Chinese Journal OF Rice Science >
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
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
Yan-xiu DU, Xin JI, Hui-jie CHEN, Ting PENG, Jing ZHANG, Jun-zhou LI, Hong-zheng SUN, Quan-zhi ZHAO . CRISPR/Cas9 System-based Editing of OsbHLH116 Gene and Its Off-target Effect Analysis[J]. Chinese Journal OF Rice Science, 2016 , 30(6) : 577 -586 . DOI: 10.16819/j.1001-7216.2016.6024
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