Optimized dCas9 Transcription Activators for Plants

Understanding how gene expression impacts plant development and physiology is important for rationally engineering improved crops. Programmable Transcriptional Activators (PTAs), including CRISPR-Cas activators, have traditionally relied on a limited number of transcriptional activation domains. Usually the VP64 domain, derived from human herpes simplex virus, is fused to a DNA-binding domain to activate target gene expression. We reasoned there was considerable space for PTA improvement by replacing VP64 with a plant-derived activation domain. To address this, we designed, built, and tested a PTA library of 38 putative plant transcriptional activation domains. Domains from HSFA6b, AvrXa10, DOF1, DREB1, and DREB2 genes function as strong activators in Setaria viridis and Arabidopsis thaliana 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 demonstrates 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.