Multi-layer CRISPRa/i circuits for dynamic genetic programs in cell-free and bacterial systems

CRISPR-Cas transcriptional circuits hold great promise as platforms for engineering metabolic networks and information processing circuits. Historically, prokaryotic CRISPR control systems have been limited to CRISPRi. Creating approaches to integrate CRISPRa for transcriptional activation with existing CRISPRi-based systems would greatly expand CRISPR circuit design space. Here, we develop design principles for engineering prokaryotic CRISPRa/i genetic circuits with network topologies specified by guide RNAs. We demonstrate that multi-layer CRISPRa/i cascades and feedforward loops can operate through the regulated expression of guide RNAs in cell-free expression systems and E. coli. We show that CRISPRa/i circuits can program complex functions by designing type 1 incoherent feedforward loops acting as fold-change detectors and tunable pulse-generators. By investigating how component characteristics relate to network properties such as depth, width, and speed, this work establishes a framework for building scalable CRISPRa/i circuits as regulatory programs in cell-free expression systems and bacterial hosts. A record of this paper's transparent peer review process is included in the supplemental information.