期刊
PLANT CELL REPORTS
卷 35, 期 7, 页码 1545-1554出版社
SPRINGER
DOI: 10.1007/s00299-016-1967-1
关键词
SSN; CRISPR/Cas9; SSCP; Rice; OsROC5; OsDEP1
资金
- National Science Foundation of China [31330017, 31271420, 31371682]
- national Transgenic Major Project [2014ZX0801003B-002]
- Fundamental Research Funds for the Central Universities [ZYGX2013J099]
- East Carolina University
- North Carolina Biotechnology Center [2016-CFG-8003]
- Syngenta
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.
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