CN104805118A - 一种苏禽黄鸡胚胎干细胞特定基因进行靶向敲除方法 - Google Patents
一种苏禽黄鸡胚胎干细胞特定基因进行靶向敲除方法 Download PDFInfo
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Abstract
本发明公开了一种苏禽黄鸡胚胎干细胞特定基因进行靶向敲除的方法：首先查询出基因的外显子序列，克隆出基因，测序，获得完整的外显子序列，并在此序列上设计CRISPR/Cas9敲除靶位点作为gRNA，构建CRISPR/Cas9双启动子敲除载体；构建完成的Cas9载体进行SSA活性检测，对照组转染空载体，检测luciferase信号，luciferase活性相对于对照组增长的越多，则证明gRNA剪切活性越高；将具有高SSA活性的CRISPR/Cas9载体转染ESCs，流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物。
Description
技术领域：
本发明涉及干细胞工程领域，尤其涉及一种基于CRISPR/Cas9技术对苏禽黄鸡胚胎干细胞特定基因进行靶向敲除的方法。
技术背景：
目前基因组编辑技术主要有三种方法，锌指核酸酶(zinc finger endonuclease，ZFN)、类转录激活因子效应物核酸酶(Transcription Activator-Like EffectorNuclease，TALEN)以及成簇的规律间隔的短回文重复序列及相关基因(clusteredregularly interspaced short palindromic repeats/CRISPR-associated，CRISPR/Cas)。这三种方法都通过在外源DAN靶位点上产生双链切口，从而诱导出同源重组修复和非同源末端连接，进而实现基因组的编辑。
ZFN结构上包括锌指蛋白(Zinc finger protein，ZFP)结构域和Fok I切割结构域，其中Fok I切割结构域源于一种IIS型限制性内切酶，当Fok I酶二聚体化，发挥其切割活性切割DNA的靶位点，形成双链断裂(double-stranded breaks，DSB)，从而诱导细胞内的修复机制，即同源重组修复(homology-directed repair，HDR)或非同源末端链接(non-homologous end-joining，NHEJ)，从而实现基因组的靶向敲除(knock-out)或敲入(knock-in)，然而ZFN目前还存在设计苦难、脱靶严重，技术垄断等问题；
而TALEN结构上包括TALE(transcription activator-like effector)结构域和Fok I核酸内切酶结构域两个部分，其作用机制与ZFN一致，目前也存在多项缺陷，例如成本高，可操作性难度大，细胞毒性等；
CRISPR/Cas系统中主要以典型的Type II CRISPR/Cas为主，结构上分为三个部分tracrRNA位于5’端，CRISPR序列位于3’端，中间的为一系列的Cas基因家族，其中Cas9为核心蛋白，因此可以称此II型系统为CRISPR/Cas9系统。当外源DNA序列进入细菌或古细菌的含有CRISPR的细胞中，CRISPR复合体中的类似于Cas1核酸结合蛋白会介导该复合体与外源DNA序列进行结合，然后该复合体中类似于Cas2核酸内切酶会对外源DNA序列进行切割，形成众多约17-84bp不均等的小片段，其中的一个片段会在相关蛋白的作用下被整合到CRISPR的前导序列和第一个重复序列之间，形成一个新的间隔序列，入侵者与间隔序列相同的序列称为protospacer。当外源DNA再次入侵时，与其protospacer互补匹配的间隔序列，及其两边的部分重复序列转录形成前体crRNA(pre-crRNA)。转录完成后，tracrRNA与pre-crRNA形成二聚体，再与Cas9蛋白结合形成核糖核蛋白复合物，与外源DNA结合，扫描、寻找靶序列，crRNA的间隔序列与靶序列进行互补配对，在外源DNA互补配对的特定位置上被核蛋白复合物剪切。
CRISPR/Cas的打靶效率比较高，最高可达80％，且靶位点设计灵活、方便，载体构建简单；不同于ZFN或TALEN中以蛋白质识别DNA的方式，CRISPR/Cas系统对靶序列的识别是以RNA与DNA碱基配对的方式，这样会降低了脱靶的几率，降低了细胞毒性，但并不是代表不会产生；此外，较于ZFN与TALEN，CRISPR/Cas的设计更为简单、廉价，一般普通的实验也可自行操作；最后，CRISPR/Cas最大的有点就是可同时打靶多个基因，且每多一个靶位点只需多一个gRNA质粒。
有优势必然也会存在局限性，例如CRISPR/Cas技术目前还尚未成熟，需要设计出特异性较高的gRNA质粒，严重的脱靶效应以及在未建系的干细胞上无成功先例等，但相信随着CRISPR/Cas的发展，这些难题终将会被解决。
发明内容：
本发明公开了一种基于CRISPR/Cas9技术对苏禽黄鸡胚胎干细胞特定基因进行靶向敲除的方法：首先查询出基因的外显子序列，克隆出基因，测序，获得完整的外显子序列，并在此序列上设计CRISPR/Cas9敲除靶位点作为gRNA，构建CRISPR/Cas9双启动子敲除载体，其中禽源U6启动子启动gRNA表达，T7启动子启动Cas9基因表达，并在载体中插入GFP蛋白作为报告基因；构建完成的Cas9载体进行SSA活性检测，将终止子和靶位点插入双荧光Luciferase载体中，靶位点位于终止子后，共转染CRISPR/gRNA载体，以及新构建的luciferase报告基因和内参renilla质粒，对照组转染空载体，检测luciferase信号，luciferase活性相对于对照组增长的越多，则证明gRNA剪切活性越高；将具有高SSA活性的CRISPR/Cas9载体转染ESCs，流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物，克隆出靶位点前后各250bp的片段，使用T7E1酶进行活性检测，具有活性的敲除载体与T载体连接，摇菌后测序，计算具体的活性数值。
本发明包括以下步骤：
(1)目的基因的获取
根据基因序列号在NCBI数据库中查询目的基因在原鸡中的CDS序列，设计特异性引物，以苏禽黄鸡组织或细胞RNA反转录形成的单链cDNA为模板克隆并获得苏禽黄鸡中目的基因完整的CDS序列，通过比对得到完整的目的基因外显子序列；
(3)gRNA设计及合成
寻找获得的外显子序列中的PAM序列，将PAM序列前20bp左右的碱基作为靶序列，所有的靶序列在全基因组比对，无同源性的靶位点作为gRNA并合成；
(4)CRISPR/Cas9基因敲除载体构建
VK001-08载体为基础载体进行载体改造，以禽源U6作为启动子启动gRNA的表达，T7启动最启动Cas9酶的表达，同时插入GFP荧光基因作为报告基因。
(5)CRISPR/Cas9基因敲除载体SSA活性检测
将终止子和靶位点插入Luciferase载体中，靶位点位于终止子后，共转染CRISPR/gRNA载体，以及新构建的luciferase报告基因和内参renilla质粒，对照组转染空载体，检测luciferase信号，luciferase活性相对于对照组增长的越多，则证明gRNA剪切活性越高；
(6)CRISPR/Cas9基因敲除载体内源活性检测
剪切活性较高CRISPR/Cas9基因敲除载体转染状态良好的第二代ESC，48小时后流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物，克隆出靶位点前后各250bp共计500bp左右片段，使用T7E1酶进行活性检测，若存在敲除活性，将产生非配对DNA片段，即能被非配对内切酶--T7核酸内切酶I剪切；若没有发生突变，将产生配对DNA片段，而无法被非配对内切酶--T7核酸内切酶I剪切；
(7)CRISPR/Cas9基因敲除载体敲除效率检测
CRISPR/Cas9基因敲除载体转染状态良好的第二代ESC，48小时后流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物，克隆出靶位点前后各250bp共计500bp左右片段，进行TA克隆，挑菌测序并与原始序列进行对比，计算CRISPR/Cas9基因敲除载体的具体效率，计算方法：(序列发生突变的菌液数/全部测序菌液数*100％)，注意测序的菌液需要足够多；
本发明的优越性在于：本方法简单易行，可重复性强，在普通实验室即可进行。运用该方法可以快速、高效在短时间内完成一个基因在鸡胚胎干细胞中基因功能的验证。
具体实施方式
一种基于CRISPR/Cas9技术对苏禽黄鸡胚胎干细胞特定基因进行靶向敲除方法，包括如下步骤：
(1)目的基因的获取
根据基因序列号在NCBI数据库中查询目的基因在原鸡中的CDS序列，设计特异性引物，以苏禽黄鸡PGC的RNA反转录形成的单链CDNA为模板克隆并获得苏禽黄鸡中目的基因完整的CDS序列，通过比对得到完整的目的基因外显子序列；
(4)gRNA设计及合成
寻找获得的外显子序列中的PAM序列，将PAM序列前20bp左右的碱基作为靶序列，所有的靶序列在全基因组比对，无同源性的靶位点作为gRNA并合成；
(5)CRISPR/Cas9基因敲除载体构建
VK001-08载体为基础载体进行载体改造，以禽源U6作为启动子启动gRNA的表达，T7启动最启动Cas9酶的表达，同时插入GFP荧光基因作为报告基因。
(6)CRISPR/Cas9基因敲除载体SSA活性检测
将终止子和靶位点插入Luciferase载体中，靶位点位于终止子后，共转染CRISPR/gRNA载体，以及新构建的luciferase报告基因和内参renilla质粒，对照组转染空载体，检测luciferase信号，luciferase活性相对于对照组增长的越多，则证明gRNA剪切活性越高；
(7)CRISPR/Cas9基因敲除载体内源活性检测
剪切活性较高CRISPR/Cas9基因敲除载体转染状态良好的第二代ESC，48小时后流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物，克隆出靶位点前后各250bp共计500bp左右片段，使用T7E1酶进行活性检测，若存在敲除活性，将产生非配对DNA片段，即能被非配对内切酶--T7核酸内切酶I剪切；若没有发生突变，将产生配对DNA片段，而无法被非配对内切酶--T7核酸内切酶I剪切；
(8)CRISPR/Cas9基因敲除载体敲除效率检测
CRISPR/Cas9基因敲除载体转染状态良好的第二代ESC，48小时后流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物，克隆出靶位点前后各250bp共计500bp左右片段，进行TA克隆，挑菌测序并与原始序列进行对比，计算CRISPR/Cas9基因敲除载体的具体效率，计算方法：(序列发生突变的菌液数/全部测序菌液数*100％)，注意测序的菌液需要足够多。
Claims (1)
1.一种苏禽黄鸡胚胎干细胞特定基因靶向敲除方法，其特征在于：包括如下步骤：
(1)目的基因的获取
根据基因序列号在NCBI数据库中查询目的基因在原鸡中的CDS序列，设计特异性引物，以苏禽黄鸡PGC的RNA反转录形成的单链CDNA为模板克隆并获得苏禽黄鸡中目的基因完整的CDS序列，通过比对得到完整的目的基因外显子序列；
(2)gRNA设计及合成
寻找获得的外显子序列中的PAM序列，将PAM序列前20bp左右的碱基作为靶序列，所有的靶序列在全基因组比对，无同源性的靶位点作为gRNA并合成；
(3)CRISPR/Cas9基因敲除载体构建
VK001-08载体为基础载体进行载体改造，以禽源U6作为启动子启动gRNA的表达，T7启动最启动Cas9酶的表达，同时插入GFP荧光基因作为报告基因。
(4)CRISPR/Cas9基因敲除载体SSA活性检测
将终止子和靶位点插入Luciferase载体中，靶位点位于终止子后，共转染CRISPR/gRNA载体，以及新构建的luciferase报告基因和内参renilla质粒，对照组转染空载体，检测luciferase信号，luciferase活性相对于对照组增长的越多，则证明gRNA剪切活性越高；
(5)CRISPR/Cas9基因敲除载体内源活性检测
剪切活性较高CRISPR/Cas9基因敲除载体转染状态良好的第二代ESC，48小时后流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物，克隆出靶位点前后各250bp共计500bp左右片段，使用T7E1酶进行活性检测，若存在敲除活性，将产生非配对DNA片段，即能被非配对内切酶--T7核酸内切酶I剪切；若没有发生突变，将产生配对DNA片段，而无法被非配对内切酶--T7核酸内切酶I剪切；
(6)CRISPR/Cas9基因敲除载体敲除效率检测
CRISPR/Cas9基因敲除载体转染状态良好的第二代ESC，48小时后流式细胞术筛选出GFP阳性细胞，提取基因组DNA，设计引物，克隆出靶位点前后各250bp共计500bp左右片段，进行TA克隆，挑菌测序并与原始序列进行对比，计算CRISPR/Cas9基因敲除载体的具体效率，计算方法：(序列发生突变的菌液数/全部测序菌液数*100％)，注意测序的菌液需要足够多。
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Cited By (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
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