利用RGR核酶结构拓展家蚕CRISPR基因组编辑系统的应用研究  

Expanding the Application of CRISPR Genome Editing System in Bombyx mori Using RGR Ribozyme Structure

作　　者：张启超 宋红生[1] 曾保胜 陈树清[2] 许军[2] 李芝倩 张忠杰[2] 陈旭 Zhang Qichao;Song Hongsheng;Zeng Baosheng;Chen Shuqing;Xu Jun;Li Zhiqian;Zhang Zhongjie;Chen Xu(College of Life Sciences, Shanghai University, Shanghai 201900, China;Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China)

机构地区：[1]上海大学生命科学学院,上海201900 [2]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《蚕业科学》2018年第2期226-232,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金国际合作项目(No.31420103918);国家自然科学基金重点项目(No.31530072)

摘　　要：针对目前家蚕基因组编辑平台在基因组特定位点的靶点选择、多靶点同时编辑等方面的技术瓶颈,利用家蚕卵巢培养细胞系（Bm N）验证含有双核酶结构的RGR（Ribozyme-gRNA-Ribozyme）在CRISPR/Cas9基因组编辑系统中的应用效果,用以拓展现有CRISPR/Cas9系统的靶点范围。研究结果显示,采用Cas9系统和Cpf1系统,RGR结构在Bm N细胞中具有良好的定点编辑效果,可成功实现对内源、外源靶基因序列的剪切,且对内外源基因具有高效编辑作用,表明RGR结构可应用于家蚕CRISPR/Cas9和CRISPR/Cpf1基因组编辑系统。利用RGR结构的自剪切功能不仅可以拓展家蚕基因组靶基因的范围,还为进一步实现多靶点编辑打下了基础。In the present study,we used BmN cell line to verify the effect of using RGR（ Ribozyme-gRNA-Ribozyme）structure on improving CRISPR/Cas9 based genome editing efficiency so as to break the technical bottlenecks such as specific genomic target selection and simultaneous editing at multiple targets in silkworm（ Bombyx mori）,and to explore the possibility of applying RGR structure in a new genome editing technology CRISPR/Cpf1 for further expanding the targets of current CRISPR system in silkworm. The results showed that the gRNA-guided Cas9 or Cpf1 released from the expression vector could successfully disrupt target exogenous gene or endogenous gene in BmN cell,demonstrating that RGR structure can be used in CRISPR/Cas9 and CRISPR/Cpf1 systems for silkworm genome editing effectively. The selfsplicing function of RGR structure can not only expand the range of target genes in silkworm genome,but also lay a foundation for further application of multiple targets editing.

关 键 词：CRISPR/Cas9系统 CRISPR/Cpf1系统 RGR核酶结构 家蚕基因组编辑 家蚕卵巢培养细胞 

分 类 号：S881.26[农业科学—特种经济动物饲养] Q789[农业科学—畜牧兽医]