(174bv) Systematic Mapping of Bacterial Crispra Design Rules and Implications for Synergistic Gene Activation

CRISPR gene activation (CRISPRa) has the potential to dramatically accelerate bacterial strain engineering, but effective and generalizable bacterial CRISPRa is still challenging. In eukaryotic cells, there are several strategies to combine multiple activators for synergistic gene activation. In principle, similar effects should be possible in bacteria because there are multiple bacterial CRISPRa effectors that could be delivered in combination. However, there have been no reports of successful synergistic CRISPRa in bacteria. To identify plausible combinations of effectors for synergistic activation, we systematically characterized bacterial activator proteins with a set of engineered, synthetic promoters. We found that optimal activator placement relative to the transcription start site (TSS) could vary up to 200 base pairs. This result is qualitatively consistent with previous findings, but performing these comparisons in the same promoter context was critical because the precise targeting rules vary between promoters. Using these guidelines, we tested multiple activator combinations with each positioned at its optimal target site. We did not find any effector combinations that produced synergistic activation, and we found that many combinations were antagonistic. This systematic investigation highlights fundamental mechanistic differences between bacterial and eukaryotic transcriptional activation systems, and suggests that alternative strategies will be necessary for strong bacterial gene activation at arbitrary endogenous targets.