Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein

Thanks to its ease, speed, and sensitivity, CRISPR-based nucleic acid detection has been increasingly explored for molecular diagnostics. However, one of its major limitations is lack of multiplexing capability because the detection relies on the trans-cleavage activity of the Cas protein, which necessitates the use of multiple orthogonal Cas proteins for multiplex detection. Here we report the development of a multiplexed CRISPR-based nucleic acid detection system with single-nucleotide resolution using a single Cas protein (Cas12a). This method, termed as CRISPR-TMSD, integrates the toehold-mediated strand displacement (TMSD) reaction, and the cis-cleavage activity of the Cas protein and multiplexed detection are achieved using a single Cas protein owing to the use of target-specific reporters. A set of computational simulation toolkits was used to design the TMSD reporter, allowing for highly sensitive and specific identification of target sequences. In combination with the recombinase polymerase amplification (RPA), the detection limit can reach as low as 1 copy/mu L. As proof of concept, CRISPR-TMSD was subsequently used to detect an oncogenic gene and SARS-CoV-2 RNA with a single-nucleotide resolution. This work represents a conceptually new strategy for designing a CRISPR-based diagnostic system and has great potential to expand the application of CRISPR-based diagnostics.

Automated summary

In this study, a multiplexed CRISPR-based nucleic acid detection system with single-nucleotide resolution using a single Cas protein was developed. This system, called CRISPR-TMSD, integrated the toehold-mediated strand displacement reaction and the cis-cleavage activity of the Cas protein. By utilizing target-specific reporters, multiplexed detection was achieved using just one Cas protein. The combination with recombinase polymerase amplification allowed for extremely sensitive detection, with a detection limit as low as 1 copy/μL. This work presents a new strategy for designing CRISPR-based diagnostic systems and has the potential to expand the applications of CRISPR-based diagnostics.