CRISPR-Based Synthetic Gene Circuits for Controllable Gene Therapies

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has opened-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, safety and controllability of CRISPR therapeutics will remain a challenge for human translation. To this end, synthetic biology can offer important solutions. We apply the design principles of synthetic biology to develop CRISPR-based genetic logic gates, layered genetic circuits and safety switches in mammalian cells. Synthetic CRISPR-based genetic circuits can be designed to generate a user-defined actuation such as spatiotemporal regulation. Here, I will review my team’s efforts to engineer CRISPR for safer and controllable gene therapies. Engineering gRNA and Pol II-driven control mechanisms enable cell and context specific regulation. This strategy combined with synthetic promoter engineering allow us to develop genetic circuits to control the timing and sequence of function of catalytically active or inactive Cas9 protein or the gRNA. In parallel, we have developed and tested a number of safety switches in mammalian cells by harnessing multi-functionality of CRISPR, including DNA cleavage or transcriptional modulation or both. For some clinical applications, transcriptional modulation by nuclease incompetent Cas9 can be a safer alternative that genetic disruption by Cas9 nuclease. We utilize the nuclease incompetent Cas9 protein to test a plethora of potent transcriptional activation and repression domains, test them both in synthetic genetic circuits and endogenous gene circuits to understand design principles of transcriptional activation and repression by CRISPR and use for therapeutic purposes. We have engineered potent and novel CRISPR repressors that can be used to generate two layered Pol II driven genetic cascades. An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. We believe that synthetic CRISPR-based circuits can push the boundaries of CRISPR technologies towards such capacity for future therapies.