Chinese Journal OF Rice Science >
Knock-out Efficiency Analysis of Pi21 Gene Using CRISPR/Cas9 in Rice
Received date: 2016-01-18
Revised date: 2016-03-03
Online published: 2016-09-10
A CRISPR/Cas9 vector, which contains two targets (Target 1 and Target 2) of Pi21gene, was constructed and transformed to rice variety Nanjing 9108. Twenty-eight T0 transgenic lines were obtained and confirmed by T-DNA specific PCR. Restriction digestion analysis revealed 78.57% mutant ratio in Target 1 site, 92.86% mutant ratio in Target 2 site, 78.57% ratio of lines containing both Target 1 and Target 2 mutant in transgenic lines. Through sequencing analysis, we found the mutant types of targets, such as deletion of bases, insertion of bases, insertion behinds deletion of bases, and long fragment deletion of DNA, etc. Moreover, except amino acid deletion, most transgenic lines displayed frameshift mutation,which caused function loss of Pi21. In this way, a series of Pi21-knock-out rice lines were obtained, and further, could be used for functional analysis of Pi21 and rice blast broad-spectrum resistant breeding.
Key words： CRISPR/Cas9; rice blast; Pi21; gene knock-out
Fang-quan WANG, Fang-jun FAN, Wen-qi LI, Jin-yan ZHU, Jun WANG, Wei-gong ZHONG, Jie YANG . Knock-out Efficiency Analysis of Pi21 Gene Using CRISPR/Cas9 in Rice[J]. Chinese Journal OF Rice Science, 2016 , 30(5) : 469 -478 . DOI: 10.16819/j.1001-7216.2016.6009
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