Endornase-Based Off- and on-Switches for RNA-Level Computation in Mammalian Cells

Many synthetic biology circuits to date have traditionally been DNA-based. However, due to complications in delivery and the possibility of integration, large efforts have turned to RNA-only circuits as a mechanism for biomedical applications. Unfortunately, the number of modules that act on RNA still falls short of DNA-acting tools such as transcription factors, resulting in limited examples of RNA-level circuits that match the complexity, robustness, and therapeutic promise of their DNA-based analogs.

As a step towards increasing the availability of RNA-acting modules we have developed and characterized a library of CRISPR-specific endoRNase-responsive RNA switches. Endonuclease cleavage typically results in transcript degradation; therefore we adapt this concept to demonstrate robust reporter knockdown for several CRISPR-specific endoRNases when their respective recognition sites are placed in the reporter’s 5’ UTR. We achieve OFF switches with dynamic ranges of over 300 fold change and limited crosstalk. We also developed a new RNA ON-switch topology termed “Programmable Endonucleolytic RNA Scission-Induced Stabilization” (PERSIST) switch, that responds to each of these same CRISPR-specific endoRNases. The PERSIST-switch design includes repeats of a transcript degradation motif, which are cleaved off by these endoRNases leading to transcript stabilization and reporter expression. Similarly, these switches show robust responses with limited crosstalk. Finally, when OFF- and ON-switches are combined we demonstrate RNA-level ultrasensitive behavior, a response that is vital for the design of more complex circuits such as bistable switches and oscillators. These switches will find use in the design of more complex RNA-based circuits and the PERSIST-switch in particular may enable sensitive detection of endogenous biomarkers such as miRNAs, proteins and endoRNases.