(182p) Overexpressing a Bottleneck Gene in Catharanthus Roseus with CRISPR Activation

The medicinal plant Catharanthus roseus is the sole source of chemotherapy drugs vinblastine (VBL) and vincristine (VCR). Shortages of these drugs made national headlines in 2019, highlighting the need for improved processes for VBL and VCR production. One major driver of drug shortages is the low concentration of the compounds in the leaves; since VBL and VCR are secondary metabolites, many genes in the ~30-step biosynthetic pathway are lowly expressed. One approach to improve VBL and VCR concentrations in C. roseus is by overexpressing important pathway genes that act as bottlenecks to VBL and VCR accumulation.

In this project, we use CRISPR activation (CRISPRa) to target catharanthine synthase (HL1), a lowly expressed gene in the pathway. Using a promoter-transactivation assay, we present insights on the optimal CRISPRa design; factors such as the number of guide RNAs (gRNAs), guide location, and percent GC content are studied to determine the optimal design for overexpression of important promoters. Notably, we found that having 4 gRNAs significantly improves promoter activation compared to 3 gRNAs or single gRNAs. Elucidating the optimal gRNA parameters will allow researchers to streamline the CRISPRa design when targeting lowly expressed genes in plants.