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CRISPR-Cas9全程实操教程：从gRNA设计到挑单克隆全程指导

发布: 2017-05-11 2,075阅读 213评论

基本原理
CRISPR-Cas9是一种细菌获得性免疫，用于降解入侵的外源DNA。CRISPR RNA (crRNA) 与转录激活crRNA (Trans-activating crRNA， tracrRNA) 退火形成的复合物能特异性识别与其互补的基因组序列，引导Cas9核酸内切酶在目的片段生成DNA双链断裂，如下图所示：

通过基因工程手段对crRNA和tracrRNA进行改造，将其连接在一起得到sgRNA (single guide RNA)。通过将表达sgRNA的原件与表达Cas9的原件相连接，得到可以同时表达两者的质粒，将其转染细胞，便能够对目的基因进行基因操作，在PAM (5’-NGG) 上游 ~3bp进行切割，如下图所示：
因此本实验的关键步骤是设计一对20bp（不包括NGG在内的）完全互补的oligo插入到如上图所示的filler。另U6启动子需要5’端的G起始转录，因此若设计的oligo第一个碱基非G，需额外增加一个G。

详细操作流程
1. 设计sgRNA
确定靶基因的序列后，可通过在线网站设计(http://tools.genome-engineering.org )，或根据靶基因的CDS序列自行设计，最方便的是在human KO Library sgRNA里选择。无论选择哪种方式，都建议进行一下blast。

附上在线设计网页截图。输入基因的name，填写email address，设计结果稍后会发送到此邮箱，选择序列的类型以及物种。如sequence type选择unique region，一次只能输入23~500bp的基因片段，最好一次只输入一个外显子，避免guide序列跨越内含子，都选择键入后点击提交，关注邮箱或者在线等。

分析中，显示你所键入的基因序列里有36对可选的guide序列。


分析结束，点击Download as genbank查看结果，此时也会收到邮件。

选择分数较高的Guide序列，以Guide#1序列为例，2条单链oligo的序列如下：oligo1：5’- caccGCGTTCAAGTACCAGTTCGTG -3’; oligo2：5’- aaacCACGAACTGGTACTTGAACGc。标粗部分与经BsmBI酶切后的载体互补配对。BsmBI又名BbsI。

※oligoDNA序列的第一个碱基必须是G，如选取的Guide序列的第一个碱基不是G，需自行添加一个额外的G。

2. Oligo退火形成duplex

PCR仪 95℃ 5 min，缓慢降温至室温1h，1：200稀释duplex。

3. 质粒酶切

4. 胶回收
大片段约11kb 回收；小片段约2kb为切下来的filler，不要。

5. 连接

室温连接4-6 h。

6. 转化（Amp+），挑克隆，摇菌，抽提质粒，测序！

7. 转染
24孔板，细胞不要过满，同实验室日常转染操作，可选择性设置三个平行。同时转染空载作为阴性对照。每孔500 ng。

8. puromycin筛选
转染稳定48-72h后加puromycin进行筛选，1~3g/mL，直至不再有细胞数量稳定，不再有细胞因不耐药死去。

9. 单细胞分离
由于篇幅原因，有限稀释法具体细节会在后面的文章单独讲解，请关注科研小助手后续文章。100个细胞铺在一个大盘或96孔板中。剩余的细胞冻存。

10. 获得单克隆细胞株
当肉眼可见类似于菌落的细胞团时，将其从大盘或96孔板消化下来转移至12或24孔板中继续扩大培养。注意不要污染到其它细胞团，不要选取过密的细胞团。

11. 扩大培养，提取细胞全基因组DNA和细胞总蛋白

12. 测序
Guide序列上游100bp左右和下游200bp左右为上下游设计一对引物，以全基因组为模板，以上述引物进行PCR。PCR产物送测序，或将产物用T7E1进行酶切消化检测突变。

13. Western
与阴性对照相比，靶基因应完全敲除

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