Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems:Improvement Strategies and Performance Comparison br

Clustered regularly interspaced short palindromicrepeats (CRISPR)-based nucleic acid-sensing systems have grownrapidly in the past few years. Nevertheless, an objective approach tobenchmark the performances of different CRISPR sensing systems islacking due to the heterogeneous experimental setup. Here, wedeveloped a quantitative CRISPR sensingfigure of merit (FOM) tocompare different CRISPR methods and explore performanceimprovement strategies. The CRISPR sensing FOM is defined asthe product of the limit of detection (LOD) and the associatedCRISPR reaction time (T). A smaller FOM means that the methodcan detect smaller target quantities faster. We found that there is atradeoffbetween the LOD of the assay and the required reactiontime. With the proposed CRISPR sensing FOM, we evaluatedfivestrategies to improve the CRISPR-based sensing: preamplification, enzymes of higher catalytic efficiency, multiple crRNAs,digitalization, and sensitive readout systems. We benchmarked the FOM performances of 57 existing studies and found that theeffectiveness of these strategies on improving the FOM is consistent with the model prediction. In particular, we found thatdigitalization is the most promising amplification-free method for achieving comparable FOM performances (similar to 1fMmiddotmin) as thoseusing preamplification. Thefindings here would have broad implications for further optimization of the CRISPR-based sensing.