CRISPR/Cas9基因编辑系统中两种gRNA活性检测方法比较  

The comparison of two methods for detection of gRNA activity in CRISPR/Cas9 system

作　　者：刘燕[1] 任卫红[1] 王绿娅[1] 张晓萍[1] 高诗娟[1] 武威[1] 李凤娟[1] 杜杰[1] 

机构地区：[1]北京市心肺血管疾病研究所-首都医科大学附属北京安贞医院心血管重塑相关疾病教育部重点实验室科研基地建设-心血管重大疾病协同创新中心,100029

出　　处：《心肺血管病杂志》2016年第7期563-568,共6页Journal of Cardiovascular and Pulmonary Diseases

基　　金：国家自然科学基金(81300120);北京市自然科学基金(7142030);北京市科技新星计划(Z151100000315067)

摘　　要：目的:比较CRISPR/Cas9系统中gRNA活性检测的两种方法,即利用Surveyor/T7E1核酸酶检测方法或利用Cas9蛋白的体外检测方法。方法:一方面构建表达Cas9和特异gRNA的质粒,转染NIH3T3细胞,然后提取DNA,利用T7E1核酸内切酶检测gRNA介导Cas9切割靶DNA并产生indel突变的效率;另一方面,设计引物并利用PCR扩增出gRNA的DNA模板片段,通过体外转录获得gRNA,利用Cas9蛋白及gRNA进行体外反应检测切割效率。结果:利用Cas9蛋白体外酶切可以检测到较高的gRNA活性,然而通过T7E1核酸酶方法检测gRNA在细胞中活性整体偏低,且在不同基因之间差异较大。结论:细胞Surveyor/T7E1法与Cas9蛋白体外酶切法检测到的gRNA活性不完全一致,体外活性检测法不能替代。Objective： The aim of this study was to compare two methods （Surveyor/T7E1 assay or in vitro assay by Cas9 protein） for detecting the activity of guide RNA （gRNA） on mediating cut of double-strand DNA by Cas9 enzyme. Methods： 20bp gRNAs of 4 genes were designed. For Surveyor/T7E1 assay, oligonucleotide sequences of gRNAs were chemically synthesized, and were inserted into plasmid which could express both of Cas9 and gRNA. Plasmid for each gRNA was transfected into NIH3T3 cells, and the ratio of indel mutation mediated by gRNA and Cas9 was determined by T7E1 endonuclease. For in vitro assay of gRNA activity by Cas9 digestion, the DNA templates for gRNA transcription were amplified by PCR technology synthesized primers containing gRNA core fragment, and then the gRNAs were transcribed in vitro. The efficiency of gRNA together with Cas9 was detected in vitro by digestion of target DNA fragments. Results： Most gRNAs showed high activity in the in vitro assay. However, some gRNAs exhibited much lower activity in T7E1 assay than in vitro assay. Conclusion： The activity of gRNA in the in vitro assay was not similar as Surveyor/T7E1 assay in cells, which indicated the in vitro assay of gRNA activity could not instead of Surveyor/T7E1 assay.

关 键 词：CRISPR/Cas9 gRNA活性 Surveyor/T7E1检测 体外检测 

分 类 号：R54[医药卫生—心血管疾病]