Fig. 1: Structure-based guide RNA design and synthetic promoters enable design space mapping with tunable CRISPRa.
From: Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling
Computational analysis of scRNA sequence identified a kinetic parameter describing the rate of conversion between the most stable structure and the active structure for CRISPRa, and scRNAs screened using this parameter predictably activated bacterial expression from a set of synthetic promoters. Tuning the activation of these promoters by truncating their scRNA spacer sequences—and again computationally verifying their efficacy—allows combinations of activation level at each promoter. The promoters can be paired with chosen output ORFs, including metabolic pathways. This method of controlling pathway gene expression allows for profiling of pathway design spaces for metabolic engineering using a combinatorial library of CRISPR-activated expression levels.
