利用CRISPR/Cas9技术制备B2M^–细胞模型

Preparation of B2M~– Cell By CRISPR/Cas9 Technology

Ⅰ型主要组织相容性复合物(major histocompatibility complex class I,MHC I)是造成免疫排斥的重要分子,该研究利用CRISPR/Cas9(clustered regularly interspersed short palindromic repeats)技术靶向其β2微球蛋白(beta-2 microglobulin,B2M)亚基实现MHC I的敲除,并在分子和细胞水平进行验证。设计针对B2M基因的g RNA(guide RNA)并构建对应的CRISPR/Cas9基因敲除质粒,通过电穿孔转染技术将同时带有Cas9蛋白与g RNA骨架的质粒瞬时转入HEK293细胞中进行蛋白表达与基因组核酸切割,流式分选得到HEK293 B2M阴性细胞,提取其基因组并对g RNA靶向区域与脱靶区域进行聚合酶链式反应(polymerase chain reaction,PCR),将PCR产物连接T载体测序检测基因打靶与脱靶情况。同时,利用反转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)与实时定量荧光聚合酶链式反应(Real-time polymerase chain reaction,Real-time PCR)测定B2M基因在RNA水平的表达。综上所述,该研究发现了一种利用CRISPR/Cas9技术快速便捷、低成本的可用于敲除B2M基因的方法,经检测可使目的细胞中B2M基因发生移码、插入、缺失、单碱基突变,且未发现脱靶现象,这在未来解决免疫排斥相关难题上意义重大。

The major histocompatibility complex I（MHC I） is an extremely important molecule that causes immune rejection. In this study, they used CRISPR/Cas9 technology to target its B2 M（beta-2 microglobulin） subunit to achieve MHC I knockout, and verify the deletion efficiency on molecular and cellular level. The guide RNA（g RNA） of B2 M gene was designed and constructed to the CRISPR/Cas9 knockout plasmid containing both Cas9 protein and g RNA. Then the plasmid was transfected into HEK293 cells by electroporation systems. After that, HEK293-B2 M– cells were sorted by fluorescence activated cell sorting, followed by extracting genomic DNA from B2 M knockout HEK293 cells. The polymerase chain reaction（PCR） was performed for amplifying either the g RNA targeted or potential off-target regions of genome. The PCR product was inserted into the T vector and analyzed of targeted and off-target efficiency using genetic sequencing. At the same time, the expression of B2 Mgene in RNA level was detected by reverse transcription-polymerase chain reaction（RT-PCR） and Real-time polymerase chain reaction（Real-time PCR）. In conclusion, this study developed a rapid and convenient methodfor knocking out B2 M gene using CRISPR/Cas9 technology. After detecting, it demonstrated to successfully introducing frameshift, insertion, deletion and single base mutations in B2 M gene, and off-target phenomenon was not found. It could be a great application in solving the problem of immune rejection in the future.