Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Here, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP–DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter and observed C-to-T conversions. We then targeted the T7 promoter-driven acetolactate synthase sequence that had been stably integrated in the rice genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the acetolactate synthase sequence, resulting in the enrichment of herbicide-responsive residues. We then validated these herbicide-responsive regions in the transgenic rice plants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance.
Bibliographical noteFunding Information:
We would like to thank members of the genome engineering and synthetic biology laboratory at KAUST for their critical discussion and technical help in this work. This work was funded by KAUST-baseline funding to M Mahfouz.
© 2022 Butt et al.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
- Plant Science
- Health, Toxicology and Mutagenesis