Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

Abstract

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. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, 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. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated in the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.