CN106399377A - 一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法 - Google Patents
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Abstract
本发明涉及一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，首先建立sgRNA文库；然后用慢病毒包装sgRNA文库，并收集病毒；之后在癌细胞系中筛选sgRNA文库；再提取筛选所得细胞及筛选前细胞的基因组DNA；最后富集基因组DNA中的sgRNA。与现有技术相比，本发明对CRISPR/Cas细胞的筛选过程做了改进，利用被感染细胞的puromycin抗性，用简便的方法确定了病毒感染效率，确定了病毒MOI值；更重要的是，本发明极大优化了病毒包装方法，将病毒包装效率提高至常规方法的5倍以上，能够大大节省大规模的药物靶点筛选成本，用于推动癌症药物靶点筛选的产业化。
Description
技术领域
本发明属于高通量测序技术领域，尤其是涉及一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法。
背景技术
致癌基因和抑癌基因都是癌症基因治疗的潜在靶点，因此其鉴定在癌症的治疗中有着巨大的应用前景，目前已经针对致癌基因EGFR、Alk等基因开发出治疗癌症的药物。但是目前有效的癌症基因靶向药物还远远不能满足临床治疗的需要。基于CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/clusteredregularly interspaced short palindromic repeats associated protein 9)的大规模功能基因筛选技术作为近几年的基因工程新兴技术，能够在全基因组层面筛选癌症相关基因，从而为癌症的治疗提供药物靶点候选基因。
目前CRISPR/Cas9 screen技术只在国内外少数实验室应用，并未推广。CRISPR/Cas9 screen技术的关键是保证sgRNA的覆盖率，因此前期的sgRAN文库的构建的有效性，sgRAN文库扩增的高效性以及病毒包装的有效性成为问题的关键。目前在国际期刊论文中长出现sgRNA文库质量不高或是后期sgRNA测序sgRNA文库分布有偏态性的问题。另外传统建库方法费时费力，并且需要大量的PCR酶，经济负担也比较大。为解决以上问题，需要一种sgRNA文库建立、扩增、病毒包装及测序的方法，以保证大规模癌症基因靶点筛选的有效性和高效性。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法。
本发明的目的可以通过以下技术方案来实现：
一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，该方法包括以下步骤：
(1)用电转的方法建立sgRNA文库：
将合成的sgRNA的oligo片段用PCR扩增的方法加接头，然后用Gibson assembly的方法连接进入lentiCRISPRv2质粒，用Bio-rad电转仪将连接产物转化至感受态中，从而获得sgRNA文库；
(2)用慢病毒包装sgRNA文库：
无菌条件下，培养293FT细胞，并用转染试剂X-tremeGENE HP DNA TransfectionReagent将Lenti CRISPRv2及另外两种病毒包装质粒psPAX2和pMD2.G包装慢病毒；
(3)sgRNA文库在细胞中的筛选：
选取合适的病毒量，感染待检测的癌细胞系，感染48小时后，用puromycin筛选2天，所得阳性细胞收取部分作为对照组，其他细胞继续培养至42-46天(优选为45天)后收取；
(4)提取筛选所得细胞及筛选前细胞的基因组DNA：
用细胞裂解液裂解细胞，吹打混匀后，加入RNase酶，65℃孵育30分钟，再加终浓度为10μg/ml的蛋白酶K，55℃孵育过夜，用纯化液抽提DNA片段；
(5)富集基因组DNA中的sgRNA：
用限制性内切酶对基因组DNA进行酶切，37℃反应过夜，将酶切后的DNA片段放置在0.8wt％的低熔点琼脂糖胶中，80V，1小时电泳，切胶回收1600-2000bp的DNA片段。
(6)sgRNA文库构建与测序：
文库按照illumina公司的建库试剂盒说明书构建，将建好文库进行Hiseq2000的50SE进行高通量测序，结果进行生物信息分析。
在第(1)步骤用电转的方法建立sgRNA文库前，先进行sgRNA文库设计，具体操作为：利用网站http://crisprscan.org和http://www.e-crisp.org在线设计感兴趣的基因群的sgRNA，针对每种基因设计10个sgRNA。
步骤(1)中，oligo的PCR扩增条件为：
步骤(1)中，Gibson assembly的条件为：
步骤(1)中，所述的病毒包装过程Lenti CRISPRv2、psPAX2、pMD2.G的质粒质量按照4：3：1的比例转染293FT细胞；以10cm的培养皿为例Lenti CRISPRv2、psPAX2、pMD2.G的质粒质量分别为6μg，4.5μg，1.5μg，X-tremeGENE HP DNA Transfection Reagent的量为30μL。转染时采用悬浮转染的方法。并于之后的48小时和72小时收取病毒液。
步骤(2)中利用感染病毒细胞对puromycin的抗性来检测病毒感染效率，具体操作为：
在六孔板中培养细胞至融合度70％，用25μL、50μL、100μL、200μL、400μL的病毒液感染细胞，取能感染48-52％(优选为50％左右)细胞的病毒量作为合适病毒量进行后续试验。
步骤(4)中，细胞裂解液的成分为400μMNaCl、0.2％SDS、2mMEDTA、10mMTris-HCl。
步骤(4)中，所述的纯化液指体积比25:24:1的酚、氯仿与异戊醇的混合液。
步骤(5)中，限制性内切酶反应体系为：50μL中，含有6μg DNA，5μL 10×NEBbuffer，限制性内切酶EcoNⅠ3μL。
待检测的癌细胞系包括肺癌细胞与肝癌细胞细胞系。
与现有技术相比，本发明具有以下优点及有益效果：
本发明对细胞的筛选过程做了改进，利用被感染细胞的puromycin抗性，用简便的方法确定了病毒感染效率，确定了病毒MOI值；限制性内切酶切方法与高通量方法的结合，而且能够降低非特异性PCR扩增产生的假阳性，提高了建库效率；采用悬浮转染包装病毒的方法大大提高了病毒滴度，转染试剂价格昂贵，此方法节约了筛选成本。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
实施例1
对乳腺癌细T47D进行药物基因靶点的筛选。具体方法如下：
(1)sgRNA文库设计：
利用网站http://crisprscan.org和http://www.e-crisp.org在线设计感兴趣的基因群的sgRNA,针对每种基因设计10个sgRNA。
(2)用电转的方法建立sgRNA文库
将合成的sgRNA的oligo片段用PCR扩增的方法加接头，然后用Gibson assembly的方法连接进入lentiCRISPRv2质粒。用Bio-rad电转仪将连接产物转化至感受态中，从而获得sgRNA文库。
(3)用慢病毒包装sgRNA文库：
无菌条件下，培养293FT细胞，并用Roche的转染试剂X-tremeGENE HP DNATransfection Reagent将Lenti CRISPRv2及另外两种病毒包装质粒psPAX2和pMD2.G包装慢病毒。细胞转染后15小时换液，并于之后的24小时和48小时收取病毒液；
(4)T47D细胞中筛选sgRNA文库
选取合适的病毒量，感染T47D，48小时后，用puromycin筛选2天,所得阳性细胞收取部分作为对照组，其他细胞继续培养至40天后收取。
(5)提取筛选所得细胞及筛选前细胞的基因组DNA
用细胞裂解液A裂解细胞，吹打混匀后，加入RNase酶，65℃孵育30分钟，再加终浓度为10μg/ml的蛋白酶K，55℃孵育过夜。用纯化液(体积比25:24:1的酚、氯仿与异戊醇的混合液)抽提DNA片段；
(6)富集基因组DNA中的sgRNA
用细胞裂解液A裂解细胞，吹打混匀后，加入RNase酶，65℃孵育30分钟，再加终浓度为10μg/ml的蛋白酶K，55℃孵育过夜。用纯化液(体积比25:24:1的酚、氯仿与异戊醇的混合液)抽提DNA片段；
用限制性内切酶对基因组DNA进行酶切，37℃反应过夜，将酶切后的DNA片段放置在0.8wt％的低熔点琼脂糖胶中，80V，1小时电泳，切胶回收1600-2000bp的DNA片段；
(7)sgRNA文库构建与测序：
文库按照illumina公司的建库试剂盒说明书构建，将建好文库进行Hiseq2000的50SE进行高通量测序，结果进行生物信息分析。
实施例2
应用于前列腺癌细胞系LNcap的CRISPR/Cas9应用于大规模筛选癌症基因靶点的CRISPR/Cas9筛选方法，包括以下步骤：
(1)sgRNA文库建立：
利用网站http://crisprscan.org和http://www.e-crisp.org在线设计与全基因组的sgRNA。sgRNA的载体采用Lenti CRISPRv2。
(2)用慢病毒包装sgRNA文库：
无菌条件下，培养293FT细胞，并用Roche的转染试剂X-tremeGENE HP DNATransfection Reagent将Lenti CRISPRv2及另外两种病毒包装质粒psPAX2和pMD2.G包装慢病毒。按照4：3：1的比例转染293FT细胞，以10cm的培养皿为例Lenti CRISPRv2、psPAX2、pMD2.G的质粒质量分别为6μg,4.5μg,1.5μg,X-tremeGENE HP DNA Transfection Reagent的量为30μL。细胞转染后15小时换液，并于之后的24小时和48小时收取病毒液；
(3)sgRNA文库在细胞中筛选：
选取合适的病毒量，感染待检测的癌细胞系，感染48小时后，用puromycin筛选2天,所得阳性细胞收取部分作为对照组，其他细胞继续培养至45天左右后收取。为确定病毒量，要确定病毒的感染效率。发明利用感染病毒细胞对puromycin的抗性进行检测。在六孔板中培养细胞至融合度70％，用50μL、100μL、200μL、400μL、500μL的病毒液感染细胞，取能感染50％左右细胞的病毒量作为合适病毒量进行后续试验；
(4)提取筛选所得细胞及筛选前细胞的基因组DNA：
用细胞裂解液A裂解细胞，吹打混匀后，加入RNase酶，65℃孵育30分钟，再加终浓度为10μg/ml的蛋白酶K，55℃孵育过夜。用纯化液(体积比25:24:1的酚、氯仿与异戊醇的混合液)抽提DNA片段；细胞裂解液A的成分为400μMNaCl2,0.2％SDS,2mMEDTA,10mMTris-HCl。
(5)富集基因组DNA中的sgRNA
用限制性内切酶对基因组DNA进行酶切，37℃反应过夜，将酶切后的DNA片段放置在0.8wt％的低熔点琼脂糖胶中，80V，1小时电泳，切胶回收1600-2000bp的DNA片段；限制性内切酶反应体系为50μL中：6μg DNA,5μL 10×NEB buffer,限制性内切酶EcoNⅠ3μL。
(6)sgRNA文库构建与测序：
文库按照illumina公司的建库试剂盒说明书构建，将建好文库进行Hiseq2000的50SE进行高通量测序，结果进行生物信息分析。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (9)
1.一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，该方法包括以下步骤：
(1)用电转的方法建立sgRNA文库：
将合成的sgRNA的oligo片段用PCR扩增的方法加接头，然后用Gibson assembly的方法连接进入lentiCRISPRv2质粒，用Bio-rad电转仪将连接产物转化至感受态中，从而获得sgRNA文库；
(2)用慢病毒包装sgRNA文库：
无菌条件下，培养293FT细胞，并用转染试剂X-tremeGENE HP DNA TransfectionReagent将Lenti CRISPRv2及另外两种病毒包装质粒psPAX2和pMD2.G包装慢病毒；
(3)sgRNA文库在细胞中的筛选：
选取合适的病毒量，感染待检测的癌细胞系，感染48小时后，用puromycin筛选2天，所得阳性细胞收取部分作为对照组，其他细胞继续培养至42-46天后收取；
(4)提取筛选所得细胞及筛选前细胞的基因组DNA：
用细胞裂解液裂解细胞，吹打混匀后，加入RNase酶，65℃孵育30分钟，再加终浓度为10μg/ml的蛋白酶K，55℃孵育过夜，用纯化液抽提DNA片段；
(5)富集基因组DNA中的sgRNA：
用限制性内切酶对基因组DNA进行酶切，37℃反应过夜，将酶切后的DNA片段放置在0.8wt％的低熔点琼脂糖胶中，80V，1小时电泳，切胶回收1600-2000bp的DNA片段。
2.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，步骤(1)中，oligo的PCR扩增条件为：
3.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，步骤(1)中，Gibson assembly的条件为：
4.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，步骤(1)中，所述的病毒包装过程Lenti CRISPRv2、psPAX2、pMD2.G的质粒质量按照4：3：1的比例转染293FT细胞。
5.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，步骤(2)中利用感染病毒细胞对puromycin的抗性来检测病毒感染效率，具体操作为：
在六孔板中培养细胞至融合度70％，用25μL、50μL、100μL、200μL、400μL的病毒液感染细胞，取能感染48-52％细胞的病毒量作为合适病毒量进行后续试验。
6.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，步骤(4)中，细胞裂解液的成分为400μMNaCl、0.2％SDS、2mMEDTA、10mMTris-HCl。
7.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，步骤(4)中，所述的纯化液指体积比25:24:1的酚、氯仿与异戊醇的混合液。
8.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，步骤(5)中，限制性内切酶反应体系为：50μL中，含有6μg DNA，5μL 10×NEB缓冲液，限制性内切酶EcoN Ⅰ 3μL。
9.根据权利要求1所述的一种基于CRISPR/Cas9高通量技术筛选药物靶点基因的方法，其特征在于，待检测的癌细胞系包括肺癌细胞与肝癌细胞细胞系。
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