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.

Construction and Application of Orthogonal T7 Expression System in Eukaryote: An Overview

“…[147,148] In addition, the application of T7 expression system in nanotechnology and nanomaterial indicates a promising potential in gene therapy. The combination of T7 system with deaminase, [149][150][151] CRISPR/Cas9 expression system, and optical genetic provides effective tools for genome editing in eukaryote. [152,153] Furthermore, in vitro T7 expression system has been applied extensively to detect various biomolecules, especially in clinical diagnosis, which facilitates the development of cell-free synthetic biology in eukaryote.…”

A dual gene-specific mutator system installs all transition mutations at similar rates in vivo

“…CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It The deaminase-T7RNAP system was first reported in bacteria (MutaT7; (Moore et al, 2018)) and further extended to mammalian cells (TRACE; (Chen et al, 2020)), yeast (TRIDENT; (Cravens et al, 2021)), and plants (Butt et al, 2021). We previously demonstrated that the mutation rate of MutaT7 could be enhanced 7-to 20-fold with a more efficient cytidine deaminase, Petromyzon marinus cytidine deaminase (PmCDA1) (Park & Kim, 2021).…”

“…Considering that most directed evolution experiments focus on a single protein, the ideal target range is a single gene of interest. Gene-specific targeting was achieved through homologous platforms that use chimeric mutator proteins, generated by fusing a nucleobase deaminase to an orthogonal T7 RNA polymerase (T7RNAP) (Alvarez et al , 2020; Butt et al , 2021; Chen et al , 2020; Cravens et al , 2021; Moore et al , 2018; Park & Kim, 2021).…”