Harnessing CRISPR-Cas systems for maximally active and multiplexed viral detection

Abstract

Originally discovered as a mechanism for prokaryotes to defend themselves against invading viruses and plasmids, the clustered regularly-interspaced short palindromic repeats (CRISPR)-Cas systems have since been repurposed into potent gene editing tools for biological research. Cas12 and Cas13 are single effector-CRISPR proteins that target DNA or RNA, respectively. These proteins activate upon crRNA-target recognition to unleash their cis- and trans-cleavage activity. Interestingly, the cleavage activities differ between Cas12, Cas13, and their respective orthologs which allows them to be used to detect, edit, or cut specific nucleic acid sequences, including viruses in vitro and in vivo. Depending on the application, different cleavage activities are preferred. For diagnostic applications, we rely on the strong collateral cleavage activities of Cas12a and Cas13a to cleave a fluorescent reporter in solution to readout the viral positive status of a sample. Yet, the COVID-19 pandemic has highlighted that these diagnostic strategies are not sufficient to meet public health demands by specifically falling short on throughput, multiplicity, and comprehensiveness. By harnessing the programmable nuclease activities of these CRISPR systems, I aimed to 1) understand the Cas13 crRNA characteristics for maximally active and species-specific guide design, 2) develop highly multiplexed CRISPR-based diagnostics for pathogen detection, and 3) use multiplexed diagnostic platforms for surveillance applications.