Genome editing in rice using CRISPR/Cas12i3

Ping Lv, Fei Su, Fangyuan Chen, Chunxue Yan, Dandan Xia, Hui Sun, Shanshan Li, Zhiqiang Duan, Changle Ma, Hui Zhang, Mugui Wang, Xiaomu Niu, Jian-Kang Zhu, Jinshan Zhang

Planr Biotechnology Journal; First published: 11 October 2023

Summary

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

See https://onlinelibrary.wiley.com/doi/full/10.1111/pbi.14192

Figure 1: Genome editing in rice using CRISPR/Cas12i3. (a) Diagrams of the genome editing construct, and crRNAs and pre-crRNA arrays. Target sequences of the OsYSA, OsNAL, OsMIR396e and OsPYL6 genes are underlined, and PAM sequences are highlighted in red. (b) Percentages of T0 plants with mutations in the target sequences. (c) Examples of OsYSA gene mutations generated by CRISPR/Cas12i3. (d) Statistics of homozygous, bi-allelic, heterozygous, or chimeric mutations in the OsYSA, OsNAL, OsMIR396e, and OsPYL6 target genes. (e) Types and frequencies of T0 generation mutations in the OsYSA, OsNAL, OsMIR396e, and OsPYL6 target genes. d: deletion; i: insertion; s: substitution. (f) Representative Sanger sequencing chromatograms of OsYSA, OsNAL, OsMIR396e and OsPYL6 gene mutations. Homo: homozygous; Het: heterozygous.