(556c) Engineered Prokaryotic Promoters Enable Multi-Input Processing Crispra/I Circuitry

CRISPRa/i transcriptional regulatory networks hold great promise as a framework for engineering complex metabolic networks and information processing circuits. Recent analysis of CRISPRa/i circuit properties suggests a high degree of composability and scalability, highlighting the possibility of building more complex regulatory programs. However, to increase the complexity of the circuits and the range of their applications, an expandable set of high-performing CRISPRa/i promoters and guide RNAs is required. To engineer a set of high-performing activatable promoters, we characterize the effect of different promoter parts on the promoter expression profiles by using CRISPRa as a promoter interrogation tool. We design promoters with up to 1000-fold dynamic range in an E. coli-based cell-free expression system (CFS), constituting a 33-fold improvement from previous synthetic promoters. These new activatable promoters enable implementation of novel ligand-responsive CRISPR activation domains and circuit topologies not accessible with previous components, such as dense overlapping regulons and deep activation cascades. Additionally, we find a high degree of transferability of the insights gained with CRISPRa to other systems, such as the light-inducible transcriptional activator EL222. We demonstrate multi-input processing by integrating both ligand-responsive CRISPRa and EL222 into a tunable CRISPRa/i pulse generator. Overall, this work provides a toolbox of high-performing activatable promoters and orthogonal inducible transcriptional activators that expand the capabilities of the CRISPRa/i regulatory framework.