Repurposing Type III CRISPR-Cas as Modular Cellular RNA Biosensors

Over the past decade, CRISPR-Cas based biosensors have emerged as sensitive, specific and programmable platforms for nucleic acid detection, with applications ranging from diagnostic tools, environmental monitoring and various applications in fundamental research. The vast majority of these biosensors are currently limited to in vitro applications, highlighting the need for cellular platforms. Here, we provide a perspective on the potential of type III CRISPR-Cas systems as a platform for real-time RNA biosensing in living cells. We discuss the unique properties that distinguish type III from other CRISPR-Cas types, including their intrinsic cyclic oligoadenylate (cOA) signal amplification cascade, PAM-free RNA targeting and the extensive biochemical diversity of their downstream CARF and SAVED effector proteins. We outline the fundamental design principles of a genetically encoded type III cellular biosensor, comprising a programmable guide RNA, a multi-subunit sensing complex and a cOA-sensing effector protein capable of generating optical or electrochemical readout signals. A remaining challenge for type III cellular biosensors is the inherent cytotoxicity of canonical cOA-activated effectors. We propose several strategies to re-engineer these outputs towards non-cytotoxic reporter systems. While cellular biosensors present a natural route toward in vivo sensing, they face many challenges beyond the choice of the output signal and hardware strategy. We conclude that type III CRISPR-Cas is a promising platform for programmable real-time RNA sensing in living cells, utilizing its diverse modular effector outputs, representing a significant expansion of the CRISPR biosensor toolkit, while emphasizing appropriate deployment scenarios that enables full potential of these biosensors.