Developing, Characterizing, and Modeling CRISPR-Based\nPoint-of-Use Pathogen Diagnostics

Recent years have\nseen intense interest in the development of point-of-care\nnucleic acid diagnostic technologies to address the scaling limitations\nof laboratory-based approaches. Chief among these are combinations\nof isothermal amplification approaches with CRISPR-based detection\nand readouts of target products. Here, we contribute to the growing\nbody of rapid, programmable point-of-care pathogen tests by developing\nand optimizing a one-pot NASBA-Cas13a nucleic acid detection assay.\nThis test uses the isothermal amplification technique NASBA to amplify\ntarget viral nucleic acids, followed by the Cas13a-based detection\nof amplified sequences. We first demonstrate an in-house formulation\nof NASBA that enables the optimization of individual NASBA components.\nWe then present design rules for NASBA primer sets and LbuCas13a guide\nRNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA\nfragments, resulting in 20–200 aM sensitivity. Finally, we\nexplore the combination of high-throughput assay condition screening\nwith mechanistic ordinary differential equation modeling of the reaction\nscheme to gain a deeper understanding of the NASBA-Cas13a system.\nThis work presents a framework for developing a mechanistic understanding\nof reaction performance and optimization that uses both experiments\nand modeling, which we anticipate will be useful in developing future\nnucleic acid detection technologies.