CRISPR/Cas9技术介导猪基因组单碱基编辑效率的研究  

Programmable base editing efficiency study of CRISPR/Cas9-guided DNA base editors in pig genome

作　　者：张婷婷 陈涛 李燕莉 杨漫漫 魏强 王然 李林 李勇 ZHANG Ting-ting;CHEN Tao;LI Yan-li;YANG Man-man;WEI Qiang;WANG Ran;LI Lin;LI Yong(BGI-Shenzhen Sanshengyuan Technology Co.,Ltd.,Shenzhen 518000,Guangdong,China;BGI-Agricultural Application Research Institute,Shenzhen 518000,Guangdong,China;Shenzhen Engineering Laboratory for Genomics-Assisted Animal Breeding,Shenzhen 518000,Guangdong,China)

机构地区：[1]深圳华大三生园科技有限公司,广东深圳518000 [2]深圳市华大农业应用研究院,广东深圳518000 [3]深圳动物基因组辅助育种工程实验室,广东深圳518000

出　　处：《湖北农业科学》2020年第18期143-149,155,共8页Hubei Agricultural Sciences

基　　金：大鹏新区产业发展专项资金项目(KY20160305);深圳市卫计委医疗卫生“三名工程”(SZSM201512003);深圳市基因组辅助育种工程实验室提升项目。

摘　　要：利用国际上最新的两种基于CRISPR/Cas9的BE3(Cytosine base editor,CBE)和ABE7.10(Adenine base editor,ABE)单碱基修饰技术对猪基因组靶基因位点进行编辑效率的分析研究。设计、合成并构建4个猪基因组靶基因位点gRNA表达载体,分别与CBE或ABE共转染PK15细胞,继续培养48 h,结合二代测序技术测定单碱基替换效率和indels发生率。结果表明,单碱基编辑系统BE3和ABE7.10对猪基因组靶位点碱基修饰的活性编辑窗口主要分别为5个核苷酸和4个核苷酸;两套单碱基编辑系统主要对编辑窗口内的目标碱基进行单碱基转换而非indel;两套单碱基编辑系统对猪基因组碱基置换有一定偏好性,其中CMAH、MC1R(1-2)、MC1R(2-1)基因编辑窗口内C→T的效率分别为2.2%、0.4%和1.3%;猪GGTA、MSTN-2窗口内A→G的效率分别为1.4%、1.4%。表明单碱基编辑系统BE3和ABE7.10均能够对猪细胞的基因组靶位点序列进行有效的单碱基置换。The CRISPR/Cas9-based BE3(Cytosine base editor,CBE)and ABE7.10(Adenine base editor,ABE)were used to analyze and study the editing efficiency of target gene loci in pig genome.Four porcine genomic target gene loci gRNA expression vectors were designed,synthesized and constructed,which were co-transfected into PK15 cells with CBE or ABE,respectively.The PK15 cells were further cultured for 48 h,and single-base replacement efficiency and indels incidence were determined by second-generation se⁃quencing technology.The results showed that the active editing Windows of the single base editing system BE3 and ABE7.10 were 5 nucleotides and 4 nucleotides,respectively;Both BE3 and ABE7.10 were mainly contributed to single base conversion but not indel;The two sets of single-base editing systems showed certain preference for base replacement in pig genome.In the CMAH,MC1R(1-2)and MC1R(2-1)gene editing Windows,the efficiency of C→T was 2.2%,0.4%and 1.3%,respectively;The efficiency of A→G in pig GGTA and MSTN-2 was 1.4%and 1.4%,respectively.It is shown that both BE3 and ABE7.10 can perform effective single base substitutions for genomic target sequences in pig cells.

关 键 词：猪 单碱基编辑 CRISPR/Cas9 BE3 ABE7.10 

分 类 号：Q812[生物学—生物工程]