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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The pBlueScript-derived constructs were kind gifts from Satoshi Ota and Atsuo Kawahara at RIKEN Institute in Japan. This work is supported by Grants including the National Science Foundation of China (31330017, 31271420 and 31371682), the national Transgenic Major Project (2014ZX0801003B-002) and the Fundamental Research Funds for the Central Universities (ZYGX2013J099) to YZ, and startup funds from East Carolina University and a Collaborative Funding Grant (2016-CFG-8003) from North Carolina Biotechnology Center and Syngenta to YQ.
Conflict of interest
The authors declare that they have no conflict interests.
X. Zheng, S. Yang and D. Zhang contributed equally to this work.
Communicated by T. Cardi.
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Supplementary material 2 (PPTX 71 kb) PAGE-based detection of heteroduplex DNA of 1-bp and 2-bp deletions. This method is not as sensitive as SSCP because it failed to detect the 1-bp deletion in this case
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Zheng, X., Yang, S., Zhang, D. et al. Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism. Plant Cell Rep 35, 1545–1554 (2016). https://doi.org/10.1007/s00299-016-1967-1