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当前位置: 首页 > 文章 > CRISPR/Cas技术可有效介导家鸡基因敲除 畜牧兽医学报 2016,47 (6) 1266-1271
Position: Home > Articles > CRISPR/Cas Techniques Can Knockout the Gene of Chicken Effectively Chinese Journal of Animal and Veterinary Sciences 2016,47 (6) 1266-1271

CRISPR/Cas技术可有效介导家鸡基因敲除

作  者：
左其生;王颖洁;赵瑞丰;程少泽;汪怡临;靳锴;王飞;纪艳芹;路镇宇;张文慧;张亚妮;李碧春
单  位：
扬州大学动物科学与技术学院
关键词：
CRISPR/Cas;基因敲除;鸡
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。
译  名：
CRISPR/Cas Techniques Can Knockout the Gene of Chicken Effectively
作  者：
ZUO Qi-sheng;WANG Ying-jie;ZHAO Rui-feng;CHENG Shao-ze;WANG Yi-lin;JIN Kai;WANG Fei;JI Yan-qin;LU Zhen-yu;ZHANG Wen-hui;ZHANG Ya-ni;LI Bi-chun;College of Animal Science and Technology,Yangzhou University;
关键词：
CRISPR/Cas;;gene knockout;;chicken
摘  要：
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.

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