CRISPRCas9基因精确靶向修饰技术.pdfVIP

CRISPR /Cas9 基因精确靶向修 饰技术 CRISPR-Cas: Highlight in 2013 CRISPR-Cas: Highlight in 2013 CRISPR 概述 CRISPR-Cas：（Clustered Regularly Interspaced Short Palindromic Repeat ）一种来源于细菌获得性免疫的由RNA指 导Cas蛋白对靶向基因进行修饰的技术。具有引导作用的单个 sgRNA(single-guide RNA)引导Cas9 内切酶对DNA 的定点切割。 DNA双链发生断裂后，其在修复的过程中可实现对基因的定 点插入、缺失、突变或修饰。 CRISPR/Cas 概述 (Andrew R. B et al ,2013) CRISPR 工作原理 ? CRISPR-Cas系统靶向识别的关键序列 PAM （protospacer-adjacent motif ）为 NGG 利用CRISPR获得基因敲除果蝇的几 种工作方法 Plasmid 1 Plasmid 2 Expresses the Cas9 gene Expresses the sgRNA, under the Hsp70 promoter under the pol III promoter injection Blastoderm stage embryos mutant overall (%) : 0.25-1.37 (Gratz et al., 2013) 利用CRISPR获得基因敲除果蝇的几 种工作方法 Cas9 mRNA sgRNA injection Early stage embryos up to 88% of injected flies gave rise to mosaic expression of the yellow gene (Bassett et al., 2013; Yu et al., 2013) 利用CRISPR获得基因敲除果蝇的几 种工作方法 Transgenic fly 1 Transgenic fly 2 Expresses Cas9 in the Expresses the sgRNA in germline under the nanos the ubiquitns under the U6 promoter promoter Cross More than 90% of flies with at least one mutant offspring