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Author:
Date:
2014-09-05
[Abstract] RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 nuclease is directed by a short single guide RNA (gRNA or sgRNA) to generate double-strand breaks (DSB) at the specific sites of chromosomal DNA, thereby introducing mutations at the DSB by error-prone non-homologous end joining repairing. Cas9-gRNA recognizes targeted DNA based on complementarity between a gRNA spacer (~ 20 nt long leading sequence of gRNA) and its targeted DNA which precedes a protospacer-adjacent motif (PAM, Figure 1). In this ...
[摘要] 使用细菌II型簇定期间隔的短回文重复序列（CRISPR）相关核酸酶（Cas）的RNA指导的基因组编辑（RGE）已经作为用于在包括植物和作物物种的许多生物体中的基因组编辑的简单和通用工具而出现。在基于化脓性链球菌CRISPR-Cas9系统的RGE中，Cas9核酸酶由短的单引导RNA（gRNA或sgRNA）引导以在染色体的特定位点产生双链断裂（DSB） DNA，从而通过易错的非同源末端连接修复在DSB处引入突变。 Cas9-gRNA基于gRNA间隔区（约20nt的gRNA的前导序列）与其在原间质体相邻基序（PAM，图1）之前的靶DNA之间的互补性识别靶向DNA。在该协议中，我们描述了使用CRISPR-Cas9系统和农杆菌介导的转化的植物RGE的一般程序。该协议包括gRNA设计，Cas9-gRNA质粒构建和水稻RGE的突变检测（基因分型），可适用于其他植物物种。
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