Custom-made design of metabolite composition in N. benthamiana leaves using CRISPR activators

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...

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Published inbioRxiv
Main Authors Sara Selma, Sanmartin, Neus, Espinosa-Ruiz, Ana, Gianoglio, Silvia, Lopez-Gresa, Maria Pilar, Vazquez-Vilar, Marta, Flors, Victor, Granell, Antonio, Orzaez, Diego
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 12.07.2021
Subjects
CRISPR
Flavonoids
Gene expression
Kaempferol
Leaves
Metabolic pathways
Metabolism
Metabolites
Naringenin
Principal components analysis
Quercetin
Transcription factors
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Summary: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. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://zenodo.org/record/5091654#.YOwVc-gzaUk
DOI:10.1101/2021.07.12.452005