CN106350540A - 一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体及其应用 - Google Patents

一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体及其应用 Download PDF

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CN106350540A
CN106350540A CN201610739487.4A CN201610739487A CN106350540A CN 106350540 A CN106350540 A CN 106350540A CN 201610739487 A CN201610739487 A CN 201610739487A CN 106350540 A CN106350540 A CN 106350540A
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秦晓峰
彭昌伟
李春峰
彭迪
张路路
程根宏
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Suzhou Institute Of Systems Medicine
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Abstract

本发明提供了一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体，所述CRISPR/Cas9包括Cas9蛋白和sgRNA。通过构建CRISPR/Cas9基因敲除载体，并将CRISPR/Cas9 用慢病毒系统高效地转导进入多种类型的细胞之中，应用于PD‑L1基因的敲除，本发明提供的载体能准确有效地完成其基因编辑的使命。

Description

一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载 体及其应用
技术领域
本发明属于基因工程领域，具体涉及一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体及其在敲除应用。
背景技术
CRISPR(Clustered Regularly Interspersed Short Palindromic Repeats)是细菌用来抵御病毒侵袭/躲避哺乳动物免疫反应的基因系统。科学家们利用RNA引导Cas9核酸酶可在多种细胞(包括iPS)的特定的基因组位点上进行切割，修饰。核酸内切酶Cas9介导的基因编辑因其简单的操作方式、低廉的构建成本、显著的切割效率迅速成为了科学家们进行基因组工程的最佳选择之一，掀起了一股CRISPR浪潮，被《Science》杂志评为2013年的年度第二大突破，在动物模型构建的应用前景非常广阔。
慢病毒(Lentivirus)是逆转录病毒科中的一个属的总称，因其在侵染宿主细胞后潜伏期(incubation time)较一般的逆转录病毒更长而得名，而慢病毒载体(Lentiviralvector)通常特指利用人类免疫缺陷I型病毒(HIV-I)所改造发展而成的一类高效的基因传递载体。由于慢病毒载体是通过稳定整合进入基因组进行表达，在表达时间和表达量上并没有特异性，因而我们通常结合诱导表达系统，使得慢病毒介导的外源基因表达成为可控的特异性表达。四环素诱导表达系统(Tetracycline-inducible,Tetinducible)就是我们最常用的诱导表达系统之一。其主要由两个部分组成：一个是含有四环素应答元件(tetracycline response element)的TRE启动子，另一个是由四环素或其类似物控制的反式激活因子(tetracycline-controlled transactivator)。根据对诱导物，即四环素或其类似物(如强力霉素)的不同反应，将这个诱导表达系统分为两种，分别为TetOn以及TetOff：TetOn系统中，反式激活因子rtTA只有在加入诱导剂后才能与启动子中的操纵子序列相结合，从而启动下游基因的表达；TetOff系统则恰恰相反。TetOn诱导表达系统中，基因表达受到严格的控制，并且表现为诱导剂剂量依赖性表达，也就是说可以通过调节诱导剂的浓度来控制基因表达量的多少。
对于CRISPR这样一个能简便高效介导细胞基因组工程的技术，除了对技术系统本身进行改良，提高编辑效率、降低脱靶效应之外，我们还需要关注其表达的控制方式。
发明内容
本发明提供了一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体及其应用，提供了如何将CRISPR/Cas9用慢病毒系统高效地转导进入多种类型的细胞之中，并且能准确有效地完成其基因编辑的使命。
本发明的目的通过以下技术方案来实现：
一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体，所述CRISPR/Cas9包括Cas9蛋白和sgRNA，所述载体包括Cas9慢病毒表达载体FG-TRE-Cas9、及sgRNA慢病毒表达载体FG12-sgRNA。
优选地，一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体的构建方法，包括如下步骤，
S1、构建Cas9慢病毒表达载体FG-TRE-Cas9；
S11、以载体pCS2(+)-hSpCas9为模板，经过PCR扩增得到含有Flag标签以及Cas9表达序列的片段BstBI-3xFALG-hSpCas9-SacII；
S12、将S11得到的片段用BstBI及SacII酶切链接到含有TRE启动子的载体FG-TRE中，得到中间载体；
S13、通过PacI、ClaI酶切将Bsd基因从载体FG-EH-DEST-PB-WPRE亚克隆到S12得到的中间载体，最终得到带有Bsd抗性基因的最终目标载体FG-TRE-Cas9；
S2、构建sgRNA慢病毒表达载体FG12-sgRNA；
S21、合成目标表达片段XbaI—U6—sgRNA scaffold—XhoI，所述目标表达片段的序列为SEQ ID NO.1，所述U6为人类U6小RNA启动子，序列为SEQ ID NO.2，所述sgRNAscaffold序列为SEQ ID NO.3；
S22、通过XbaI、XhoI双酶切目标表达片段连接到FG12载体，即为FG12-SgRNA载体。
优选地，所述S11中PCR扩增时所需的引物为，正向引物为Cas9_BstBI_F：CGGATTCGAA TTCATGGACTATAAGGACCAC；
反向引物为Cas9_SacII_R：TCCCCGCGG TCATTTCTTTTTCTTAGCTTG。
优选地，以上一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体的应用。
优选地，一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体在敲除PD-L1基因中的应用。
本发明的有益效果体现在：能实现对多种细胞靶基因的高效、可控敲除，从而使之成为在各种哺乳动物细胞以及动物模型中具有广泛应用价值的研究基因功能的工具。
附图说明
图1为构建诱导型慢病毒表达载体的流程示意图。
图2为用诱导型慢病毒表达载体构建基因敲除细胞系的流程示意图。
图3可诱导基因敲除的细胞系H441/TetOn/TRE-Cas9/CD274sgRNA表达CD274SgRNA的流式细胞术检测。
图4可诱导基因敲除的细胞系H441/TetOn/TRE-Cas9/CD274sgRNA，经多西霉素诱导后，CAS9蛋白表达情况。
图5为敲除CD274的效率与诱导剂多西霉素的浓度之间的关系示意图。
图6用测序方法检测敲除CD274的突变方式。
图7用流式细胞术检测敲除CD 274单克隆细胞的基因敲除效率。
图8为加入多西霉素诱导后，单克隆细胞中CAS9蛋白的表达水平。
具体实施方式
以下结合实施例具体说明本发明的制备方法，如无特别说明，均为常规方法：
构建Cas9慢病毒表达载体FG-TRE-Cas9：
首先以载体pCS2(+)-hSpCas9为模板，通过PCR得到含有Flag标签以及Cas9表达序列的片段BstBI-3xFALG-hSpCas9-SacII，正向引物：Cas9_BstBI_F：CGGA TTCGAATTCATGGACTATAAGGACCAC；反向引物：Cas9_SacII_R：TCC CCGCGG TCATTTCTTTTTCTTAGCTTG。将该片段用BstBI及SacII酶切链接到含有TRE启动子的载体FG-TRE中，得到中间载体FG-TRE-Cas9。接着通过PacI，ClaI酶切将Bsd基因从载体FG-EH-DEST-PB-WPRE插入 到中间载体中，即得到带有Bsd抗性基因的最终目标载体FG-TRE-Cas9。
构建sgRNA慢病毒表达载体FG12-sgRNA：
由于FG12载体本身就是为表达小RNA—shRNA所设计的，因而可以直接以其作为起点构建目标载体FG12-sgRNA。将GFP基因连接到FG12载体上，用来检测载体的转染及RNA的表达。在FG12中通过XbaI—XhoI双酶切来插入shRNA的启动子和表达序列。
由于FG12载体上已经含有了多个sgRNA克隆时所使用的BbsI位点，为了能够更方便地在目的载体上直接克隆sgRNA，选择将sgRNA的克隆位点更改为BsmBI。由于sgRNA的骨架序列本身不长，只有76bp，加之酶切位点有各种限定，所以采用直接合成XbaI—U6—sgRNA scaffold—XhoI,并将所需合成的sgRNA表达片段XbaI—U6—sgRNA scaffold—XhoI序列送至上海捷瑞生物工程有限公司进行基因合成。然后通过XbaI，XhoI双酶切连接到FG12载体，即为FG12-SgRNA载体，如图1所示。
所述sgRNA表达片段XbaI—U6—sgRNA scaffold—XhoI其DNA序列为SEQ IDNO.1,如下：
TCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACGGCATTTCGTCTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGGGCCCATCGGTACCCTCGAG
其中人类U6为小RNA启动子序列为SEQ ID NO.2；sgRNA
scaffold为sgRNA的骨架,序列为SEQ ID NO.3；所述U6启动子的终止序列为SEQID NO.4。
SEQ ID NO.2
CCCCCAGTGGAAAGACGCGCAGGCAAAACGCACCACGTGACGGAGCGTGACCGCGCGCCGAGCGCGCGCCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAAT
SEQ ID NO.3
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC
SEQ ID NO.4
TTTTTTT
以下阐述下CRISPR/Cas9基因敲除载体及其应用
CD274靶位点sgRNA的设计及其SgRNA表达载体的构建
借助MIT Zhang FengLab开发的CRISPR Design(http://crispr.mit.edu/)进行设计针对CD274的SgRNA，具体步骤如下：
首先，输入所选择的CD274(即PD-L1)Target Sequence(第一个外显子23-500nt的序列，超过长度的可分两次进行)，选择对应物种的基因组，sequence type选择uniqueregion。在Scan completed后选择Guides&off-targets查看设计结果。所显示的结果依照其脱靶的可能性进行打分排序(100分为不脱靶，分越低表示脱靶的可能性越高)，且列出所有可能的off-targets位点。选择得分最高的sgRNA序列TCCAGATGACTTCGGCCTTG。由于使用人类U6小RNA启动子表 达sgRNA，同等条件下可以优先选择以碱基G开头的sgRNA序列；若是所选序列第一位不为碱基G，则在所选择的序列前加入一个碱基G，构成以G+20nt sgRNA。将所设计的sgRNA按照以下Oligo序列按照引物的形式进行合成：
5’-CACC gTCCAGATGACTTCGGCCTTG-3’
3’-cAGGTCTACTGAAGCCGGAAC CAAA-5’sgRNA Sequence(G+20nt without PAM)
将合成好的DNA oligo复链，使用BsmBI对FG12-sgRNA载体进行酶切消化，使其线性化，连接FG12-sgRNA载体与shRNA，连接产物转化TOP10感受态细胞，测序鉴定正确后，得到FG12-CD274SgRNA载体。
构建诱导型CRISPR细胞系并用于敲除CD274基因
构建好了表达CRISPR必须组件的载体质粒之后，我们要将其包装并产生出慢病毒颗粒，对靶细胞进行侵染，使慢病毒所携带的遗传信息稳定整合于细胞基因组内。由于整个可诱导CRISPR系统是由两个携带了不同的抗性标签以及一个荧光蛋白标签的慢病毒载体构成。因而我们需要用病毒进行依次分步侵染整合，并使用对应的抗性药物筛选阳性细胞。
用磷酸钙转染法对慢病毒载体进行转染包装，收获后浸染细胞。
(1)将293T-FQ细胞传入24孔板中，每个孔约加入0.5mL新鲜的DMEM培养基以及1.5～2×105个细胞，放入37培养箱中培养14～16h后可进行转染。
(2)将慢病毒包装质粒pSL3(VSV-G)、pSL4(gag,pol/RRE)、pSL5(Rev)以及慢病毒载体(以FG为例)按照以下比例加入至20μL的Distilled Water中混合均匀，pSL3：pSL4：pSL5：FG＝100ng：400ng：200ng：200ng。加入5μL、1.25M、CaCl2溶液；加入25μL 2×HBS溶液，并立即用调至50μL的移液器上下吹打混匀；混合液室温下静止2～3min，然后将其全部逐滴地加入细胞培养基中。转染6～8h之后，更换新鲜的DMEM培养基1mL，继续培养48h。
根据可诱导CRISPR系统的作用原理，如图2所示，我们首先向目标细胞H441细胞中侵染TetOn作用原件病毒FG-UbiC-TetOn，并使用Zeocin进行筛选，获得H441/TetOn细胞系；接着用表达Cas9的慢病毒FGTRE-Cas9进行侵染，并用Blasticidin进行筛选，获得H441/TetOn/TRE-Cas9细胞系；最后用表达FG12-CD274-sgRNA的慢病毒进行侵染，获得最后的可诱导基因敲除的细胞系H441/TetOn/TRE-Cas9/CD274sgRNA。结果如图3、图4所示，用病毒侵染的细胞可以检测到明显的GFP信号，而没有病毒侵染的细胞，检测不到GFP信号。在没有多西霉素加入时，检测不到Cas9蛋白的表达，随着加入多西霉素量的升高，可以看到CAS9蛋白表达水平随之升高，说明该细胞系能被多西霉素诱导表达Cas9。
构建诱导型敲除CD274的细胞系
将细胞系H441/TetOn/TRE-Cas9/CD274sgRNA用多西霉素诱导，消化收集到1.5mLEP管中，用CD274的抗体(eBioScience)检测在细胞表面的CD274表达情况。如图5所示，在加入多西霉素后，细胞表面的CD274表达水平明显降低。提取细胞的基因组，用Indel实验检测发现，随着多西霉素浓度的升高，Indel的比例逐渐升高，说明敲除CD274的效率依赖于诱导剂多西霉素的浓度，如图6所示。而用其他不相关SgRNA进行敲除(如EMX1)，并未发现CD274基因被编辑，说明该诱导型CRISPR载体具有特异性。
提取敲除CD274的细胞系基因组，将对应片段PCR扩增出来后，并克隆到T载体上，送到北京金唯志公司测序，检测突变类型，可以看 到突变分为插入及缺失两种，以插入1个碱基造成移码突变的最多，如图7所示，最多可以插入60多个碱基。同时将消化的细胞，用新鲜的培养基稀释，按照0.3～0.5个细胞/孔，于96孔板中铺孔，每孔100μL培养基(即取10mL培养基加入30～50个细胞即可铺板约100孔)。铺板后培养7d左右即可看到由单个细胞长出的细胞团克隆，此时需留意观察是否有一个孔内存在多个克隆的情况，若出现则废弃该孔。培养期间注意对细胞进行补液、换液操作。约14d，单细胞可以长成较大的群落，再传代到24孔板中扩大培养，从而获得了细胞系的单个细胞克隆。将单克隆细胞系用流式细胞仪检测细胞表面的CD274蛋白表达水平，如图8所示，在加入多西霉素诱导后，CD274的表达水平明显降低，同时能检测到CAS9蛋白的表达。以上结果显示，我们构建的诱导型CRISPR载体可以用于哺乳动物细胞CD274基因或其他基因的有效及可控的敲除。
本发明尚有多种具体的实施方式。凡采用等同替换或者等效变换而形成的所有技术方案，均落在本发明要求保护的范围之内。

Claims (5)

1.一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体，所述CRISPR/Cas9包括Cas9蛋白和sgRNA，其特征在于：所述载体包括Cas9 慢病毒表达载体FG-TRE-Cas9、及sgRNA 慢病毒表达载体FG12-sgRNA。
2.如权利要求1所述的一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体的构建方法， 其特征在于：包括如下步骤，
S1、构建Cas9 慢病毒表达载体FG-TRE-Cas9；
S11、以载体pCS2(+)-hSpCas9 为模板，经过PCR扩增得到含有Flag 标签以及Cas9 表达序列的片段BstBI-3xFALG-hSpCas9-SacII；
S12、将S11得到的片段用BstBI及SacII酶切链接到含有TRE启动子的载体FG-TRE中，得到中间载体；
S13、通过PacI、ClaI酶切将Bsd基因从载体FG-EH-DEST-PB-WPRE亚克隆到S12得到的中间载体，最终得到带有Bsd抗性基因的最终目标载体FG-TRE-Cas9；
S2、构建sgRNA 慢病毒表达载体FG12-sgRNA；
S21、合成目标表达片段XbaI—U6—sgRNA scaffold —XhoI，所述目标表达片段的序列为SEQ ID NO.1，所述 U6为人类U6 小RNA 启动子，序列为SEQ ID NO.2，所述sgRNAscaffold序列为SEQ ID NO.3；
S22、通过XbaI、XhoI 双酶切目标表达片段连接到FG12 载体，即为FG12-SgRNA载体。
3.如权利要求2所述的一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体的构建方法，其特征在于：所述S11中PCR扩增时所需的引物为，正向引物为Cas9_BstBI_F：CGGATTCGAA TTCATGGACTATAAGGACCAC；
反向引物为Cas9_SacII_R：TCCCCGCGG TCATTTCTTTTTCTTAGCTTG。
4.如权利要求1所述的一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体的应用。
5.如权利要求1所述的一种由慢病毒介导的高效可诱导型CRISPR/Cas9基因敲除载体在敲除PD-L1基因中的应用。
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