Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy

Yiqiu Cheng; Lei Zhang; Jing Li; Xiaofei Dang; Jian-Kang Zhu; Hiroaki Shimada; Daisuke Miki

doi:10.3389/fpls.2024.1360925

Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy

Front Plant Sci. 2024 Mar 13:15:1360925. doi: 10.3389/fpls.2024.1360925. eCollection 2024.

Authors

Yiqiu Cheng^{1

2}, Lei Zhang^{1

2}, Jing Li^{1

2}, Xiaofei Dang¹, Jian-Kang Zhu^{3

4}, Hiroaki Shimada⁵, Daisuke Miki¹

Affiliations

¹ Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China.
² University of Chinese Academy of Sciences, Beijing, China.
³ Institute of Advanced Biotechnology and School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.
⁴ Center for Advanced Bioindustry Technologies, Chinese Academy of Agricultural Sciences, Beijing, China.
⁵ Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan.

Abstract

Gene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at target sites in plants. Here we report an all-in-one approach of GT in Arabidopsis by combining a transcriptional and a translational enhancer for the Cas expression. We find that facilitating the expression of Cas9 and Cas12a variant by using enhancers can improve DSB and subsequent knock-in efficiency in the Arabidopsis genome. These results indicate that simply increasing Cas protein expression at specific timings - egg cells and early embryos - can improve the establishment of heritable GTs. This simple approach allows for routine genome engineering in plants.

Keywords: CRISPR/Cas9; CRISPR/ttCas12a; RPS5a; all-in-one; gene targeting (GT); genome engineering; transcriptional and translational enhancers.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Shanghai Science and Technology Innovation Plan (20ZR1467000 and 23WZ2500800), the Foreign Expert Project (G202201355L), the Chinese Academy of Sciences to DM, by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (No. 21570050) to HS, and by the National Key R&D Program of China (2021YFA1300404), National Natural Science Foundation of China (32188102) to JZ.