CN105567692B - 一种特异性靶向QKI基因的sgRNA及其应用 - Google Patents

一种特异性靶向QKI基因的sgRNA及其应用 Download PDF

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CN105567692B
CN105567692B CN201610108864.4A CN201610108864A CN105567692B CN 105567692 B CN105567692 B CN 105567692B CN 201610108864 A CN201610108864 A CN 201610108864A CN 105567692 B CN105567692 B CN 105567692B
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郑良荣
赵文婷
孙泽玮
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Abstract

本发明提供制备特异性靶向目标基因QKI的sgRNA，其序列如SEQ ID NO.1所示。利用本发明提供的sgRNA，合成双链sgRNA寡聚核苷酸，将sgRNA构建至pSpCas9(BB)‑2A‑Puro载体，鉴定并扩增，转染HEK293T细胞获得QKI基因敲除细胞。本发明能够在较短时间内精确靶向人QKI基因并且实现基因敲除，免去了购买QKI基因敲除小鼠分离原代细胞的复杂操作，且价格低廉，有助于进一步深入研究QKI的生物学功能。本发明方法步骤简单、sgRNA靶向性好，QKI基因敲除效率高。

Description

一种特异性靶向QKI基因的sgRNA及其应用
技术领域
本发明属于基因工程领域，更具体地说涉及一种特异性靶向QKI基因的sgRNA，以及CRISPR-Cas9特异性敲除人HEK293T细胞QKI基因的方法。
背景技术
规律成簇间隔短回文重复系统(clustered regularly interspaced shortpalindromic repeat；CRISPR-associated，CRISPR-Cas9)是一种具有核酸内切酶活性的复合体，识别特定的DNA序列，进行特定位点切割造成双链DNA断裂(Double-strand breaks,DSB)，在没有模板的条件下，发生非同源重组末端连接(Non-homologous end joining,NHEJ),造成移码突变(frameshift mutation)，导致基因敲除。
Cas9靶向切割DNA是通过向导核糖核酸(guide RNA，sgRNA)和靶序列互补识别的原理实现的。sgRNA的特异性决定了基因编辑的精确程度。因此，设计、制备出精确性和特异性靶向目标基因的sgRNA成为CRISPR-Cas9基因敲除的关键技术。
QKI是一类RNA结合蛋白，具有调控RNA剪切、运输等多个生物学功能。
CRISPR-Cas9快速、简便、高效、特异性靶向敲除基因，通过靶向敲除QKI基因，有助于深入研究其功能。而能否设计、制备出精确性和特异性靶向QKI基因的sgRNA以及通过分子生物学方法制备出靶向QKI基因的CRISPR-Cas9系统成为实现QKI基因敲除的关键技术。
参考文献：
Ran,F.A.et al.,Genome engineering using the CRISPR-Cas9system.NATPROTOC 8 2281(2013)。
Lauriat,T.L.et al.,Developmental expression profile of quaking,acandidate gene for schizophrenia,and its target genes in human prefrontalcortex and hippocampus shows regional specificity.J NEUROSCI RES 86 785(2008)。
发明内容
本发明的目的是提供制备特异性靶向目标基因QKI的sgRNA，序列如SEQ ID NO.1所示。
本发明的另一个目的是提供CRISPR-Cas9特异性敲除人HEK293T细胞QKI基因的方法，通过以下步骤实现。
一、sgRNA寡核苷酸的设计和选择
靶向QKI基因的sgRNA的设计按如下原则：
1.在QKI基因上选择特征为5’-N(19)GG或5’-N(20)GG-3’或5’-N(21)GG-3’的序列。
2.sgRNA在QKI基因上的靶向位点位于不同的各种剪切形式的共有外显子上。
3.sgRNA在QKI基因上的靶向位点位于整个基因的前半段，尤其宜在基因的功能结构域中；
按以上原则，设计得到sgRNA，序列为SEQ No.1。
二、合成双链sgRNA寡聚核苷酸
根据选择的sgRNA，去除3’端NGG三个碱基，在其5’端加上CACCG得到正向寡核苷酸；根据选择的sgRNA，去除3’端NGG三个碱基，获得对应DNA的互补链，并且在其5’加上AAAC,3’端加上C得到反向寡核苷酸。上述正向、反向寡核苷酸序列见SEQ NO.2和SEQ NO.3。
分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸变性、退火，形成可以连入pSpCas9(BB)真核表达载体的双链。
三、将sgRNA构建至pSpCas9(BB)-2A-Puro载体，鉴定并扩增
1.将退火的sgRNA寡聚核苷酸双链与pSpCas9(BB)-2A-Puro载体(序列见SEQNO.4)连接获得pSpCas9(BB)-2A-Puro-QKIsg质粒(序列见SEQ NO.5)。
2.转化并涂Amp+平板。
3.用SEQ ID NO.6的通用引物U6测序的方法鉴定阳性克隆。
4.37℃摇床摇菌过夜并抽提pSpCas9(BB)-2A-Puro-QKIsg质粒。
四、转染HEK293T细胞获得QKI基因敲除细胞
1、按照DNA&siRNA Transfection Reagent(Polyplus transfection，114-01)的操作手册，将pSpCas9(BB)-2A-Puro-QKIsg质粒转染至HEK293T细胞。
2.转染后细胞加入10μg/ml Puromycin(Merck,540411)药筛，消化存活细胞，将单个细胞接种于96孔板。
3、待细胞扩增后，提取基因组DNA，RT-PCR扩增靶向QKI区域片段，所用引物序列见SEQ NO.7和SEQ NO.8，用Sanger法测序检测sgRNA靶向区域附近基因序列，测序引物见SEQNO.8，确认是否发生移码突变。用Western Blot检测QKI基因已经被敲除。
本发明可以在较短时间内实现QKI基因敲除，免去了购买QKI基因敲除小鼠分离原代细胞的复杂操作，且价格低廉，有助于进一步深入研究QKI的生物学功能。利用本发明制备的特异性靶向人QKI基因的sgRNA能够精确靶向人QKI基因并且实现基因敲除。该制备方法步骤简单、sgRNA靶向性好，QKI基因敲除效率高。
附图说明
Sanger法测序确认QKI阅读框发生移码突变,与参考序列相比缺失22个碱基(图1和图2)。
Western Blot确认QKI基因敲除(图3)。
具体实施方式
下面结合附图和具体的实施例对本发明的技术方案做进一步介绍。
实施例1靶向人QKI基因的sgRNA的设计和合成
1.靶向人QKI基因的sgRNA的设计。
(1)在QKI基因上选择特征为5’-N(19)GG或5’-N(20)GG-3’或5’-N(21)GG-3’的序列。
(2)sgRNA在QKI基因上的靶向位点位于不同的各种剪切形式的共有外显子上。
(3)sgRNA在QKI基因上的靶向位点位于整个基因的前半段，尤其宜在基因的功能结构域中。
按以上原则，设计得到sgRNA，序列为SEQ No.1。
3.靶向人QKI基因的sgRNA寡聚核苷酸的合成和构建。
根据选择的sgRNA，去除3’端NGG三个碱基，在其5’端加上CACCG得到正向寡核苷酸；根据选择的sgRNA，去除3’端NGG三个碱基，获得对应DNA的互补链，并且在其5’加上AAAC,3’端加上C得到反向寡核苷酸。序列见SEQ NO.2和SEQ NO.3。分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸变性、退火，形成可以连入pSpCas9(BB)-2A-Puro真核表达载体的双链sgRNA寡聚核苷酸。
变性、退火反应体系为：
1μl正向寡核苷酸(100μM)
1μl反向寡核苷酸(100μM)
1μl T4ligation buffer(New England BioLabs公司，货号：B0202S)
1μl T4PNK(New England BioLabs公司，货号：M0201S)
6μl灭菌水。
在PCR仪中按照以下程序运行：37℃，30min；95℃,5min；0.1℃/sec由95℃降至25℃。
实施例2将双链sgRNA构建至pSpCas9(BB)-2A-Puro载体
将变性、退火后所得双链sgRNA按1：200比例稀释。
将双链sgRNA构建至pSpCas9(BB)-2A-Puro载体(序列见SEQ NO.4)，反应体系如下：
100ng pSpCas9(BB)-2A-Puro质粒
2μl稀释后双链sgRNA
2μl Tango buffer，10X(Thermo Scientific,货号BY5)
1μl DTT，10mM(Thermo Scientific,货号R0862)
1μl ATP，10mM(Thermo Scientific,货号AM8110G)
1μl FastDigest BbsⅠ(Thermo Scientific,货号FD1014)
0.5μl T7ligase(Enzymatics,货号L602L)
灭菌水，补足至20μl。
反应条件：37℃5min，21℃5min。共6个循环.
将上述步骤获得的连接产物转化Stbl3感受态细胞(Thermo Scientific,货号C7373-03)并涂Amp+平板(50μg/ml)，并挑取克隆。
用如序列表SEQ ID NO.6所示的通用引物U6，用Sanger法测序的方法鉴定获得阳性克隆。
37℃摇床摇菌过夜培养阳性克隆，抽提质粒，获得pSpCas9(BB)-2A-Puro-QKIsg质粒(如序列表SEQ ID NO.5所示)。
实施例3构建人HEK293T细胞QKI基因敲除
1、细胞培养与转染。
(1)HEK293T细胞接种培养于DMEM高糖培养液中(Thermo Scientific,货号11995-065)，其中含10％FBS(Thermo Scientific,货号10099-141)，penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)在转染前将细胞接种至6孔板中，待70％～80％密度时进行转染。
(3)按照DNA&siRNA Transfection Reagent(Polyplus transfection，114-01)操作手册，将2μg pSpCas9(BB)-2A-Puro-QKIsg质粒转染至每孔细胞中，6～8小时后换液，并加入10μg/ml Puromycin(Merck,540411)药筛，48小时后收取细胞。
2、挑选单克隆：
将Puromycin筛选后细胞消化后计数，取60个细胞接种至一块96孔板上，接种24h后，显微镜下观察，挑选只含单个细胞的孔，继续培养至90％密度。
将上述细胞消化后接种至24孔板，扩大培养，继续培养至90％密度。
3、Sanger法测序鉴定基因突变：
消化细胞，离心后取一半细胞继续培养，一半细胞提取基因组DNA(生工，货号B518401)，RT-PCR扩增包含sgRNA靶向QKI的片段，所用引物见SEQ NO.7和SEQ NO.8。
Sanger法测序，测序引物即SEQ NO.8，并与Ensemble数据库参考序列比对，鉴定QKI阅读框有无发生移码突变，结果见图1和图2。
4、Western Blot鉴定QKI敲除情况：
提取对照组和敲除组细胞蛋白，进行蛋白电泳，随后转膜，封闭，加入抗QKI一抗孵育(Abcam，货号ab126742)过夜，次日去除一抗并洗涤30min，加入二抗(Abcam，ab6721)孵育1小时，随后再次洗涤30min后显影，结果见图3。

Claims (1)

1.一种制备特异性靶向目标基因QKI的sgRNA，其特征在于，其序列如SEQ ID NO.1所示。
CN201610108864.4A 2016-02-26 2016-02-26 一种特异性靶向QKI基因的sgRNA及其应用 Expired - Fee Related CN105567692B (zh)

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