Methodologies in visualizing the activation of CRISPR/Cas: The last mile in developing CRISPR-Based diagnostics and biosensing – A review

CRISPR/Cas-based analytical procedures have revolutionized the sensing platform to fulfill the requirements of the current era in terms of sensitivity, selectivity, robustness, user-friendly feature, and cost-effectiveness for the detection of nucleic acid as well as non-nucleic acid analytes. Molecular target monitoring and transduction of the signals is a crucial prerequisite for precise molecular sensing tools. Besides, the reporting systems have become the last milestone for fabricating Cas-based molecular probes to visualize the activation of CRISPR/Cas enzymes. In this review, we have highlighted various CRISPR/Cas reporters, their mechanisms, sensing strategies, merits, and demerits. Moreover, signal transducers, i.e. fluorescent, colorimetric, and electrochemical, have also been discussed in detail along with various sensing strategies to generate recordable signals. It was concluded that there is still a need to overcome issues offered by the reported sensing devices, such as off-target effect, target sequence limitation, multiplexed quantitative detection, the influence of the inhibitor, and reaction kinetic constraint. Additionally, it is required to make them available for commercial use by validating their stability, robustness, safety profile in an off-lab environment as most of the probes have been tested in the controlled atmosphere of the laboratories. We believe that this novel critical interpretation and summary will assist the researchers in designing and validating new CRISPR/Cas reporters and probes for practical applications on a commercial scale.