利用CRISPR/Cas9n系统编辑绵羊成纤维细胞生长因子5(FGF5)基因  

Editing the FGF5 gene in the fibroblasts of ovis aries using the CRISPR/Cas9n system

作　　者：蒙亚琦 姚旭东 任秀美奥 郭延华[2] 唐红[2] 张译元[2] 王立民[2] 周平[1,2] MENG Yaqi;YAO Xudong;REN Xiumeiao;GUO Yanhua;TANG Hong;ZHANG Yiyuan;WANG Liming;ZHOU Ping(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;State Key Laboratory of Sheep Genetic Improvement and Healthy Production,Xinjiang Academy of Agricultural and Reclamation Sciences,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]省部共建绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院,新疆石河子832000

出　　处：《畜牧与兽医》2022年第1期8-15,共8页Animal Husbandry & Veterinary Medicine

基　　金：转基因生物新品种培育重大专项(2016ZX08008001);省部共建绵羊遗传改良与健康养殖重点实验室(2013KLS05);国家自然科学基金(31660341)。

摘　　要：成纤维细胞生长因子5(FGF5)是影响毛囊周期性活动及毛发生长的重要生长因子。本研究利用CRISPR/Cas9n系统靶向编辑绵羊成纤维细胞中FGF5基因。首先利用Gibson Assembly法将U6启动子引导表达单导向RNA(sgRNA)的原件构建至pX461质粒中,获得pX461-U6质粒;再将设计并合成好的1对靶向FGF5基因第3外显子的sgRNA及其互补链,经退火连接形成sgRNA-sg1和sgRNA-sg2双链后,分别克隆至带有BbsⅠ和BsaⅠ黏性末端的pX461-U6质粒。构建好的重组质粒pX461-U6-sg1+sg2经测序鉴定后,以电转染的方式转入绵羊成纤维细胞,72 h后收集细胞进行检测分析。经T7E1酶切检测分析表明,成功获得1对具有靶向效果的sgRNA,PCR扩增的FGF5基因片段经TA克隆后进行测序鉴定,结果sgRNA-sg1+sg2在靶位点的打靶效率为100%;缺失的核苷酸数从10到64个不等;基于预测的3D模型和RT-PCR结果显示,FGF5基因的突变将会影响FGF5蛋白与其受体的结合,导致FGF5蛋白失去其生物学功能。本研究构建了可以在同一载体中同时表达2条sgRNA和Cas9n以及绿色荧光蛋白(GFP)的质粒,瞬时转染至绵羊成纤维细胞后,成功获得了高效靶向绵羊FGF5基因的sgRNA位点,为精确和高效的靶向基因工程提供了科学依据。Fibroblast growth factor 5 is an important growth factor that affects the cyclic activity of hair follicles and hair growth.Based on the rapid development of the gene modification technology,gene editing is expected to be an effective tool for the improvement of economic traits in livestock and poultry.In this study,the CRISPR/Cas9n system was used to target FGF5 gene editing in sheep fibroblasts.The pX461-U6 plasmid was obtained by first constructing the U6 promoter-guided single guide RNA into the pX461 plasmid using the Gibson Assembly method.Then,the designed and synthesized pair of sgRNAs targeting exon 3 of the FGF5 gene and their complementary strands were annealed.sgRNA-sg1 and sgRNA-sg2 were ligated to form double-stranded sgRNA-sg1 and sgRNA-sg2,and were then cloned into the pX461-U6 plasmid with the BbsⅠand BsaⅠsticky ends,respectively.The recombinant plasmid pX461-U6-sg1+sg2 was identified by sequencing and was then transfected into sheep fibroblasts by electrotransfection.72 h later,the cells were collected for analysis.The PCR-amplified FGF5 gene fragment was cloned by T-A and sequenced for identification.The sequencing results showed that the targeting efficiency of sgRNA-sg1+sg2 at the target site was 100%;and the number of missing nucleotides ranged from 10 to 64.The prediction-based 3D model and RT-PCR results showed that mutations in the FGF5 gene affected the binding of the FGF5 protein to its receptor,resulting in the loss of the biological function of the FGF5 protein.In this study,two plasmids expressing sgRNA,Cas9n and green fluorescent protein simultaneously in the same vector were constructed and transiently transfected into sheep fibroblasts,and sgRNA loci were successfully obtained for targeting the FGF5 gene in sheep.This provided a scientific basis for precisely and efficiently targeted genetic engineering.

关 键 词：绵羊 成纤维细胞生长因子5 CRISPR/Cas9n 基因敲除 

分 类 号：S826[农业科学—畜牧学]