。
（3）采用Lipofectamine™ 3000转染试剂将CRISPR/Cas9重组质粒共转染至人食管癌Eca-109细胞，提取细胞基因组DNA进行PCR扩增，扩增产物重组连接入pGEM-T Easy载体中，测序鉴定gRNA活性。
（4）CRISPR/Cas9重组质粒转染食管癌细胞48 h后，利用嘌呤霉素筛选，直至细胞在嘌呤霉素中能形成单细胞克隆。将单细胞克隆的PCR产物重组连接入pGEM-T Easy载体中，测序鉴定突变等位基因的基因型，筛选纯合突变的单克隆细胞株，获得敲除人ezrin基因增强子的食管癌细胞株。
附图说明
图1为gRNA靶向人ezrin基因位点示意图。
图2为CRISPR/Cas9重组质粒序列比对分析图。
图3为CRISPR/Cas9重组质粒测序部分截图。
图4为转染重组质粒pX459-sgRNA-L1/2的食管癌细胞基因组DNA亚克隆测序比对分析图。
图5为转染重组质粒pX459-sgRNA-L1/2的食管癌细胞基因组DNA亚克隆测序部分截图。
图6为转染重组质粒pX459-sgRNA-R1/2的食管癌细胞基因组DNA亚克隆测序的序列比对分析图。
图7为转染重组质粒pX459-sgRNA-R1/2的食管癌细胞基因组DNA亚克隆测序部分截图。
图8为单克隆细胞株突变型等位基因序列分析图。
图9为单克隆细胞株突变型等位基因测序部分截图。
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基因增强子的gRNA设计以及载体构建
1、靶向人ezrin基因增强子的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（–1541/–706）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gRNA-L1和gRNA-L2分别靶向人ezrin基因增强子的上游（–1541/–1522和–1673/–1654），gRNA-R1和gRNA-R2分别靶向人ezrin基因增强子的下游（–723/–704和–714/–695）。gRNA靶向人ezrin基因位点见图1。在gRNA-L1序列SEQ ID NO.1和gRNA-L2序列 SEQ ID NO.2的互补链的5′端加上CACCG，合成得到正向寡核苷酸分别为SEQ ID NO.5和SEQ ID NO.7；在gRNA-L1和gRNA-L2序列的5′端加上AAAC，3′端加上C，合成得到反向寡核苷酸链分别为SEQ ID NO.6和SEQ ID NO.8。在gRNA-R1序列SEQ ID NO.3和gRNA-R2序列SEQID NO.4的5′端加上CACCG，合成得到正向寡核苷酸分别为SEQ ID NO.9和SEQ ID NO.11；在gRNA-R1和gRNA-R2序列的互补链的5′端加上AAAC，3′端加上C，合成得到反向寡核苷酸链分别为SEQ ID NO.10和SEQ ID NO.12。
2、靶向人ezrin基因增强子的CRISPR/Cas9重组质粒构建
将上述寡核苷酸链稀释至终浓度为100 μM，进行退火反应。反应体系如下：两条互补Oligo DNA各0.5 μl，2 μl Annealing Buffer（10×），17 μl ddH2O。将以上体系瞬时离心后，置于65℃水浴中孵育10 min，随后取出，室温下缓慢冷却1~2 h。退火后形成的双链如下：
将载体pX459进行BbsⅠ（NEB，Code No.R0539S）酶切反应，纯化回收目的片段，与上述gRNA-L1、gRNA-L2、gRNA-R1和gRNA-R2杂交双链DNA分别连接，构建CRISPR/Cas9重组质粒pX459-sgRNA-L1、pX459-sgRNA-L2、pX459-sgRNA-R1和pX459-sgRNA-R2。连接反应体系如下：2 μl杂交双链DNA，2 μl pX459酶切片段，1 μl T4 DNA ligation buffer，1 μl T4 DNALigase（TAKARA，Code No.2011B），4 μl ddH2O。将以上体系瞬时离心后，置于16℃水浴中孵育2 h。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.13。
四种重组质粒的测序结果与载体pX459的序列比对见图2（阴影部分为载体序列，阴影之间为重组的gRNA序列），测序部分截图见图3（图中，A：重组质粒pX459-sgRNA-L1；B：重组质粒pX459-sgRNA-L2；C：重组质粒pX459-sgRNA-R1；D：重组质粒pX459-sgRNA-R2。方框内为gRNA序列，方框两侧为pX459载体序列），测序结果表明，分别靶向人ezrin基因增强子上游和下游的gRNA序列在载体pX459上的连接位置和方向完全正确，重组质粒构建成功。
实施例2 CRISPR/Cas9重组质粒转染人食管癌细胞及gRNA活性验证
1、CRISPR/Cas9重组质粒转染人食管癌细胞
人食管癌Eca-109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。将对数生长期的细胞消化接种到六孔板中，次日，当细胞汇合率达60%~80%时即可用于转染。采用脂质体法共转染CRISPR/Cas9重组质粒pX459-sgRNA-L1、pX459-sgRNA-L2、pX459-sgRNA-R1和pX459-sgRNA-R2，转染步骤参照Lipofectamine™ 3000（Invitrogen，Code No.L3000008）转染试剂说明进行。
2、gRNA活性验证
CRISPR/Cas9重组质粒转染食管癌细胞48 h后，将终浓度为5 μg/ml的嘌呤霉素加入到细胞培养液中进行筛选，提取细胞基因组DNA。检测gRNA-L活性时使用第一轮PCR引物对SEQID NO.14-15和巢式PCR引物对SEQ ID NO.16-17；检测gRNA-R活性时使用第一轮PCR引物对SEQ ID NO.18-19和巢式PCR引物对SEQ ID NO.20-21。将巢式扩增产物重组连接入pGEM-TEasy载体中，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。挑取克隆进行测序鉴定。测序引物为SEQ ID NO.22。
验证gRNA-L活性的5个克隆的测序结果，以未突变序列EZR-L为对照，进行序列比对，结果见图4（图中，EZR-L为未缺失的对照序列，C-L-1~5为转化克隆测序。 方框内为gRNA-L1/2序列），其中1个克隆（C-L-2）未发生突变，3个克隆（C-L-1、3、4）缺失突变87 bp发生在sgRNA-L2上游，1个克隆（C-L-5）缺失突变150 bp发生在sgRNA-L1上游，可以确定gRNA-L1和gRNA-L2均具有活性。克隆C-L-1和C-L-5测序的部分截图见图5（图中，A：克隆C-L-1，目标序列为正向插入，方框内为残存的部分gRNA-L2序列；B：克隆C-L-5，目标序列为反向插入，方框内为残存的部分gRNA-L1序列。箭头所指为缺失片段连接位点）。测序比对结果中在gRNA识别位点以外的个别碱基不同可能源于PCR引入的突变或单核苷酸多态性（singlenucleotide polymorphism，SNP）。
验证gRNA-R活性的5个克隆的测序结果，以未突变序列EZR-R为对照，进行序列比对，结果见图6（图中，EZR-R为未缺失的对照序列，C-R-1~5为转化克隆测序。方框内为gRNA-R1/2序列），检测的5个克隆均出现了大小不同的缺失突变，突变发生在gRNA-R1和gRNA-R2识别位点附近，可以确定gRNA-R1和gRNA-R2具有活性。克隆C-R-4测序的部分截图见图7（图中，目标序列为正向插入，方框内为gRNA-R1和残存的部分gRNA-R2序列。箭头所指为缺失片段连接位点）。
由此可见，转染CRISPR/Cas9重组质粒pX459-sgRNA-L1、pX459-sgRNA-L2、pX459-sgRNA-R1和pX459-sgRNA-R2，能够实现在预定位点对目标DNA进行的切割。
实施例3、单细胞克隆的筛选及ezrin基因增强子的敲除鉴定
1、单细胞克隆筛选
CRISPR/Cas9重组质粒转染食管癌细胞48 h后，将终浓度为5 μg/ml的嘌呤霉素加入到细胞培养液中进行筛选。筛选72 h后，更换正常培养基，待细胞长到一定融合度后进行第二次筛选，直至细胞在嘌呤霉素中能形成单细胞克隆。
2、单克隆细胞株ezrin基因增强子的敲除鉴定
选取12个单细胞克隆，分别提取基因组DNA，以SEQ ID NO.14和SEQ ID NO.19为引物进行第一轮PCR，以SEQ ID NO.16和SEQ ID NO.21为引物进行巢式PCR，将获得的12个PCR产物分别测序，根据测序结果选择1号单克隆细胞株做进一步亚克隆测序，以确认其携带的突变等位基因的基因型。将1号单细胞克隆的巢式PCR产物重组连接入pGEM-T Easy载体中，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进行测序鉴定。测序引物为SEQ ID NO.22。
测序结果以引物对SEQ ID NO.16和SEQ ID NO.21之间未突变序列EZR为对照，进行序列比对，结果见图8（图中，EZR为未缺失的对照序列，C-A为突变型A等位基因测序结果，C-B为突变型B等位基因测序结果，方框内为gRNA序列），表现为两种不同的突变型等位基因。突变型A等位基因序列SEQ ID NO.23为EZR缺失826 bp，位于gRNA-L1和gRNA-R2之间，突变型B等位基因序列SEQ ID NO.24为EZR缺失959 bp，位于gRNA-L2和gRNA-R2之间，两个等位基因的缺失区域均包含ezrin基因增强子。突变型等位基因测序的部分截图见图9（图中，A：突变型A等位基因，目标序列为反向插入，箭头所指为缺失片段连接位点，箭头左侧方框为残存的gRNA-R2序列，箭头右侧方框为残存的gRNA-L1序列；B：突变型B等位基因，目标序列为反向插入。箭头所指为缺失片段连接位点，箭头左侧方框为残存的gRNA-R2序列，箭头右侧方框为残存的gRNA-L2序列）。
由上述实验结果可见，本发明成功筛选出纯合突变单克隆细胞系, 获得敲除人ezrin基因增强子的食管癌细胞株。
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Claims (3)

1.一种在CRISPR/Cas9靶向敲除人ezrin基因增强子中用于特异性靶向人ezrin基因增强子的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基因增强子的上游或下游。
2.如权利要求1所述的在CRISPR/Cas9靶向敲除人ezrin基因增强子中用于特异性靶向人ezrin基因增强子的gRNA，其特征为：其对应的DNA序列如序列表SEQ ID NO.1-4任意一条序列所示。
3.一种在CRISPR/Cas9靶向敲除人ezrin基因增强子的方法，该方法用于非诊断或治疗目的，其特征为包括如下步骤：
（1）权利要求1-2任意一项所述的gRNA，在gRNA序列SEQ ID NO.1和SEQ ID NO.2的互补链的5′端加上CACCG，合成得到正向寡核苷酸链；在SEQ ID NO.1和SEQ ID NO.2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.3和SEQ ID NO.4的5′端加上CACCG，合成得到正向寡核苷酸链；在SEQ ID NO.3和SEQ ID NO.4的互补链的5′端加上AAAC，3′端加上C，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BbsⅠ酶切反应线性化，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CRISPR/Cas9重组质粒pX459-sgRNA-L1、pX459-sgRNA-L2、pX459-sgRNA-R1和pX459-sgRNA-R2；
（3）采用Lipofectamine™ 3000转染试剂将CRISPR/Cas9重组质粒共转染至人食管癌Eca-109细胞，提取细胞基因组DNA进行PCR扩增，扩增产物重组连接入pGEM-T Easy载体中，测序鉴定gRNA活性；
（4）CRISPR/Cas9重组质粒转染食管癌细胞48 h后，利用嘌呤霉素筛选，直至细胞在嘌呤霉素中能形成单细胞克隆；
将单细胞克隆的PCR产物重组连接入pGEM-T Easy载体中，测序鉴定突变等位基因的基因型，筛选纯合突变的单克隆细胞株, 获得敲除人ezrin基因增强子的食管癌细胞株。
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