Figure 1.

Schematic representation of the flavonoid biosynthetic pathway in plants. The different metabolites are represented in boxes. The genes that integrate the pathway are: ANS (anthocyanidin synthase), CHS (Chalcone synthase), CHI (Chalcone isomerase), CCoAOMT (Caffeoyl‐CoA O‐methyltransferase), C3H (4‐coumarate 3‐hydroxylase), C3’H (4‐coumaroyl shikimate/quinate 3’‐hydroxylase), C4H (Cinnamate 4‐hydroxylase), 4CL (4‐coumaroyl CoA ligase), DFR (Dihydroflavonol 4‐reductase), F3H (Flavanone 3‐hydroxylase), F3’H (flavonoid 3’‐hydroxylase), F3’‐5H (Flavonoid 3’ 5‐hydroxylase), F3’‐5’H (Flavonoid 3’ 5’‐hydroxylase), FLS (Flavonol synthase), HCT (4‐hydroxycinnamoyl CoA: shikimate/quinate hydroxycinnamoyltransferase), PAL (Phenylalanine ammonia‐lyase). The genes marked in red are those selected for transcriptional activation with dCasEV2.1 in this work. The metabolites targeted for accumulation are shown in bold. A representation of each candidate gene promoter with the targeted gRNA positions is also shown. gRNAs assayed in the first round of optimization are shown in red, and those assayed in the second round are shown in blue. Red/blue striped boxes represent activation sites that were included in activation programmes assayed in both optimization rounds. The asterisks represent the gRNA position recalculated with updated information on the TSS available in databases (https://www.nbenth.com/).