Inducible CRISPR:Cas transcription regulators for de novo assembly of gene regulatory networks (360G-Wellcome-102425_Z_13_Z)

The CRISPR:Cas9 system has proven to be a simple and versatile tool to engineer biology. Initially used for genome editing, CRISPR:Cas9 was rapidly repurposed to carryout programmed transcriptional regulation. The next frontier in expanding its potential will be set off by the design of inducible systems responsive to exogenous cues and/or endogenous metabolites. Leveraging the sensor/actuator capabilities of allosteric self-cleaving ribozymes (aRz), which can be modulated by various ligands, I propose to re-engineer theRNA component of the system to create inducible CRISPR transcriptional regulators (iCRISPR-TR). This conceptual framework is expected to play a pivotal role in synthetic biology, empowering scientists to create complex devices able to rewire cell behavior upon sensinga disease state. Throughout the project, aRz sequences will be tested and their activity optimized via in vivo evolution in human cell lines (Aim 1). Promising candidates will then be implemented to power our iCRISPR-TR strategies (Aim 2). A systematic characterization of all devices at the transcript/protein level will provide novel insight into CIRSPR:Cas dynamics, which will feedback into our designs. Finally, to demonstrate the potential of this innovative platforms, microRNA sensing iCRISPR-TR will be used to reprogram latent phase EBV infected B-cells(Aim 3).