Versatile Genome Editing Using Type I-E CRISPR-Cas3 in Rice

Hiroaki Saika, Naho Hara, Shuhei Yasumoto, Toshiya Muranaka, Kazuto Yoshimi, Tomoji Mashimo, Seiichi Toki

Plant & Cell Biology; 28 October 2025; pcaf138, https://doi.org/10.1093/pcp/pcaf138

Abstract

Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because 7 CRISPR-Cas3 components (6 Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39–71% of transformed calli by PCR analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the PAM sequence was estimated as 21–61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1–7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice; base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells.

Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.

See https://academic.oup.com/pcp/advance-article/doi/10.1093/pcp/pcaf138/8305110?login=false