Journal Article

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

Tang, Xu, Yang, Shixin, Zhong, Zhaohui, Qi, Yiping, Zhang, Dengwei, Deng, Kejun, Zhou, Jianping, Zheng, Xuelian, Zhang, Yong

2016

Overview

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.

• Publisher: Springer Berlin Heidelberg,Springer Nature B.V
• Subject: Base Sequence / Biomedical and Life Sciences / Biotechnology / callus / Cell Biology / Cereal crops / CRISPR-Cas Systems / DNA / Gene Frequency / genome / Genome editing / Genotype / genotyping / Genotyping Techniques - methods / homozygosity / INDEL Mutation / Life Sciences / Models, Genetic / Mutagenesis, Site-Directed - methods / Mutants / Mutation / Original Article / Oryza - genetics / Plant Biochemistry / Plant Proteins - genetics / Plant Sciences / Plant species / Polymorphism, Single-Stranded Conformational / rapid methods / Reproducibility of Results / rice / screening / Seedlings / single-stranded conformational polymorphism / site-directed mutagenesis