中国水稻科学2016，Vol.30Issue(6)：577-586,10.DOI:10.16819/j.1001-7216.2016.6024
基于 CRISPR/Cas9系统的 OsbHLH116基因编辑及其脱靶效应分析
CRISPR/Cas9 System-based Editing of OsbHLH116 Gene and Its Off-target Effect Analysis
摘要
Abstract
With OsbHLH1 1 6 as the editable object,the 1 9-bp sgRNA was designed in the exon 1 site according to the coding sequence (CDS)and the oligonucleotides of sgRNA were chemically synthesized and inserted into linearized plasmid pBUN41 1 . The transgenic lines were obtained by Agrobacterium-mediated transformation method. Identification of mutants and off-target effects analysis of OsbHLH1 1 6 were conducted by combing restriction enzyme digestion with sequencing.The results showed that the recombinant vector of pBUN41 1-gRNA succeeded in oriented editing of OsbHLH1 1 6 .The restriction enzyme analysis results indicated that we got 6 mutants from 10 randomly selected transgenic lines.Sequence analysis of the six mutants indicated that homozygous mutation,biallelic mutation and heterozygous mutation occured.After searching the rice genome using the target sequence with PAM,two highly identical sites were found.However,we did not observe any mutation at these sites by restriction enzyme.关键词
水稻/基因编辑/CRISPR/Cas9/脱靶效应/OsbHLH1 16Key words
rice/gene editing/CRISPR/Cas9/off-target effects/OsbHLH1 1 6分类
生物科学引用本文复制引用
杜彦修,季新,陈会杰,彭廷,张静,李俊周,孙红正,赵全志..基于 CRISPR/Cas9系统的 OsbHLH116基因编辑及其脱靶效应分析[J].中国水稻科学,2016,30(6):577-586,10.基金项目
河南省现代农业技术体系专项经费资助项目(S2012-04-02) （S2012-04-02）
