靶向剪切AAVS1位点CRISPR/Cas9腺病毒系统的构建  

Construction of Adenovirus-Mediated CRISPR/Cas9 System Targeting AAVS1 Locus

作　　者：于声[1] 杨玲凤[1] 姜伯劲[1] 张安文[1] 陈美琳[1] 陈晓丹[1] 段斯亮[1] 

机构地区：[1]广西科技大学医学院,柳州545005

出　　处：《基因组学与应用生物学》2017年第4期1355-1360,共6页Genomics and Applied Biology

基　　金：国家级大学生创新创业训练计划项目;广西大学生创新创业训练计划项目(项目编号:201610594051);广西科技大学校级项目(校科医1307206)共同资助

摘　　要：本研究拟使用腺病毒介导的CRISPR/Cas9系统靶向剪切AAVS1位点,为实现AAVS1位点基因定点插入奠定基础。设计针对AAVS1位点的gRNA序列,连接到pENTRY-U6-h EF1a-Cas9载体,通过Gateway技术重组到腺病毒骨架质粒pAD,转染293A细胞包装腺病毒。腺病毒感染Hela细胞系,使用T7E1酶切及测序检测AAVS1位点的打靶效率。T7EI酶切结果显示腺病毒介导的CRISPR/Cas9系统剪切效率达到28.5%。腺病毒介导的CRISPR/Cas9系统对AAVS1位点成功实施了剪切,为下一步在Hela细胞内进行基因定点敲入及基因治疗奠定了基础。In this study, we intended to use the adenovirus-mediated CRISPR/Cas9 system to cut the AAVS1 locus so as to lay the foundation for the fixed-point insertion of gene in AAVS 1 locus. The gRNA of the AAVS 1 locus was designed, which was connected to the pENTRY-U6-hEFla-Cas9 vector, and then it was connected to recombinant adenovirus vector pAD through the Gateway technology to transfect 293A cell to generate adenovirus. Then, adenovirus infected Hela cell. What＇s more, the TTE1 enzyme digestion and sequencing were used to detect targeting efficiency of AAVS 1 locus. The results of T7E 1 enzyme digestion showed that the shearing efficiency of adenovirus-mediated CRISPR/Cas9 system reached 28.5%. In addition, adenovirus-mediated CRISPR/Cas9 system successfully shear AAVS 1 locus, which laid the foundation for the next gene knock-in and gene therapy in Hela cells.

关 键 词：AAVS1位点 CRISPR-CAS9 基因敲除 腺病毒系统 

分 类 号：Q78[生物学—分子生物学] R450[医药卫生—治疗学]