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当前位置: 首页 > 文章 > CRISPR/Cas9体外酶切检测猪生长抑素基因定点修饰靶点活性的研究 中国畜牧兽医 2016,43 (1) 31-38
Position: Home > Articles > Study on Detection of Pig SSTGene Site-directed Modification Activity by CRISPR/Cas9 Systemin vitro China Animal Husbandry & Veterinary Medicine 2016,43 (1) 31-38

CRISPR/Cas9体外酶切检测猪生长抑素基因定点修饰靶点活性的研究

作  者：
李晓敏;任红艳;毕延震;刘西梅;郑新民
单  位：
中国农业科学院北京畜牧兽医研究所;湖北省农业科学院畜牧兽医研究所;湖北省农业科学院畜牧兽医研究所动物胚胎工程及分子育种湖北省重点实验室
关键词：
猪;生长抑素基因;Cas9酶;单链导向RNA(sgRNA);体外转录
摘  要：
本试验旨在通过CRISPR/Cas9体外酶切法检测猪生长抑素(SST)基因定点修饰靶点的活性。试验设计5个长20bp的单链导向RNA(sgRNA),即SST-sgRNA-g1、SST-sgRNA-g2、SST-sgRNA-g3、SST-sgRNA-g4和SST-sgRNA-g5。化学合成sgRNA寡核苷酸序列,将寡核苷酸序列连接到可同时表达Cas9和sgRNA的质粒中,挑选正确克隆的质粒作为模板进行体外转录形成SST-sgRNA。利用CRISPR/Cas9体外酶切含靶点的DNA片段,根据酶切条带的灰度换算成sgRNA活性。结果显示目的sgRNA寡核苷酸双链成功插入到质粒中且序列正确,以质粒为模板体外转录SST-sgRNA成功。靶位点经Cas9蛋白酶切后与标准sgRNA1和sgRNA2酶切活性作比较,确定靶点SST-sgRNA-g1、SST-sgRNA-g4和SST-sgRNA-g5活性较高,可为在细胞水平、胚胎水平做基因定点修饰提供依据。
译  名：
Study on Detection of Pig SSTGene Site-directed Modification Activity by CRISPR/Cas9 Systemin vitro
作  者：
LI Xiao-min;REN Hong-yan;BI Yan-zhen;LIU Xi-mei;ZHENG Xin-min;WU Min-yao;College of Life Sciences,Shaanxi Normal University;Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province,Institute of Animal and Veterinary Sciences,Hubei Academy of Agricultural Sciences;
关键词：
pig;;SSTgene;;Cas9 enzyme;;single-guide RNA(sgRNA);;transcription in vitro
摘  要：
This study was aimed to detect the targets activities which were modified in pig SST gene through the CRISPR/Cas9 enzyme digestion method in vitro.Five 20 bp sgRNAs SSTsgRNA-g1,SST-sgRNA-g2,SST-sgRNA-g3,SST-sgRNA-g4 and SST-sgRNA-g5 of SST were designed.Oligonucleotide sequences of sgRNA were chemically synthesized,and then inserted into linearized plasmid which could express Cas9 and sgRNA together.Then,the correct sgRNA plasmids were selected as a template for transcription of SST-sgRNAin vitro.Target DNA fragments were digested by CRISPR/Gas9 in vitro and then the gray bands of enzyme digestion were converted into sgRNA activity.The results indicated that the target nucleotide sequences were successfully inserted into the expected sites of vector and sequences were correct.The vector was successfully transcribed into sgRNAin vitro.It showed that compared with standard sgRNA1 and sgRNA2enzyme activity,the target SST-sgRNA-g1,SST-sgRNA-g4 and SST-sgRNA-g5 hadhigh activities,they could provide basis for the site-directed modification of gene in cell and embryo.

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