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

• Published in: Plant cell reports Vol. 35; no. 7; pp. 1545 - 1554
• Main Authors: Zheng, Xuelian, Yang, Shixin, Zhang, Dengwei, Zhong, Zhaohui, Tang, Xu, Deng, Kejun, Zhou, Jianping, Qi, Yiping, Zhang, Yong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.07.2016; Springer Nature B.V
• Subjects: 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; SSCP; CRISPR/Cas9; SSN; Rice; OsROC5; OsDEP1
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-016-1967-1

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.