绵羊布鲁氏杆菌抗性基因IRF3上游调控区克隆及其基因编辑单克隆细胞株的筛选  

Cloning of Upstream Regulatory Region of Brucella Resistance Gene IRF3 and Screening of Gene Edited Cell Colonies of Sheep(Ovis aries)

作　　者：王媛媛 叶丽君 朱妍妍 陈玉欣 尚菲菲 李亭亭 王海涛 刘秋月 WANG Yuan-Yuan;YE Li-Jun;ZHU Yan-Yan;CHEN Yu-Xin;SHANG Fei-Fei;LI Ting-Ting;WANG Hai-Tao;LIU Qiu-Yue(School of Life Science,Bengbu Medical College,Bengbu 233030,Anhui,China;Innovation Academy for Seed Design,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]蚌埠医学院生命科学学院,蚌埠233030 [2]中国科学院遗传与发育生物学研究所,种子创新研究院,北京100101

出　　处：《农业生物技术学报》2023年第8期1555-1566,共12页Journal of Agricultural Biotechnology

基　　金：中国科学院战略性先导科技专项(A类)(XDA24030205);国家大学生创新创业训练计划项目(202110367057)。

摘　　要：布鲁氏菌病(Bruollosis)是对羊养殖业危害最严重的传染病之一,在山羊(Capra hircus)中,干扰素调节因子3(interferon regulatory factor 3,IRF3)基因调控区的突变对抵抗布鲁氏杆菌(Brucella)的感染起到一定抑制作用。绵羊与山羊的IRF3基因高度同源,可通过基因编辑技术在绵羊(Ovis aries)中引入相同类型的IRF3突变,获得IRF3基因编辑的绵羊单克隆细胞株,并为后续利用体细胞核移植技术制备具有抗布鲁氏菌病的基因修饰绵羊提供供体细胞。本研究利用CRISPR/Cas9及先导编辑(prime editing,PE)技术对绵羊IRF3基因的上游开放阅读框(upstream open reading frame,uORF)进行编辑。首先根据NCBI数据库绵羊的IRF3参考序列设计引物,经过IRF3克隆和测序获得湖羊的IRF3序列;在此基础上设计靶向的小向导RNA(small guide RNA,sgRNA)及先导编辑向导RNA(primer editing sgRNA,pegRNA)序列,并将其构建到表达载体上,结合CRISPR/Cas9或PE表达载体转染湖羊胎儿成纤维细胞株,经单克隆细胞筛选,成功利用CRISPR/Cas9获得2株IRF3-uORF靶点位置精准缺失10 bp的阳性单克隆细胞株。而利用PE技术未获得IRF3-uORF精确编辑的单克隆细胞株。本研究为结合体细胞克隆技术在个体水平上获得抗布鲁氏菌病绵羊育种新材料和探究与IRF3相关抗病的机制提供新思路。Brucellosis is one of the most serious infectious diseases in sheep(Ovis aries)and goats(Capra hircus)husbandry.In goats,mutations located at upstream regulatory region of interferon regulatory factor 3(IRF3)gene have benefit for their resistance to brucellosis infection.IRF3 gene is highly homologous between sheep and goats,and same mutations of IRF3 can be obtained by gene editing in sheep.Aiming to obtain IRF3 edited cell colonies and supply donor cells to generate cloned Brucella resistant sheep by nuclear tranfer.The upstream open reading frame(uORF)region of sheep IRF3 gene was modified by gene editing mediated by CRISPR/Cas9 and prime editing(PE)technology.According to the reference sequence of sheep IRF3 in NCBI database,primers were designed,and the DNA sequence of IRF3 gene in Hu sheep was cloned;Small guide RNA(sgRNA)targeting IRF3-uORF and primer editing sgRNA(pegRNA)sequence were designed based on cloned sequences.All sgRNAs were constructed into the expression vector.Fetal fibroblast cell lines derived from Hu sheep were transfected by CRISPR/Cas9 or PE expression vectors.After monoclonal cell screening,two positive monoclonal cell lines with 10 bp deletion in IRF3-uORF region were successfully obtained by CRISPR/Cas9 but no positive colonies were obtained by PE.This study provide new ideas for combining somatic cloning to obtain new Brucella-resistant sheep germplasm at animal level.

关 键 词：CRISPR/Cas9 先导编辑(PE) 干扰素调节因子3(IRF3) 布鲁氏杆菌 绵羊 

分 类 号：S826[农业科学—畜牧学]