Fluorescence Signal-Readout of CRISPR/Cas Biosensors for Nucleic Acid Detection

The CRISPR/Cas system is now being used extensively in nucleic acid detection applications, particularly after the trans-cleavage activity of several Cas effectors was found. A CRISPR/Cas system combined with multiple signal-readout techniques has been developed for various molecular diagnostics applications. Fluorescence is now a widely utilized dominant read-out technique in CRISPR biosensors. An in-depth understanding of various fluorescence readout types and variables affecting the fluorescence signals can facilitate better experimental designs to effectively improve the analytical performance. There are the following two commonly used types of CRISPR/Cas detection modes: the first is based on binding activity, such as Cas9 and dCas9; the second is based on cleavage activity, such as Cas12a, Cas12b, Cas13, and Cas14. In this review, fluorescence signal-readout strategies from the last 5 years based on the binding activity and cleavage activity of the CRISPR/Cas system with fundamentals and examples are fully discussed. A detailed comparison of the available fluorescent reporter sequences and design principles is summarized. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.