title: Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.
abstract: 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.
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
authors.forename: Xuelian
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
authors.forename: Shixin
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
authors.forename: Dengwei
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Zhong
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
authors.forename: Xu
authors.initials: X
authors.lastname: Tang
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
authors.forename: Kejun
authors.initials: K
authors.lastname: Deng
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Biology, East Carolina University, Greenville, NC, 27858, USA. qiy@ecu.edu.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu, 610054, People's Republic of China. zhangyong916@uestc.edu.cn.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D005787:Gene Frequency; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods; D054643:INDEL Mutation; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed / Q000379:methods; D012275:Oryza / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results
chemical_list: D010940:Plant Proteins
keywords: CRISPR/Cas9; OsDEP1; OsROC5; Rice; SSCP; SSN