Custom-made design of metabolite composition inN. benthamianaleaves using CRISPR activators

Abstract

Transcriptional regulators based on CRISPR architecture expand our ability of reprogramming endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely naringenin, eriodictyol, kaempferol and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programs for each of the four metabolites, each program activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation program showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples in five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for custom-made design of metabolic contents in plants.

Efficient gene activation in plants by the MoonTag programmable transcriptional activator

“…Because this multi-gene targeting requires only the addition of relatively small sgRNA sequences to genetic constructs, CRISPR/Cas-based activators show promise in modulating multi-locus traits. Applications in plants include addressing basic research in plant biology, metabolic reprogramming, engineering beneficial traits in crops, or creating barriers to sexual reproduction and gene flow between engineered and wild type plants (15,(23)(24)(25)(26)(27)(28). The MoonTag activators described here perform well in stable transgenic plants and are a suitable platform for developing such applications.…”

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

“…In this regard, a strong VIGA effect not only serves in research to elucidate gene function, but in the case of N. benthamiana, VIGA can also be envisioned as a tool for shaping plant metabolic content. Recently our group showed how CRISPR-based programable transcriptional factors can be employed to channel metabolic fluxes toward the accumulation of selected metabolites in N. benthamiana leaves (Selma et al 2021). In that work, gRNA-encoded activation programs were delivered in the form of multiplex T-DNA constructs via agroinfiltration, a strategy that limits the scalability of the approach.…”

“…A possible constraint of PVX_VIGR is that cargo limitations restrict the complexity of the gene activation/repression programs that can be delivered to the plant. In metabolic reprogramming using T-DNA delivery via agroinfiltration, we previously employed up to fifteen tandemly arrayed gRNAs to successfully redirect a metabolic pathway (Selma et al 2021). However, it is unlikely that such large gRNA tandem repeats could work as efficient programs in PVX_VIGR.…”

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

“…DFR plays an important role in the synthesis of anthocyanins [35], while PAL is the key enzyme catalyzing the conversion of aromatic amino acids into secondary metabolites [25]. A dCasEV2.1 construct in Cu/Cu conformation (CBS4:minDFR:dCas9:EDLL:tNOS-CBS4:minDFR:MS2:VPR:tNOS) was targeted with a pol-III expressed multiplexing gRNA to three positions of either the NbDFR promoter [31] or the NbPAL2 promoter [32]. A gRNA targeting the S. lycopersicum MTB gene was included as a negative control of the activation.…”