CRISPRCas9系统建立HtrA2敲除细胞株.pdfVIP

网络出版时间：2014-12-01 19:24 网络出版地址：/kcms/doi/10.11844/cjcb.2014.12.0270.html 中国细胞生物学学报 Chinese Journal of Cell Biology 2014, 36(12): DOI: 10.11844/cj cb.2014.12.0270 运用改良的CRISPR/Cas9系统建立HtrA2敲除细胞株 胡 袁 张 婷 姜长安* ( 四川大学生物治疗国家重点实验室/生物治疗协同创新中心, 成都61004 1) 摘要 能识别特定基因组DNA序列的核酸酶是基因编辑的重要工具。在单链RNA 引导下, 来源于一种产脓链球菌的成簇规律间隔短回文重复(clustered regularly interspaced short palindromic repeats, CRISPR)关联蛋白(CRISPR-associated protein 9, Cas9)能够发挥其核酸酶功能对基因组特定 序列进行剪切。这种功能依赖于单链引导RNA(single-guide RNAs, sgRNAs或gRNAs) 中20 nt 的核 心靶向序列。在该研究中, 作者对已有的CRISPR/Cas9 系统载体进行了优化, 简化了gRNA表达载 体的构建。运用优化后的CRISPR/Cas9 系统, 我们敲除了HEK293T细胞中HtrA2基因的第一个外显 子。这一改进大幅度降低了CRISPR/Cas9技术的使用成本。 关键词 CRISPR/Cas9; HtrA2 ; 基因敲除 Generation of HtrA2 Knockout Cell Line with An Improved CRISPR/Cas9 System Hu Yuan, Zhang Ting, Jiang Changan* (State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu 610041, China) Abstract Sequence-specific nucleases are powerful tools for genome editing. The RNA-guided Cas9 nuclease from Streptococcus pyogenes can be effectively targeted to genomic loci and generate DNA breaks. The site-specific DNA cleavages of Cas9 rely on the core 20 nt targeting sequences within its guide RNA (sgRNA or gRNA). In this paper, we describe a modified version of the CRISPR/Cas9 system, which significantly simplifies the construction of gRNA expression vectors. Using this system, we have efficiently generated HtrA2 knockout HEK293T cells by removing the first exon of the gene. This improvement significantly reduces the cost associated with the genome editing