Issue 7, 2022

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

Abstract

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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
09 Okt 2021
Accepted
17 Jan 2022
First published
17 Jan 2022
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2022,13, 2011-2020

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

S. Chen, R. Wang, S. Peng, S. Xie, C. Lei, Y. Huang and Z. Nie, Chem. Sci., 2022, 13, 2011 DOI: 10.1039/D1SC05558E

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