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T7 Endonuclease I

T7核酸内切酶I

Company: New England Biolabs
Catalog#: M0302S
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Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9
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Date:
2017-07-05
[Abstract]  The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species. [摘要]  CRISPR / Cas9系统已成为植物及其以外基因编辑的强大工具。 我们已经开发了植物载体系统,用于拟南芥中多达两个不同靶位点的基因的目标Cas9依赖性诱变。 该方案描述了对于含有Cas9和sgRNA的基因的单个T-DNA载体的简单的1周克隆程序,以及植物中诱导突变的检测。 该方法可能适用于其他可转化植物物种。
【背景】CRISPR / Cas9系统(Cas9)提供了一种简单且广泛适用的方法来修改感兴趣的基因组区域,因此成为植物和其他生物体基因组编辑的首选工具(Schiml和Puchta,2016)。该系统依赖于可以通过短的人造单指导RNA分子(sgRNA)向基因组DNA序列引导的化脓性链球菌(Cas9)的细菌Cas9核酸酶(Jinek等人, ,2012),在那里它创建一个双链断裂(DSB)。然后通过植物细胞的固有DNA修复机制修复这些DSB。在这里,可以区分两个主要途径(Salomon和Puchta,1998)。 (i)与DSB位点高度同源的DNA分子可用作修复模板。可以利用这种同源性定向修复(HDR)方法在DSB的现场引入特定的序列(Schiml等人,2014; Baltes and Voytas,2015)。然而,由于这些序列的低融合率,植物中HDR介导的基因编辑仍然具有挑战性。 ...

Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System
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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