PAM-less conditional DNA substrates leverage trans-cleavage of CRISPR-Cas12a for versatile live-cell biosensing

The CRISPR-Cas system has been repurposed as a powerful live-cell imaging tool, but its utility is limited to genomic loci and mRNA imaging in living cells. Here, we demonstrated the potential of the CRISPR-Cas system as a generalizable live-cell biosensing tool by extending its applicability to monitor diverse intracellular biomolecules. In this work, we engineered a CRISPR-Cas12a system with a generalized stimulus-responsive switch mechanism based on PAM-less conditional DNA substrates (pcDNAs). The pcDNAs with stimulus-responsiveness toward a trigger were constructed from the DNA substrates featuring no requirement of a protospacer-adjacent motif (PAM) and a bubble structure. With further leveraging the trans-cleavage activity of CRISPR-Cas12a for signal reporting, we established a versatile CRISPR-based live-cell biosensing system. This system enabled the sensitive sensing of various intracellular biomolecules, such as telomerase, ATP, and microRNA-21, making it a helpful tool for basic biochemical research and disease diagnostics.

Automated summary

In this study, the CRISPR-Cas system was engineered as a versatile live-cell biosensing system for monitoring diverse intracellular biomolecules. By leveraging the trans-cleavage activity of CRISPR-Cas12a, this system enabled sensitive sensing of various biomolecules such as telomerase, ATP, and microRNA-21.