Optimized dCas9 Programmable Transcription Activators for Plants

Abstract

Understanding how the expression of genes impacts plant development and physiology is important for rationally engineering crop improvements. Programmable Transcription Activators (PTAs), including CRISPRa activators, have traditionally relied on a limited number of transcription activation domains, namely the VP64 domain derived from human herpes simplex virus, to control gene expression. We reasoned there was considerable space for PTA improvement by replacing this domain with a plant-derived activation domain. To address this, we designed, built, and tested a PTA library of 38 putative plant transcription activation domains. Domains from HSFA6b, AvrXa10, DOF1, DREB1, and DREB2 genes function as strong activators in Setaria viridis and Arabidopsis thaliana both in protoplast assays and in transgenic plants. Overexpression of multiple endogenous genes (FT, PAP1, WUS) reached levels similar to the highly expressed housekeeping gene, PP2A, regardless of basal expression level. Further, these domains were effective in different PTA architectures, including the dCas9-SunTag, dCas9-Moontag, and TALE-SunTag systems. Lastly, we demonstrate the ability of these improved PTAs to map enhancer regions that promote gene expression in plants. This work showcases the effective and flexible nature of PTAs to activate target genes in plants, providing tools that can be used to improve agronomically relevant traits of interest.