Deletion of a target gene in Indica rice via CRISPR/Cas9
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Using CRISPR/Cas9, we successfully deleted large fragments of the yield-related gene DENSE AND ERECT PANICLE1 in Indica rice at relatively high frequency and generated gain-of-function dep1 mutants.
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a rapidly developing technology used to produce gene-specific modifications in both mammalian and plant systems. Most CRISPR-induced modifications in plants reported to date have been small insertions or deletions. Few large target gene deletions have thus far been reported, especially for Indica rice. In this study, we designed multiple CRISPR sgRNAs and successfully deleted DNA fragments in the gene DENSE AND ERECT PANICLE1 (DEP1) in the elite Indica rice line IR58025B. We achieved deletion frequencies of up to 21% for a 430 bp target and 9% for a 10 kb target among T0 events. Constructs with four sgRNAs did not generate higher full-length deletion frequencies than constructs with two sgRNAs. The multiple mutagenesis frequency reached 93% for four targets, and the homozygous mutation frequency reached 21% at the T0 stage. Important yield-related trait characteristics, such as dense and erect panicles and reduced plant height, were observed in dep1 homozygous T0 mutant plants produced by CRISPR/Cas9. Therefore, we successfully obtained deletions in DEP1 in the Indica background using the CRISPR/Cas9 editing tool at relatively high frequency.
KeywordsCRISPR/Cas9 sgRNA Target gene deletion Indica rice DEP1
We are indebted to our colleagues in the Transformation and Analysis Group who contributed to this study. We would especially like to acknowledge our colleagues Qiudeng Que and Zhongying Chen for their advice. Many thanks to Michael Nuccio, Qiudeng Que, and Zhongying Chen for reviewing our manuscript. We sincerely thank the BTP platform group and Liang Shi for their support. Funding was provided by Biotechnology platform project of syngenta biotechnology Co. Ltd (Grant No: PI0008619).
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Conflict of interest
The authors declare that they have no conflict of interest.