CN108559760A - 基于CRISPR靶向基因组修饰技术建立荧光素酶knock-in细胞系的方法 - Google Patents

基于CRISPR靶向基因组修饰技术建立荧光素酶knock-in细胞系的方法 Download PDF

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CN108559760A
CN108559760A CN201810020095.1A CN201810020095A CN108559760A CN 108559760 A CN108559760 A CN 108559760A CN 201810020095 A CN201810020095 A CN 201810020095A CN 108559760 A CN108559760 A CN 108559760A
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夏海滨
李子航
赵俊丽
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Abstract

本发明涉及一种基于CRISPR靶向基因组修饰技术建立荧光素酶knock‑in细胞系的方法，采用CRISPR/Cas9技术在基因组上SREBP1基因的3’端原位整合入T2A‑Luciferase报告基因，建立了SREBP1‑T2A‑Luciferase的knock‑in细胞系，并验证了此细胞系中外源基因在基因组上的定点整合。同时利用已报道的对SREBP1有激活作用的转录因子LXRα对SREBP1‑T2A‑Luciferase细胞系进行转录激活，结果表明SREBP1‑T2A‑Luciferase细胞系内的Luciferase表达水平可以准确灵敏地反映细胞系内SREBP1分子表达水平。该细胞系的建立将有助于SREBP1基因功能研究及筛选影响SREBP1表达的小分子化学药物，为脂代谢及其相关研究提供一种新的实验思路及解决方案。

Description

基于CRISPR靶向基因组修饰技术建立荧光素酶knock-in细胞 系的方法
技术领域
本发明属于分子生物学领域，涉及利用CRISPR/Cas9介导的靶向基因组***技术建立细胞系，具体涉及一种基于CRISPR/Cas9靶向基因组修饰技术建立KI-T2A-Luciferase细胞系的新方法。
背景技术
CRISPR/Cas9是细菌和古细菌长期演化形成的免疫防御***，该***是利用CRISPR-derivedRNA(crRNA)通过碱基配对与trans-activating RNA结合形成复合物，Cas9内切酶在此复合物引导下对与crRNA配对的序列进行定点切割。所以，通过人工设计具有引导作用的sgRNA(short guide RNA)，可以引导Cas9对宿主细胞DNA进行定点切割，然后通过非同源末端连接(non-homologous end joining，NHEJ)或同源重组(homologousrecombination，HR)两种修复途径的机制进行修复，实现基因编辑。
目前检测基因的表达调控主要是通过RT-PCR、Western Blot和将目标基因启动子克隆到携带报告基因的表达检测载体中等手段来实现。RT-PCR过程繁琐且不稳定，WesternBlot抗体标记费时昂贵，这些都不利于高通量筛选；由于基因组本身存在表观遗传学修饰，将启动子克隆到携带报告基因的表达检测载体中的方法无法模拟基因组的真实状态，因此难以准确反映基因组上基因表达情况。
随着今年来报告基因技术的不断发展，荧光蛋白报告基因、荧光素酶报告基因等已成为基础研究中常用的细胞标记与示踪的方法，由于其具有方便快捷、安全直观的优点，不仅普遍应用于探索构建分子通路的分子生物学实验，而且越来越广泛用于筛选药物所构建的细胞系。
发明内容
本发明的目的在于，利用CRISPR/Cas9技术，提供一种基于CRISPR/Cas9靶向基因组修饰技术建立KI-T2A-Luciferase细胞系的新方法。
为了实现上述任务，本发明采取如下的技术解决方案：
一种基于CRISPR/Cas9靶向基因组修饰技术建立KI-T2A-Luciferase细胞系的方法，其特征在于，按下列步骤实施：
1)在目的基因SREBP1的终止密码子下游设计并合成相应的sgRNA，室温退火后连入pU6-sgRNA1.0，筛选后获得sgRNA表达组件，并检测其打靶效率；
2)将检测过的打靶效率高的sgRNA表达组件与Cas9基因克隆至一个表达载体，获得pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA；
3)构建靶向SREBP1基因的打靶载体pUC19/SREBP1-donor，该打靶载体pUC19/SREBP1-donor的结构为两部分，第一部分是与断裂位点上下游分别具有相同序列的上下游同源臂；第二部分是位于上下游同源臂之间的待重组入基因组靶位点的T2A-Luciferase-CMV-eGFP-T2A-Neomycin-SV40pA DNA片段；
4)将pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA与pUC19/SREBP1-donor共转HEK293细胞系，待细胞系稳定后，加1.0mg/mL的G418筛选10天，待细胞系稳定后，再加10μg/mL的GCV筛选3周，待细胞系稳定过后，对细胞经有限稀释法进行克隆化，再对克隆化后的细胞进行PCR鉴定并测序，证实携带荧光素酶报告基因的打靶载体在目标位点处的正确重组，最终获得单一稳定的HEK293-SREBP1-T2A-luciferase-KI细胞系；
5)克隆并构建SREBP1基因的转录激活因子LXRα表达载体pUC19/CMV-LXRα，该表达载体pUC19/CMV-LXRα被用于SREBP1基因的转录激活实验研究；
6)验证HEK293-SREBP1-T2A-luciferase-KI细胞系中Luciferase表达变化是否可以真实反映内源性SREBP1基因的相对表达量及表达变化；使用所构建的SREBP1基因的转录激活因子LXRα表达载体pUC19/CMV-LXRα分别转染HEK293-SREBP1-T2A-luciferase-KI细胞系和野生型HEK293细胞系，并对HEK293-SREBP1-T2A-luciferase-KI细胞系中的Luciferase活性及HEK293细胞系中SREBP1分子的mRNA表达水平分别进行检测；通过对knock-in细胞系中Luciferase表达活性与HEK293细胞中的SREBP1mRNA表达水平及变化的比较，进一步验证HEK293-SREBP1-T2A-luciferase-KI细胞系中的Luciferase活性是否可以准确反映SREBP1分子的相对表达变化。
根据本发明，步骤1)中所述的合成相应的sgRNA为针对SREBP1的终止密码子下游设计的sgRNA，其中：
sgRNA1序列为：GTCGAAGCTTTGAAGGCCGA；
sgRNA2序列为：GATCTTGACCCTAAGACCGG；
sgRNA3序列为：G TGGCCGATCGGGGCACTGC；
sgRNA4序列为：GCTTTCCCGGACTGCAAGCA。
进一步地，步骤4)中所述的HEK293细胞系为人胚肾细胞系HEK293。
本发明的基于CRISPR/Cas9靶向基因组修饰技术建立KI-T2A-Luciferase细胞系的新方法，具有如下优点：
采用CRISPR/Cas9技术在基因组上SREBP1基因的3’端原位整合入T2A-Luciferase报告基因，建立了SREBP1-T2A-Luciferase的knock-in细胞系，并验证了此细胞系中外源基因在基因组上的定点整合。同时利用已报道的对SREBP1有激活作用的转录因子LXRα对SREBP1-T2A-Luciferase细胞系进行转录激活，结果表明SREBP1-T2A-Luciferase细胞系内的Luciferase表达水平可以准确灵敏地反映细胞系内SREBP1分子表达水平。该细胞系的建立将有助于SREBP1基因功能研究及筛选影响SREBP1表达的小分子化学药物，为脂代谢及其相关研究提供一种新的实验思路及解决方案。同时，通过在靶基因下游整合入报告基因来检测靶基因表达的方法也可广泛应用于其它各种基因的相关研究。
附图说明
图1是携带sgRNA及Cas9的表达载体结构示意图。
图2是打靶Donor载体结构示意图。
图3是所建立的KI-T2A-Luciferase细胞系的结构示意图。
图4是细胞正负筛选稳定后倒置荧光显微镜下观察结果图，其中左图为白光图，右图为荧光图。
图5是对细胞经有限稀释法进行克隆化后每个克隆的Luciferase活性检测图。
图6是对有Luciferase活性的单克隆细胞PCR检测电泳图。
图7是对SREBP1-T2A-Luciferase细胞克隆化后所得27号克隆的PCR产物中野生型大小条带和整合型大小条带分别进行胶回收测序结果。
图8是转录激活因子LXRα表达载体pUC19/CMV-LXRα转染HEK293-SREBP1-T2A-luciferase-KI细胞系后Luciferase的检测结果。
图9是转录激活因子LXRα表达载体pUC19/CMV-LXRα转染野生型HEK293细胞系后，SREBP1分子的mRNA表达水平的检测结果。
下面结合附图和实施例对本发明作进一步的详细说明。
具体实施方式
本实施例给出一种基于CRISPR/Cas9靶向基因组修饰技术建立KI-T2A-Luciferase细胞系的方法，利用CRISPR/Cas9***在特定位置产生双链切口(DSBs)，在Cas9-sgRNA组分中加入一个供体载体(Donor DNA)，Donor DNA上带有靶向位点侧翼的同源序列，DSBs的修复会以供体DNA为模板进行，进而将特定片段***到目的基因的基因组特定位置上。
具体按以下步骤实施：
1)在目的基因SREBP1的终止密码子下游设计并合成相应的sgRNA，室温退火后连入pU6-sgRNA1.0，筛选后获得sgRNA表达组件，并检测其打靶效率；
2)将检测过的打靶效率高的sgRNA表达组件与Cas9基因克隆至一个表达载体，获得pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA；
3)构建靶向SREBP1基因的打靶载体pUC19/SREBP1-donor，该打靶载体pUC19/SREBP1-donor结构为两部分，第一部分是与断裂位点上下游分别具有相同序列的上下游同源臂；第二部分是位于上下游同源臂之间的待重组入基因组靶位点的T2A-Luciferase-CMV-eGFP-T2A-Neomycin-SV40pADNA片段；
4)将pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA与pUC19/SREBP1-donor共转HEK293细胞系，待细胞系稳定后，加1.0mg/mL的G418筛选10天，待细胞系稳定后，再加10μg/mL的GCV筛选3周左右，待细胞系稳定过后，对细胞经有限稀释法进行克隆化，再对克隆化后的细胞进行PCR鉴定并测序，证实携带荧光素酶报告基因的打靶载体在目标位点处的正确重组，最终获得单一稳定的HEK293-SREBP1-T2A-luciferase-KI细胞系；
5)克隆并构建SREBP1基因的转录激活因子LXRα表达载体pUC19/CMV-LXRα，该表达载体pUC19/CMV-LXRα将被用于SREBP1基因的转录激活实验研究；
6)验证HEK293-SREBP1-T2A-luciferase-KI细胞系中Luciferase表达变化是否可以真实反映内源性SREBP1基因的相对表达量及表达变化；使用所构建的SREBP1基因的转录激活因子LXRα表达载体pUC19/CMV-LXRα分别转染HEK293-SREBP1-T2A-luciferase-KI细胞系和野生型HEK293细胞系，并对HEK293-SREBP1-T2A-luciferase-KI细胞系中的Luciferase活性及HEK293细胞系中SREBP1分子的mRNA表达水平分别进行检测；通过对knock-in细胞系中Luciferase表达活性与HEK293细胞中的SREBP1mRNA表达水平及变化的比较，进一步验证HEK293-SREBP1-T2A-luciferase-KI细胞系中的Luciferase活性是否可以准确反映SREBP1分子的相对表达变化。
本实施例中，步骤1)中所述的合成相应的sgRNA为针对SREBP1的终止密码子下游设计的sgRNA，其中：
sgRNA1序列为：GTCGAAGCTTTGAAGGCCGA；
sgRNA2序列为：GATCTTGACCCTAAGACCGG；
sgRNA3序列为：GTGGCCGATCGGGGCACTGC；
sgRNA4序列为：GCTTTCCCGGACTGCAAGCA。
所述的打靶Donor和Cas9-sgRNA表达载体共转染的细胞系为人胚肾细胞系HEK293。
本实施例给出的基于CRISPR/Cas9靶向基因组修饰技术建立KI-T2A-Luciferase细胞系的方法，涉及两个关键载体构建，一个是表达sgRNA及Cas9的表达载体，另一种是包含上下游同源臂的打靶Donor载体。
其中：
所提供的表达sgRNA及Cas9的表达载体是将sgRNA表达组件和Cas9基因克隆至同一个表达载体所构建。
所提供的打靶Donor载体，是将打靶断裂位点上游909bp和其下游887bp的两段序列分别作为打靶载体的上下游同源臂，再将其与申请人实验室保存的T2A-Luciferase报告基因、正筛元件CMV-eGFP-T2A-Neomycin-SV40pA及负筛元件PGK-TK-T2A-mCherry-SV40pA分别连接到pU19表达载体中所构建。
以下是发明人给出的具体实施例。
实施例1：靶向目的基因SREBP1的终止密码子下游sgRNA的设计、合成及载体构建
(1)选取SREBP1基因的终止密码子下游作为打靶区(TSF)，长度大约为1000bp；
(2)在TSF区找出所有NGG及其前12位碱基在NCBI进行Blast，筛选出与目标序列完全匹配并且唯一匹配的序列(若无符合要求的NGG，反向查找CCN)，减少潜在脱靶位点；
本实施例设计了4个sgRNA，其中：
sgRNA1序列为：GTCGAAGCTTTGAAGGCCGA；
sgRNA2序列为：GATCTTGACCCTAAGACCGG；
sgRNA3序列为：GTGGCCGATCGGGGCACTGC；
sgRNA4序列为：GCTTTCCCGGACTGCAAGCA。
分别在其5’加上ACCG得到正向寡核苷酸，获得其互补链，并且在其5’加上AAAC得到反向寡核苷酸。将合成的正向和反向寡核苷酸室温退火后连入pU6-sgRNA1.0，获得sgRNA表达组件。
实施例2：表达sgRNA组件与Cas9基因的载体构建
(1)通过T7E1检测后，筛选出打靶效率高的sgRNA表达载体；
(2)将打靶效率高的sgRNA表达载体和Cas9的表达载体分别用KpnⅠ和SpeⅠ酶切后，经质量浓度为1％的琼脂糖凝胶电泳回收后，将得到的片段与载体连接，经过酶切及测序鉴定获得阳性克隆。将获得的克隆命名为pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA(如图1所示)。
实施例3：构建靶向SREBP1基因的打靶载体pUC19/SREBP1-donor的构建
所构建的打靶载体包含打靶断裂位点上游909bp和其下游887bp的两段序列作为打靶载体的上下游同源臂，其中：
上游同源臂使用引物SREBP1up arm ClaⅠfor序列TATCGATGTCAGGCAGTGGTGGAGATG、SREBP1up arm SpeⅠreverse序列GACTAGTGCTGGAAGTGACAGTGGTCC得到。
下游同源臂使用引物SREBP1down arm SalⅠfor序列CGTCGACGGCCACAAGGTACACAACTTT、SREBP1down arm BglⅡreverse reverse序列AAGATCTCTGTCCGTCCGTGTCCTCA得到。
再将其与本实验室保存的T2A-Luciferase报告基因、正筛元件CMV-eGFP-T2A-Neomycin-SV40pA及负筛元件PGK-TK-T2A-mCherry-SV40pA分别连接到pU19表达载体，将获得的载体命名为pUC19/SREBP1-donor(如图2所示)。
实施例4：对克隆化后的细胞系进行PCR测序
将实施例2和实例3得到的载体以质量比4μg：8μg配置，使用磷酸钙法共同转染HEK293细胞，待细胞系稳定后，加1.0mg/mL的G418筛选10天，待细胞系稳定后，再加10μg/mL的GCV筛选3周，待细胞系稳定过后，用倒置荧光显微镜观察细胞eGFP的表达情况(图4)，之后对细胞经有限稀释法进行克隆化后每个克隆的Luciferase活性检测(图5)，再对克隆化后的细胞进行PCR鉴定。PCR引物SREBP1integration detection for序列TGTGACCTGCTTCTTGTGGT、SREBP1integration detection reverse序列GGAGCGCAAAACCCAAGAAG(引物在基因组上位置如图3箭头所示)。PCR结果显示克隆化后所得高Luciferase活性的阳性克隆分别有野生型大小条带和整合型大小条带两条带(如图6所示)。
实施例5：TA克隆对克隆化后的细胞系进行测序
(1)分别将实例4获得的克隆27号的野生型大小条带和整合型大小条带两条带的纯化产物3μL与0.5μLpGEM-T连接并转化大肠杆菌DH 5α感受态细胞；
(2)挑取单克隆用引物T7序列TAATACGACTCACTATAGGG、引物SP6序列ATTTAGGTGACACTATAG测序，测序结果表明携带荧光素酶报告基因的打靶载体在目标位点处的正确重组，最终获得单一稳定的HEK293-SREBP1-T2A-luciferase-KI细胞系(如图7所示)。
实施例5：克隆并构建SREBP1基因的转录激活因子LXRα表达载体
使用引物LXRαCDS XhoⅠfor序列CCTCGAGATGTCCTTGTGGCTGGGG、引物LXRαCDS XbaⅠreverse序列CTCTAGATTCGTGCACATCCCAGAT得到SREBP1基因的转录激活因子LXRα，并构建转录激活因子LXRα的表达载体，经过酶切及测序鉴定获得阳性克隆。将获得的克隆命名为pUC19/CMV-LXRα。
实施例6：验证所构建的细胞系中Luciferase表达变化是否可以真实反映内源性SREBP1基因的相对表达量及表达变化
使用所构建的SREBP1基因的转录激活因子LXRα表达载体pUC19/CMV-LXRα分别转染所构建的knock-in细胞系和野生型HEK293细胞系，所构建的knock-in细胞系中的Luciferase活性检测(如图8所示)和HEK293细胞系中SREBP1分子的mRNA表达水平检测(如图9所示)显示，与对照组相比，实验组均有显著性增强，证实了所构建的knock-in细胞系中的Luciferase活性可以准确反映SREBP1分子的相对表达变化，所建立的细胞系命名为HEK293-SREBP1-T2A-luciferase-KI细胞系。

Claims (4)

1.一种基于CRISPR靶向基因组修饰技术建立荧光素酶knock-in细胞系的方法，其特征在于，按下列步骤实施：
1)在目的基因SREBP1的终止密码子下游设计并合成相应的sgRNA，室温退火后连入pU6-sgRNA1.0，筛选后获得sgRNA表达组件，并检测其打靶效率；
2)将检测过的打靶效率高的sgRNA表达组件与Cas9基因克隆至一个表达载体，获得pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA；
3)构建靶向SREBP1基因的打靶载体pUC19/SREBP1-donor，该打靶载体pUC19/SREBP1-donor的结构为两部分，第一部分是与断裂位点上下游分别具有相同序列的上下游同源臂；第二部分是位于上下游同源臂之间的待重组入基因组靶位点的T2A-Luciferase-CMV-eGFP-T2A-Neomycin-SV40pA DNA片段；
4)将pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA与pUC19/SREBP1-donor共转HEK293细胞系，待细胞系稳定后，加1.0mg/mL的G418筛选10天，待细胞系稳定后，再加10μg/mL的GCV筛选3周，待细胞系稳定过后，对细胞经有限稀释法进行克隆化，再对克隆化后的细胞进行PCR鉴定并测序，证实携带荧光素酶报告基因的打靶载体在目标位点处的正确重组，最终获得单一稳定的HEK293-SREBP1-T2A-luciferase-KI细胞系；
5)克隆并构建SREBP1基因的转录激活因子LXRα表达载体pUC19/CMV-LXRα，该表达载体pUC19/CMV-LXRα被用于SREBP1基因的转录激活实验研究；
6)验证HEK293-SREBP1-T2A-luciferase-KI细胞系中Luciferase表达变化是否可以真实反映内源性SREBP1基因的相对表达量及表达变化；使用所构建的SREBP1基因的转录激活因子LXRα表达载体pUC19/CMV-LXRα分别转染HEK293-SREBP1-T2A-luciferase-KI细胞系和野生型HEK293细胞系，并对HEK293-SREBP1-T2A-luciferase-KI细胞系中的Luciferase活性及HEK293细胞系中SREBP1分子的mRNA表达水平分别进行检测；通过对knock-in细胞系中Luciferase表达活性与HEK293细胞中的SREBP1 mRNA表达水平及变化的比较，进一步验证HEK293-SREBP1-T2A-luciferase-KI细胞系中的Luciferase活性是否可以准确反映SREBP1分子的相对表达变化。
2.如权利要求1所述的方法，其特征在于，步骤1)中所述的合成相应的sgRNA为针对SREBP1的终止密码子下游设计的sgRNA，其中：
sgRNA1序列为：GTCGAAGCTTTGAAGGCCGA；
sgRNA2序列为：GATCTTGACCCTAAGACCGG；
sgRNA3序列为：GTGGCCGATCGGGGCACTGC；
sgRNA4序列为：GCTTTCCCGGACTGCAAGCA。
3.如权利要求1所述的方法，其特征在于，步骤4)中所述的HEK293细胞系为人胚肾细胞系HEK293。
4.如权利要求1所述的方法，其特征在于，所述的pCMV-Cas9-SV40pA-U6-sgRNAs-SV40pA与pUC19/SREBP1-donor以质量比4μg：8μg配置。
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