应用SSA报告载体提高ZFN和CRISPR/Cas9对猪IGF2基因的打靶效率  

Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system

作　　者：吴金青[1] 梅瑰[2] 刘志国[1] 陈瑶生[1] 丛佩清[1] 何祖勇[1] 

机构地区：[1]中山大学生命科学学院,有害生物控制与资源利用国家重点实验室,广州510006 [2]广东省农业科学院动物科学研究所,畜禽育种国家重点实验室,广州510640

出　　处：《遗传》2015年第1期55-62,共8页Hereditas（Beijing)

基　　金：国家转基因重大专项(编号:2013ZX08006005-005);国家重点基础研究发展计划(973计划)项目(编号:2010CB945404)资助

摘　　要：IGF2(Insulin-like growth factor 2)基因作为最复杂多样的生长因子之一,对猪胎儿发育以及出生后生长发育和肌肉生成起着非常重要的作用。通过基因组编辑技术对我国本地猪种的IGF2基因作精确的遗传修饰,对于提高本地猪种的瘦肉率具有重要的育种意义。文章在蓝塘猪胎儿成纤维细胞(Porcine fetal fibroblasts,PEF)中检测了锌指核酸酶(Zinc finger nucleases,ZFN)和CRISPR/Cas9对IGF2基因的打靶效率,结果表明CRISPR/Cas9对IGF2基因的切割效率最高可达9.2%,显著高于ZFN的切割效率(<1%),但两者均未达到作为体细胞核移植(Somatic nuclear transfer,SCNT)供体细胞所需的打靶效率。应用SSA(Single-strand annealing)报告载体筛选技术来富集IGF2基因被ZFN和CRISPR/Cas9修饰过的PEF细胞,结果表明,该技术可使CRISPR/Cas9的打靶效率提高5倍左右,对ZFN的打靶效率具有更大的增强作用。IGF2 （Insulin-like growth factor 2） is a major growth factor affecting porcine fetal and postnatal de-velopment. We propose that the precise modification of IGF2 gene of Chinese indigenous pig breed——Lantang pig by genome editing technology could reduce its backfat thickness, and increase its lean meat content. Here, we tested the genome editing activities of zinc finger nucleases （ZFNs） and CRISPR/Cas9 system on IGF2 gene in the Lantang porcine fetal fibroblasts （PEF）. The results indicated that CRISPR/Cas9 presented cutting efficiency up to 9.2%, which was significantly higher than that generated by ZFNs with DNA cutting efficiency lower than 1%. However, even by using CRISPR/Cas9, the relatively lower percentage of genetically modified cells in the transfected popula-tion was not satisfied for somatic nuclear transfer （SCNT）. Therefore, we used a SSA （Single-strand annealing） re-porter system to enrich genetically modified cells induced by ZFN or CRISPR/Cas9. T7 endonuclease I assay re-vealed that this strategy improved genome editing activity of CRISPR/Cas9 by 5 folds, and was even more effective for improving genome editing efficiency of ZFN.

关 键 词：IGF2基因 CRISPR/Cas9 SSA报告系统 CRISPR/Cas9 

分 类 号：S828[农业科学—畜牧学]