Potato virus X-delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X-derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus-induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show that gRNA-mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X-based virus-induced gene reprogramming strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.