Affiliation:
1. Laboratory for Genome Engineering and Synthetic Biology, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
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. 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 inNicotiana benthamianatransient assays to target a transgene expressingGFPunder the control of the T7 promoter and observed C-to-T conversions. We then targeted the T7 promoter-drivenacetolactate synthasesequence 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 theacetolactate synthasesequence, 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.
Publisher
Life Science Alliance, LLC
Subject
Health, Toxicology and Mutagenesis,Plant Science,Biochemistry, Genetics and Molecular Biology (miscellaneous),Ecology
Cited by
7 articles.
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