Article

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

Zheng, X; Yang, S; Zhang, D; Zhong, Z; Tang, X; Deng, K; Zhou, J; Qi, Y; Zhang, Y

Plant cell reports 2016 7

PMID: 27007717

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

KEY MESSAGE: A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

Keyword Gene Uniprot Accession
OsDEP1 Os09g0441900 Q67UU9 A0A0P0XNX6 A0A0P0XNP8 A0A0P0XNX1