Crispr Logic Circuits to Enable Next Generation Crispr-Based Gene Therapies

Cas9 is an RNA guided DNA endonuclease of the bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system that has been adopted for programmable genome editing and transcriptional regulation. Since the complexity of genetic interrogations with the CRISPR system is rapidly increasing, there is a need for novel genetic tools to gain control over Cas9 activity or expression or regulate guide RNA (gRNA) function. To this end, we apply the design principles of synthetic biology to develop genetic logic gates, layered genetic circuits and kill switches in mammalian cells. RNA Pol II driven gRNAs enable cell and context specific expression of gRNA. 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 devised a strategy to readily switch between catalytically active and null Cas9 protein, allowing knocking out and activating target genes in the same cells using distinct gRNAs. By truncating the gRNA from 5’ end, we have shown that gRNAs with 14nt guide sequences ablate the nuclease activity of Cas9, while leaving its DNA targeting capacity intact. By fusing a Cas9 nuclease protein to a potent activation domain, VPR or application of gRNAs with MS2 binding loops, we can use a same Cas9 protein for transcriptional activation (14nt gRNAs) or gene disruption (20nt gRNAs). By incorporating this capacity within our layered circuits, we have developed and tested several synthetic gene circuits that can incorporate and modulate the desired functionality of this Cas9 complex, including DNA cleavage or transcriptional modulation or both.  Considering the growing clinical application of DNA-based gene therapies and vaccines, the developed CRISPR logic circuits pave the way towards developing safer and more specific gene therapies and allow us to perform more sophisticated biological discoveries.