CN106047877A - 一种靶向敲除FTO基因的sgRNA及CRISPR/Cas9慢病毒系统与应用 - Google Patents

一种靶向敲除FTO基因的sgRNA及CRISPR/Cas9慢病毒系统与应用 Download PDF

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CN106047877A
CN106047877A CN201610487032.8A CN201610487032A CN106047877A CN 106047877 A CN106047877 A CN 106047877A CN 201610487032 A CN201610487032 A CN 201610487032A CN 106047877 A CN106047877 A CN 106047877A
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fto
cell strain
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纪卫东
苏甲林
阙彪
张海青
王敏
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First Affiliated Hospital of Sun Yat Sen University
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Abstract

本发明公开了一种靶向敲除FTO基因的sgRNA及CRISPR/Cas9慢病毒系统与应用。该sgRNA选自DNA序列如下的FTOsgRNAsp2或FTOsgRNAsp3。该sgRNA对FTO基因的切割效率高。将含有该sgRNA的CRISPR/Cas9慢病毒系统转染SV‑HUC‑1，得到的细胞株FTO蛋白表达水平显著降低。因此，本发明提供的sgRNA能有效靶向敲除FTO基因，将其构建入CRISPR/Cas9慢病毒系统，该系统能敲除FTO基因，得到敲除FTO基因的细胞株，从而有利于研究细胞株中的FTO的作用机制。

Description

一种靶向敲除FTO基因的sgRNA及CRISPR/Cas9慢病毒系统与 应用
技术领域
本发明涉及生物技术领域，特别涉及一种靶向敲除FTO基因的sgRNA及CRISPR/Cas9慢病毒系统与应用。
背景技术
FTO基因是一种与肥胖相关的等位基因，也称肥胖基因。FTO基因位于第16号染色体(16q12.2)，含有9个外显子，基因长度为410.50kb，广泛表达于人体组织的各发育阶段，且在下丘脑、骨骼肌及脂肪等组织中高表达。FTO基因参与细胞的能量代谢。由于肿瘤是细胞过度增殖造成的，其能量代谢异于正常细胞，因此，有必要研究FTO在不同细胞中的作用机制。
膀胱癌是我国泌尿外科最常见的尿路上皮肿瘤，绝大多数源于上皮组织，其中移行上皮细胞癌占90％以上。目前，研究人员在膀胱癌的发生机制上做了大量研究，发现了众多参与膀胱癌进展的途径和机制，如原癌基因激活、抑癌基因失活(点突变、重排、缺失)及染色体异常等。但目前膀胱癌发生与进展中所涉及的分子机制仍有许多尚未明确。
CRISPR/Cas9是近几年发现的由RNA向导的Cas9核酸酶靶向目的基因进行编辑的新兴技术。CRISPR/Cas9是最先在古细菌中发现的，是研究者发现古细菌对外来生物信息不断攻击而演化来的一种获得性防御机制。在CRISPR/Cas9系统中，crRNA(CRISPR-derivedRNA)通过DNA碱基配对原理与tracrRNA(trans-activating RNA)结合形成双链RNA，指导Cas9蛋白在crRNA向导的DNA序列靶定目的位点切断双链DNA。CRISPR/Cas9技术的的优点是操作简便，工作效率高。
目前，CRISPR/Cas9基因敲除技术已经广泛使用于果蝇、大鼠、小鼠、斑马鱼等实验模型。但是，在研究膀胱癌的相关基因仍然均使用RNA干扰技术，其主要缺点是在RNA水平的干扰，只能敲低，不能彻底敲除，效率低下，不适用于长期抑制研究。在研究膀胱癌发生与进展中所涉及的分子机制中仍有很大缺陷。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种靶向敲除FTO基因的sgRNA。
本发明的另一目的在于提供所述一种靶向敲除FTO基因的CRISPR/Cas9慢病毒系统的应用。
本发明的再一目的在于提供靶向敲除FTO基因的人膀胱上皮SV-HUC-1细胞的应用。
本发明的目的通过下述技术方案实现：一种靶向敲除FTO基因的sgRNA，选自DNA序列如下的FTOsgRNAsp2或FTOsgRNAsp3：
FTOsgRNAsp2的序列如下：
FTOsgRNAsp2oligo1：5’-caccgCCAATGAGGATGCGAGATAC-3’；
FTOsgRNAsp2oligo2：5’-aaacGTATCTCGCATCCTCATTGGc-3’；
FTOsgRNAsp3的序列如下：
FTOsgRNAsp3oligo1：5’-caccgCCGGTATCTCGCATCCTCAT-3’；
FTOsgRNAsp3oligo2：5’-aaacATGAGGATGCGAGATACCGGc-3’。
一种靶向敲除FTO基因的CRISPR/Cas9慢病毒系统，含有上述靶向敲除FTO基因的sgRNA的DNA序列。
所述的靶向敲除FTO基因的CRISPR/Cas9慢病毒系统的构建，包括如下步骤：
(1)使用BbsI酶切CRISPR/Cas9慢病毒载体LentiCRISPRV2，得到酶切后的CRISPR/Cas9慢病毒载体；
(2)将上述靶向敲除FTO基因的sgRNA的DNA序列磷酸化后与酶切后的CRISPR/Cas9慢病毒载体连接，得到靶向敲除FTO基因的CRISPR/Cas9慢病毒系统。
步骤(2)中所述靶向敲除FTO基因的sgRNA的DNA序列优选为FTOsgRNAsp2。
步骤(2)中所述的DNA序列是将寡核苷酸链1(oligo1)和寡核苷酸链2(oligo2)退火得到双链序列。
所述的靶向敲除FTO基因的CRISPR/Cas9慢病毒系统在制备敲除FTO基因的细胞株中的应用。
一种敲除FTO基因的细胞株，是将所述的靶向敲除FTO基因的CRISPR/Cas9慢病毒系统转染目的细胞株得到的。
所述的敲除FTO基因的细胞株，具体是通过如下步骤构建得到：
1)将所述的靶向敲除FTO基因的CRISPR/Cas9慢病毒系统通过包装细胞进行包装，得到慢病毒颗粒；
2)将慢病毒颗粒感染目的细胞株，得到敲除FTO基因的细胞株。
所述的目的细胞株优选为肿瘤细胞株。
所述的肿瘤细胞株优选为膀胱癌细胞株。
所述的膀胱癌细胞株优选为人膀胱上皮永生化细胞SV-HUC-1。
所述的敲除FTO基因的细胞株，是SV-HUC-1细胞中的FTO基因缺失或插入核苷酸得到的细胞株，为如下的细胞株1-4中的任一株：
细胞株1是在野生型FTO基因的靶向敲除位点上插入1个碱基；细胞株2是在野生型FTO基因的靶向敲除位点上有6个碱基缺失；细胞株3是在野生型FTO基因的靶向敲除位点上有40个碱基缺失；细胞株2是在野生型FTO基因的靶向敲除位点上有6个碱基缺失；FTO基因的具体靶向敲除位点如下，以下序列依次对应：野生型、细胞株1、细胞株2、细胞株3和细胞株4：
CTCACTCCGGTATCTCGCATCCTCATTGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT；
CTCACTCCGGTATCTCGCATCCTTCATTGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT；
CTCACTCCGGTATCTCGCA----------- TTGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT；
CTCACTCCGGTATCTCGCA-------------------------------------------------------------------------------CACCAGGCTCTTT；
CTCACTCCGGTATCTCGCAT----------- TGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT；
其中，划横线部分为靶向位点。
本发明相对于现有技术具有如下的优点及效果：
本发明提供FTO基因的sgRNA能有效靶向FTO基因，将其构建入CRISPR/Cas9慢病毒系统，该系统能敲除FTO基因，得到敲除FTO基因的细胞株，从而有利于研究细胞株中的FTO的作用机制。
附图说明
图1是检测FTO的三个靶向敲除效率的琼脂糖凝胶电泳图；其中，泳道M为DNAMarker，泳道sp1、sp2和sp3分别代表FTO的靶向敲除序列sgRNAsp1、sgRNAsp2和sgRNAsp3，无酶代表未加入T7E1酶，空载代表未敲除FTO的对照组。
图2是敲除FTO的SV-HUC-1单细胞克隆株的显微照片图。
图3是野生型SV-HUC-1细胞株中FTO基因的测序图。
图4是敲除FTO基因的单克隆SV-HUC-1细胞株1中FTO基因的测序图。
图5是敲除FTO基因的单克隆SV-HUC-1细胞株2中FTO基因的测序图。
图6是敲除FTO基因的单克隆SV-HUC-1细胞株3中FTO基因的测序图。
图7是敲除FTO基因的单克隆SV-HUC-1细胞株4中FTO基因的测序图。
图8是野生型SV-HUC-1细胞株和敲除FTO基因的单克隆SV-HUC-1细胞株1-4中FTO基因的比对结果图；其中，WT代表野生型SV-HUC-1细胞株，1、2、3和4代表敲除FTO的单克隆SV-HUC-1细胞株，虚线代表缺失片段，下划线代表插入碱基，+代表插入，-代表缺失。
图9是稳定细胞株中FTO蛋白的表达检测图；其中，SV-HUC-1-WT代表野生型膀胱上皮细胞株，SV-HUC-1-V2代表感染慢病毒lentiCRISPRV2病毒液的膀胱上皮细胞株，SV-HUC-1-V2-FTO代表感染敲除FTO病毒液的膀胱上皮细胞株。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
1.使用CRISPR/Cas9技术构建敲除FTO质粒
1.1sgRNA寡核苷酸链合成
使用CRISPR在线设计工具(http://crispr.mit.edu/)根据评分系统，分别在FTO的外显子3上设计3个20bp的sgRNA(sp1、sp2和sp3)，并通过BLAST验证无非特异性基因。根据这两条标准找到外显子上的核心序列：FTOsgRNAsp1、FTOsgRNAsp2和FTOsgRNAsp3。编码链模板5′端添加CACC，非编码链模板3′端添加AAAC，与BbsI酶切后形成的粘性末端互补，设计3对CRISPR寡核苷酸链，见表1。
表1FTO靶向位点及sgRNA寡核苷酸序列
1.2载体构建
1.21使用BbsI酶切1μg PX458质粒(购于Addgene公司)，30min，37℃:
1.22使用QIAGEN胶回收试剂盒纯化酶切质粒产物，按说明书进行操作。
1.23磷酸化并退火sgRNAoligos：
PCR仪器退火程序：
37℃30min
95℃维持5min，每分钟降低5℃至25℃，4℃维持
1.24将退火形成的oligo双链和酶切后的PX458载体直接连接，室温下，10min。
1.25将连接后的质粒转化至感受态细胞STBL3(TaKaRa公司)中，均匀涂至LB
固体培养基平板中，置于37℃培养箱中培养12-16小时，单个菌落即可出现。
1.3挑取单个菌落扩大培养并质粒小提。
1.4测序鉴定质粒构建成功，并命名为PX458-sgRNAsp1、PX458-sgRNAsp2和PX458-sgRNAsp3。
1.5敲除效率验证。
用含10％胎牛血清的高糖DMEM培养基于5％CO2，37℃恒温培养293T细胞(购于美国ATCC细胞库)。取对数期细胞以5×105/孔接种到六孔板培养，将其分组为：PX458(阴性对照组)、PX458-sgRNAsp1、PX458-sgRNAsp2和PX458-sgRNAsp3。待细胞融合度达到70％～80％时更换新鲜培养基，1小时后将相应的敲除质粒及阴性对照质粒各2μg经2000试剂，转染到293T细胞中，转染48小时候，荧光显微镜下观察转染效果，消化收集各组细胞，采用基因组DNA提取试剂盒提取各组细胞基因组DNA。
采用T7E1实验进一步验证切割效率：以各组细胞基因组DNA为模板，利用表2的引物PCR扩增3条靶向序列(表1)。取400ng PCR纯化产物加入10×Buffer2(NEB)变性退火，95℃，5min；以2℃/s下降至85℃；再以0.1℃/s下降至25℃，4℃保持。加入10单位T7核酸内切酶1，37℃水浴15分钟，加入2μl 0.5M EDTA终止反应。T7E1酶在不完全匹配的双链DNA处切割产生了两条片段。用表2的引物进行PCR得到的产物，sp1若有酶切，酶切片段应为120bp和220bp；sp2若有酶切，酶切片段应为200bp和190bp；sp3若有酶切，酶切片段应为310bp和80bp。使用2％的琼脂糖凝胶DNA电泳，检测切割效率发现FTO的靶向序列sgRNAsp2切割效率最高(图1)。
表2
1.6将FTO基因的sgRNAsp2连接到lentiCRISPRV2慢病毒敲除载体LentiCRISPRV2上(步骤同1.2)，并测序验证，得到LentiCRISPRV2-FTO。
2.包装慢病毒
预先将六孔板用多聚赖氨酸包被30min；种入293T细胞，培养24h(37℃,5％CO2)后，至六孔板中细胞融合度约为70％；转染包装细胞：
a.混匀以下3个质粒：(购于Addgene公司)
包装质粒PAX2 1.2μg
辅助质粒VSV-G 600ng
表达质粒LentiCRISPRV2-FTO 1.2μg
加入OPTI-MEM至总体积为50μl，室温放置5min；
b.混匀以下试剂室温放置5min：
LipofectamineTM 2000 9μl
OPTI-MEM 41μl
c.把a中50μl三质粒混合液加入b中，轻轻混匀，室温下，放置25min；
d.小心的将c中混合液加入种有293T的六孔板中，补加无抗生素培养基至1ml；
培养12h后移除感染液，每孔加入3ml高浓度血清培养基生产病毒液；培养48h后，收获病毒液至5ml离心管中，并重新向六孔板中补加3ml高浓度血清培养基再次生产病毒；培养24h后，再次收获病毒液，室温离心，1000rpm，10min；使用4.5μm的滤器过滤病毒上清液；分装后，-80℃保存。
3.病毒感染
1)将要感染的SV-HUC-1细胞(购于美国ATCC细胞库)接种到六孔板中，过夜，待细胞融合度约为50％时，移去培养基，更换500μl新鲜培养基；
2)每孔加入1ml病毒液，再加入1.6μl聚凝胺polybrene，至终浓度为8μg/ml；
3)37℃培养12h后，移除培养液，更换新鲜培养液后继续培养48h；
4)重复上述1)至3)步骤增加感染效率。
4.筛选稳定细胞株
感染结束48h后，向每孔加入嘌呤霉素(1.0μg/ml)，隔天换液，并保持培养基的嘌呤霉素浓度恒定，筛选阳性克隆细胞。所得细胞株分别命名为SV-HUC-1-V2-FTO。并使用有限稀释法挑选SV-HUC-1-V2-FTO单克隆敲除细胞株，并使用活细胞动态观察系统，确定单细胞来源(图2)。
5.稳定细胞株鉴定
提取各组敲除FTO的单克隆细胞株基因组DNA测序，鉴定出4株具有FTO基因缺失或插入突变的稳定细胞株(图3-8)。并收集FTO基因缺失最多的细胞株蛋白质(即图8中的第3株细胞)，进行Western blot方法检测FTO蛋白在各组细胞中的表达情况(图9)。结果显示，敲除FTO的膀胱上皮细胞SV-HUC-1-V2-FTO的FTO基因靶序列有缺失；稳定细胞株SV-HUC-1-V2-FTO比SV-HUC-1-V2对照组和SV-HUC-1-WT野生型FTO蛋白表达水平显著降低。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.一种靶向敲除FTO基因的sgRNA，其特征在于：选自DNA序列如下的FTOsgRNAsp2或FTOsgRNAsp3：
FTOsgRNAsp2的序列如下：
FTOsgRNAsp2oligo1：5’-caccgCCAATGAGGATGCGAGATAC-3’；
FTOsgRNAsp2oligo2：5’-aaacGTATCTCGCATCCTCATTGGc-3’；
FTOsgRNAsp3的序列如下：
FTOsgRNAsp3oligo1：5’-caccgCCGGTATCTCGCATCCTCAT-3’；
FTOsgRNAsp3oligo2：5’-aaacATGAGGATGCGAGATACCGGc-3’。
2.一种靶向敲除FTO基因的CRISPR/Cas9慢病毒系统，其特征在于：含有权利要求1所述的靶向敲除FTO基因的sgRNA的DNA序列。
3.权利要求2所述靶向敲除FTO基因的CRISPR/Cas9慢病毒系统的构建，其特征在于包括如下步骤：
(1)使用BbsI酶切CRISPR/Cas9慢病毒载体LentiCRISPRV2，得到酶切后的CRISPR/Cas9慢病毒载体；
(2)将权利要求1所述靶向敲除FTO基因的sgRNA的DNA序列磷酸化后与酶切后的CRISPR/Cas9慢病毒载体连接，得到靶向FTO基因的CRISPR/Cas9慢病毒系统。
4.权利要求2所述的靶向敲除FTO基因的CRISPR/Cas9慢病毒系统在制备敲除FTO基因的细胞株中的应用。
5.一种敲除FTO基因的细胞株，其特征在于：是将权利要求2或3所述的靶向敲除FTO基因的CRISPR/Cas9慢病毒系统转染目的细胞株得到的。
6.根据权利要求5所述的敲除FTO基因的细胞株，其特征在于是通过如下步骤构建得到：
1)将权利要求2或3所述的靶向敲除FTO基因的CRISPR/Cas9慢病毒系统通过包装细胞进行包装，得到慢病毒颗粒；
2)将慢病毒颗粒感染目的细胞株，得到敲除FTO基因的细胞株。
7.根据权利要求5或6所述的敲除FTO基因的细胞株，其特征在于：所述的目的细胞株为肿瘤细胞株。
8.根据权利要求7所述的敲除FTO基因的细胞株，其特征在于：所述的肿瘤细胞株为膀胱癌细胞株。
9.根据权利要求8所述的敲除FTO基因的细胞株，其特征在于：所述的膀胱癌细胞株为人膀胱上皮永生化细胞SV-HUC-1。
10.根据权利要求9所述的敲除FTO基因的细胞株，其特征在于：是SV-HUC-1细胞中的FTO基因缺失或插入核苷酸得到的细胞株，为如下的细胞株1-4中的任一株：
细胞株1是在野生型FTO基因的靶向敲除位点上插入1个碱基；细胞株2是在野生型FTO基因的靶向敲除位点上有6个碱基缺失；细胞株3是在野生型FTO基因的靶向敲除位点上有40个碱基缺失；细胞株2是在野生型FTO基因的靶向敲除位点上有6个碱基缺失；
FTO基因的具体靶向敲除位点如下，以下序列依次对应：野生型、细胞株1、细胞株2、细胞株3和细胞株4：
CTCACTCCGGTATCTCGCATCCTCATTGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT；
CTCACTCCGGTATCTCGCATCCTTCATTGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT；
CTCACTCCGGTATCTCGCA----------- TTGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT；
CTCACTCCGGTATCTCGCA-------------------------------------------------------------------------------CACCAGGCTCTTT；
CTCACTCCGGTATCTCGCAT----------- TGGTAATCCAGGCTGCACCTACAAGTACCTGAACACCAGGCTCTTT。
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