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FokI-dCas9

公司名称: Addgene
产品编号: 52970
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A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
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2017-06-05
[Abstract]  The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 ... [摘要]  聚类规则间隔短回文重复(CRISPR)/ CRISPR相关蛋白9(Cas9)系统是使用最广泛的基因组编辑工具。一个常见的CRISPR / Cas9系统由两个组成部分组成:单导RNA(sgRNA)和Cas9。在特定靶序列引入双链断裂(DSB)需要两种成分。该系统的一个缺点是实验室中sgRNA的生产是费力的,因为它需要在体外​​转录反应和sgRNA纯化之间克隆sgRNA序列。通过sgRNA靶向Cas9活性的替代方案是用两种小RNA:CRISPR RNA(crRNA)和反式激活性crRNA(tracrRNA)进行靶向。这两种小RNA可以化学合成,这使得与sgRNA相比,这些RNA的产生不那么困难。 CRISPR / Cas9系统的另一个缺点是已经报告了脱靶效应。然而,已经开发了改进形式的Cas9以最小化离靶效应。例如,仅当两个引导RNA在规定的距离内结合相对的链时,切口酶型Cas9(nCas9)和FokI结构域融合的催化无活性的Cas9(FokI-dCas9; fCas9)才诱导DSB。在本协议中,我们描述了使用结合crRNA,tracrRNA和Cas9修饰形式的CRISPR / Cas9系统来生产突变小鼠的实验系统。该方法不仅有利于制备用于基因组编辑系统的试剂,而且可以降低脱靶效应的风险。

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