基于CRISPR/Cas9系统的OsbHLH116基因编辑及其脱靶效应分析
收稿日期: 2016-02-19
修回日期: 2016-06-03
网络出版日期: 2016-11-10
基金资助
河南省现代农业技术体系专项经费资助项目(S2012-04-02)
CRISPR/Cas9 System-based Editing of OsbHLH116 Gene and Its Off-target Effect Analysis
Received date: 2016-02-19
Revised date: 2016-06-03
Online published: 2016-11-10
以水稻OsbHLH116 基因为编辑对象,根据基因编码区序列(CDS)在第一外显子区域设计长度为19 bp的sgRNA,化学合成sgRNA的寡核苷酸序列,然后与CRISPR/Cas9系统表达载体pBUN411连接,再用农杆菌介导法获得水稻转基因株系,最后利用酶切和测序相结合的方法对OsbHLH116 突变体进行了筛选鉴定和脱靶效应分析。结果表明,所构建的pBUN411-gRNA载体成功实现了对基因OsbHLH116 的定向编辑。酶切分析表明在选取的10株T0代转基因苗中得到了6个OsbHLH116 突变单株。对6个突变单株进行了TA克隆测序分析,发现了纯合突变、双等位突变和杂合突变3种类型。酶切分析表明2个潜在脱靶位点均未发生脱靶效应。
关键词： 水稻; 基因编辑; CRISPR/Cas9; 脱靶效应; OsbHLH116
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 . DOI: 10.16819/j.1001-7216.2016.6024
With OsbHLH116 as the editable object, the 19-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 pBUN411. The transgenic lines were obtained by Agrobacterium-mediated transformation method. Identification of mutants and off-target effects analysis of OsbHLH116 were conducted by combing restriction enzyme digestion with sequencing. The results showed that the recombinant vector of pBUN411-gRNA succeeded in oriented editing of OsbHLH116. 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.
Key words： rice; gene editing; CRISPR/Cas9; off-target effects; OsbHLH116
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