CRISPR/Cas技术可有效介导家鸡基因敲除  

CRISPR/Cas Techniques Can Knockout the Gene of Chicken Effectively

作　　者：左其生[1] 王颖洁[1] 赵瑞丰[1] 程少泽 汪怡临 靳锴 王飞[1] 纪艳芹 路镇宇 张文慧[1] 张亚妮[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2016年第6期1266-1271,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31301959;31272429;31472087);高等学校博士学科点专项科研基金资助课题(20123250120009);中国博士后基金(2012M511326;2014T70550);江苏高校优势学科建设工程资助项目;大学生创新创业训练计划项目(201411117033Z)

摘　　要：本研究旨在家鸡上建立一种高效、稳定的基因组定点编辑技术体系,实现目的基因定点敲除,为后续家鸡基因编辑提供操作依据。基于NCBI数据库提供的CDS序列克隆C2EIP(chr2,Expression In PGC)基因全长,并根据基因序列中APM的位置设计特异性gRNA1、gRNA2和gRNA3,并构建cas9/gRNA载体;将设计好的cas9/gRNA转染状态良好的DF-1,利用Luciferase SSA重组检测法、T7E1酶切法以及TA克隆测序法检测gRNA在DF-1细胞中基因的敲除效率。Luciferase SSA重组检测结果表明,只有cas9/gRNA3载体具有基因敲除活性,荧光活性比对照组高两倍,T7E1酶切结果显示cas9/gRNA3基因的敲除活性为27%,TA克隆测序结果表明,30个测序菌液中有8个菌液出现不同数目的碱基缺失或增加,初步估计基因敲除效率为26%。通过本研究在家鸡中初步建立了cas9介导的基因敲除技术,该技术能够稳定的在鸡的细胞DF-1上介导基因敲除。This study aimed to establish a highly efficient and stable gene editing technique mediated by CRISPR/Cas to knock out the targeted gene,and explore its application in chicken preliminarily,and to provide the operation basis for the subsequent poultry genetic edition.We cloned the full-length of C2EIP（chr2,Expression in PGC）gene according to sequence in NCBI Database,and designed 3gRNAs named gRNA1,gRNA2 and gRNA3based on the location of APM in the sequence to construct the cas9/gRNA vector.SSA activity assay,T7E1 digestion method and sequencing of TA cloning were used to detect the knock-out efficiency of the gRNA after the cas9/gRNA vector transfected into DF-1.Results of SSA activity assay showed that only cas9/gRNA3 vector could knock out the gene effectively,fluorescence activity which transfected cas9/gRNA3 was twice higher than the control group;Result of T7E1 digestion showed that activity of cas9/gRNA3 was 27%,sequencing of TA cloning results showed there were 8mutational samples in 30 samples,and the efficiency of gene-knockout was 26%.In this study we established the technology of gene knockout mediated by CRISPR/Cas in chicken,which can be used to konck out genes in DF-1stably.

关 键 词：CRISPR/Cas 基因敲除 鸡 

分 类 号：S831.2[农业科学—畜牧学]