Catharanthus roseus (Madagascar periwinkle) is the only commercial source of several medicinally important terpenoid indole alkaloids (TIAs). The most notable TIAs from C. roseus are vinblastine and vincristine. These unique chemotherapeutics are limited in supply due to inconsistent production and low yield from plants. The ~30 genes that comprise the TIA pathway have been identified (Fig 1). To increase the accumulation of these desired alkaloids, we are using CRISPR-based activation tools to target known transcription factors that govern the TIA pathway.
Here, we present the initial results of screening guide RNAs (gRNAs) in CRISPR-based activation (CRISPRa) for the transcriptional activator, ORCA3. ORCA3 regulates multiple TIA genes both upstream and downstream in the pathway. We compare how the design of the CRISPRa gRNA system (guide location and the use of multiple guides) affected gene activation. Specifically, for ORCA3, we demonstrate that multiple guides that are 60-90% overlappinghave a detrimental effect on CRISPR activation compared to multiple guides that are spread out. We screened the gRNAs using our transient expression method paired with qPCR analysis to assess guides in an endogenous context. This work is translatable to other plant species and provides an effective approach to screen guides for gene activation.
