四种单碱基编辑器在羊成纤维细胞上的编辑效率  

Editing Efficiency of Four Single Base Editors in Sheep(Ovis aries) and Goat(Capra hircus) Fibroblasts

作　　者：孙嘉媛 孙珂欣 丁一格 周世卫 高亚伟 陈玉林[1] 王小龙[1] SUN Jia-Yuan;SUN Ke-Xin;DING Yi-Ge;ZHOU Shi-Wei;GAO Ya-Wei;CHEN Yu-Lin;WANG Xiao-Long(College of Animal Science and Technology/Shaanxi Key Laboratory of Animal Genetic Breeding and Reproduction,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学,动物科技学院/陕西省动物遗传育种与繁殖重点实验室,杨凌712100

出　　处：《农业生物技术学报》2021年第1期169-177,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972526;31772571);陕西省科技新星项目(2018KJXX-009);宁夏自治区滩羊育种专项(2019NXTS-001)。

摘　　要：单碱基编辑器作为一种新型的CRISPR衍生工具,具有精准、高效及低脱靶等优点,现已应用于多种生物体中。单碱基编辑器经过不断改造目前已开发出多个版本。为探究不同版本碱基编辑器在羊成纤维细胞上的编辑效率,本研究选定滩羊(Ovis aries)的绵羊多胎基因(fecundity booroola, FecB)和陕北白绒山羊(Capra hircus)的成纤维细胞生长因子5 (fibroblast growth factor 5, FGF5)基因,利用xCas9-ABE(腺嘌呤碱基编辑器, adenine base editor)、ABEmax4、xCas9-BE4和BE4max 4种新型单碱基编辑器,在滩羊和陕北白绒山羊胎儿成纤维细胞上进行单碱基定点编辑。结果显示,在绵羊细胞中,ABEmax编辑效率为46.15%,xCas9-ABE的编辑效率为38.46%,比普通编辑器ABE7.10分别高出27.4%与19.71%;在山羊细胞上,BE4max的编辑效率为92.86%,比普通编辑器BE3高56.5%,但xCas9-BE4未产生编辑。综上所述,本研究在羊胎儿成纤维细胞的碱基编辑应用中筛选出最优碱基编辑器,证明了高效定点编辑羊基因组的可行性,同时也为碱基编辑器应用于大型哺乳动物基因编辑提供技术支撑。As a new type of CRISPR-derived tool, the base editors have the advantages of accuracy,efficiency, and low off-target, and have been used in many organisms. Single base editors are mainly divided into 2 types-adenine base editor(ABEs) and cytosine base editor(CBE), which can realize single base mutation of A: G and C: T, respectively. The base editor has been continuously modified and has been developed with multiple versions. The Booroola fecundity(FecB) gene is the major gene of sheep(Ovis aries)prolificacy. FecB gene is derived from A746 G site mutation of bone morphogenetic protein receptor 1 B(BMPR1 B) of Booroola sheep, resulting in 249 amino acids encoded being converted from glutamine(Q) to arginine(R), further increasing ovulation and lambing. The fibroblast growth factor 5(FGF5) gene can regulate the hair follicle cycle, and C-T base mutation on FGF5 gene can significantly affect the length of cashmere fiber, which is crucial for improving cashmere yield of cashmere goat(Capra hircus). In order to explore the editing efficiency of different base editors in sheep and goat, the Booroola fecundity(FecB) of Tan sheep and the fibroblast growth factor 5(FGF5) of Shaanbei white cashmere goat were selected. By constructing sgRNA-U6 vector, cell culture, transfection, drug screening, Four single base editors, xCas9-ABE(adenine base editor), ABEmax4, xCas9-BE4 and BE4 max, were used to perform single base fixed-point editing on Tan sheep and Shaanbei white cashmere goat fetal fibroblasts. The editing efficiency of xCas9-ABE, ABEmax4, xCas9-BE4 and BE4 max in Tan sheep and Shaanbei white cashmere goat were determined.The results showed that in sheep fibroblasts, the editing efficiency of ABEmax4 was 46.15%, and the editing efficiency of xCas9-ABE was 38.46%, which was 27.4% and 19.71% higher than that of ordinary editor ABE7.10. On goat fibroblasts, the editing efficiency of BE4 max It was 92.86%, which was 56.5% higher than the ordinary editor BE3, but no editing is generated by xCas9-BE4. In summary, the base ed

关 键 词：碱基编辑器 胎儿成纤维细胞 多胎基因(FecB) 成纤维细胞生长因子5基因(FGF5) 绵羊 山羊 

分 类 号：S813.3[农业科学—畜牧学]