Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems.

Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs). These tools are especially useful for studying gene function and interaction within various regulatory networks. First generation CRISPR-ATFs are nuclease-deactivated (dCas9) CRISPR systems where dCas9 proteins are fused to known transcriptional activator domains (VP64) or repressor domains (SRDX). When multiple chimeric dCas9-effector fusions are guided to gene regulatory regions via CRISPR gRNAs, they can modulate expression of transcript levels in planta. The protocol presented here provides a detailed procedure for activating AtPAP1 and repressing AtCSTF64 in Arabidopsis thaliana. This protocol makes use of our plant CRISPR toolbox to streamline the assembly and cloning of multiplex CRISPR-Cas9 transcriptional regulatory constructs.