(27ak) Multiplexing of Cas Orthologs for Disease Detection

Various diseases display similar symptoms, but typically, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics and more traditional forms of diagnostics only test for the presence of a single type of pathogen. During the COVID-19 pandemic, the overlapping of symptoms between SARS-CoV-2, Respiratory Syncytial Virus (RSV), and the flu demonstrated a need for a single diagnostic test that could differentiate between multiple targets. We explored the feasibility of combining multiple Cas (CRISPR-associated protein) orthologs and Cas predecessors based on cis-/trans-cleavage target type and temperature of activity, allowing for detection of up to four targets. The goal was a reaction that could be carried out partly at room temperature and then proceed at a high temperature, requiring only isothermal heating. Based on structures predicted using AlphaFold, DeepDDG analysis, and rational reasoning, we introduced mutations to the thermophilic endonucleases to achieve further thermostability. Additionally, for the endonucleases to be used at lower temperatures, we introduced mutations to increase activity and lower thermostability. To further limit cross-over activity and widen the threshold between positive and negative samples, we also investigated a variety of blockers that could potentially block cleavage activity at lower temperatures for the more stable endonucleases. With the increasing threat of emergent pathogens, we believe that the multiplexing of Cas orthologs for disease detection can have a broad impact on diagnostics for human health as well as diagnostics of plant and animal pathogens within the agricultural industry.