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

Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid-sensing systems have grown rapidly in the past few years. Nevertheless, an objective approach to benchmark the performances of different CRISPR sensing systems is lacking due to the heterogeneous experimental setup. Here, we developed a quantitative CRISPR sensing figure of merit (FOM) to compare different CRISPR methods and explore performance improvement strategies. The CRISPR sensing FOM is defined as the product of the limit of detection (LOD) and the associated CRISPR reaction time (T). A smaller FOM means that the method can detect smaller target quantities faster. We found that there is a tradeoff between the LOD of the assay and the required reaction time. With the proposed CRISPR sensing FOM, we evaluated five strategies 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 the effectiveness of these strategies on improving the FOM is consistent with the model prediction. In particular, we found that digitalization is the most promising amplification-free method for achieving comparable FOM performances (∼1 fM·min) as those using preamplification. The findings here would have broad implications for further optimization of the CRISPR-based sensing.