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

Abstract

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.

Signal Amplification by the trans-Cleavage Activity of CRISPR-Cas Systems: Kinetics and Performance

“…Nouri et al 67 defined a figure of merit (FOM), the LOD multiplied by the reaction time (LOD × T ), for the quantitative evaluation of CRISPR-based detection techniques ( eq 10 ). Using the FOM, they also discussed how different factors improved assay performance, including improving the cleavage rate by each active Cas enzyme , the preamplification ( A ), high sample volume ( V 0 ) over reaction volume ( V r ) without affecting the assay, and low LOD of the detector ( C min ) ( eq 10 ).…”

Smartphone‐compatible, CRISPR‐based platforms for sensitive detection of acute hepatopancreatic necrosis disease in shrimp

“…To achieve low‐copy detection of target pathogens, the sensitivity of a CRISPR assay must first be enhanced by incorporating a DNA preamplification step, which can be any of the aforementioned PCR and isothermal techniques (Nouri et al, 2022). In this study, we were specifically interested in integrating the Cas12a assay with AP4 nested PCR and RPA.…”

Chemiluminescence resonance energy transfer as a simple and sensitive readout mode for a CRISPR/Cas12a-based biosensing platform

“…So, it is necessary to find some other readout modes for CRISPR/ Cas12a-based biosensing. 17,18 Chemiluminescence (CL), light emission triggered by a chemical reaction, is increasingly regarded as a powerful analytical technique. 19,20 The CL instrumentation does not require a light source and spectroscopic system, and is very simple, portable and cheap.…”