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。
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.
出处
《中国畜牧兽医》
CAS
北大核心
2016年第1期31-38,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家转基因生物新品种培育重大专项(2014ZX08010-003、2014ZX08006-003)
湖北省农业科技创新中心课题(2011-620-001-003)
湖北省农业科学院青年基金(2014NKYJJ02)
