AUTHOR=Cheng Yiqiu , Zhang Lei , Li Jing , Dang Xiaofei , Zhu Jian-Kang , Shimada Hiroaki , Miki Daisuke TITLE=Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy JOURNAL=Frontiers in Plant Science VOLUME=15 YEAR=2024 URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1360925 DOI=10.3389/fpls.2024.1360925 ISSN=1664-462X 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.