植物CRISPR/Cas9系统载体基因敲除/基因组DNA编辑技术

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CRISPR-Cas系统是继锌指核酸酶(ZFNs)和TALEN核酸酶之后的另一个可精确定点编辑基因组DNA的新技术，具有设计构建简单快速等优点。已在人类细胞系、斑马鱼、小鼠、果蝇和酵母等多个物种中利用。最近，国内外研究人员纷纷利用CRISPR-Cas系统定点突变了水稻、小麦、拟南芥等多种作物的特定基因，都证实CRISPR-Cas系统能够用于植物的基因组编辑。与ZFN 和TALEN 的设计复杂和各种实验室试验相比，crispr或许相对简单点。crispr／Cas系统允许个性化的小的非编码RNA来介导靶标基因组DNA的切割，从而使基因能够通过非同源末端结合和同源接到的修复机制来修饰基因。图1是利用CRISPR系统敲除植物特定基因的流程简图。

图1

CRISPR/Cas由20个碱基的guide RNA 通过Waston-Crick配对来识别DNA靶位点序列，从而介导基因的编辑。但是20个碱基guide RNA也可能与高度同源的DNA序列结合，从而造成脱靶的问题，这种潜在问题会限制该技术在实际农业生产上的应用。但中科院等研究团队在转基因植株中细致的分析了靶位点的高度同源位点，并未发现他们跟靶位点一样发生突变。同时，经过高通量测序，在整个基因组水平上也未发现有脱靶问题。据此，他们认为在植物系统中CRISPR/Cas并不像鸟枪或者集束炸弹那样会有脱靶问题，而是像巡航导弹一般准确命中目标，同时避免了伤害无辜。

植物CRISPR专家系统服务于广大农业科研人员，本系统基于农杆菌转化系统，含一个表达Cas9的二元穿梭载体和一个表达sgRNA的中间载体，二元穿梭载体质粒架构基于植物表达载体pCambia 1303，sgRNA表达质粒启动子为水稻U3 promoter，Cas9基因为水稻密码子优化且带两个NLS。

根癌农杆菌是一种能诱发植物产生肿瘤的细菌，根癌农杆菌中诱导植物产生肿瘤的质粒（Tumor inducing plasmid），简称为 Ti 质粒。野生型农杆菌的 Ti 质粒，含有两个与致瘤有关的区域：一个是 T－DNA 区（transferred DNA region），含致瘤基因；另一个是毒性区（Virulence region），在 T－DNA 的切割、转移与整合过程中起作用。用于植物基因转化的农杆菌 Ti 质粒载体系统的构建，是将野生 Ti质粒中的致瘤基因删除，并在 T－DNA 区域内插入适当的选择标记和多克隆位点。图2、3是sgRNA表达质粒载体图；图4是植物表达载体 pPL-rCas9的 DNA 图谱，以 CaMV35S 启动子驱动的水稻密码子优化的Cas9基因以潮霉素抗性为选择标记基因，以β－葡萄糖苷酶（β－glucuronidase,Gus）为报告基因，经此质粒转化的获得的转基因细胞、组织或植株，具有抗卡那霉素的特性，经组织化学染色呈蓝色。当构建好sgRNA质粒后只要用Kpn I单酶切后一步连接入pPL-rCas9质粒即可用于下游的植物CRISPR基因敲除工程操作。

1、构建sgRNA表达载体

图2

2、Kpn I单酶切线性化pPL-sgRNA载体

图3

3、插入到经Kpn I 单酶切线性化的pPL-rCas9的载体，得到靶向特定基因的CRISPR基因敲除质粒。

图4

4、转化农杆菌及其下游基因工程筛选操作

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