Journal
ANALYTICAL CHEMISTRY
Volume 94, Issue 9, Pages 3987-3996Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c05124
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Funding
- National Natural Science Foundation of China [22104077, 22074088]
- Program for Changjiang Scholars and Innovative Research Team in University [IRT_15R43]
- Fundamental Research Funds for the Central Universities [GK202101001, GK202103044]
- Innovation Capability Support Program [2021TD-42]
- Natural Science Foundation of Shaanxi Province [2020JQ-407]
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This study demonstrates that a simple ssDNA probe can efficiently block the trans-cleavage activity of the CRISPR-Cas13a system without any modification, under low Mg2+ concentration. The blocked activity can be recovered by specific release of the DNA blocker from crRNA in a target-responsive manner, providing a convenient method for quantifying various biomarkers. This discovery enriches the CRISPR-Cas13a sensing toolbox and greatly expands its application scope.
The precise and controllable programming of the trans-cleavage activity of the CRISPR-Cas13a systems is significant but challenging for fabricating high-performance biosensing systems toward various kinds of biomolecule targets. In this work, we have demonstrated that under a critical low Mg2+ concentration, a simple and short single-stranded DNA (ssDNA) probe free of any modification can efficiently prevent the assembly of crRNA and LwaCas13a only by partially binding with the crRNA repeat region, thereby blocking the trans-cleavage activity of the LwaCas13a system. Furthermore, we have demonstrated that the blocked trans-cleavage activity of the LwaCas13a system can be recovered by various kinds of biologically important substances as long as they could specifically release the blocker DNA from the crRNA in a target-responsive manner, providing a facile route for the quantification of diverse biomarkers such as enzymes, antigens/proteins, and exosomes. To the best of our knowledge, this is reported for the first time that a simple ssDNA can be employed as the switch element to control the crRNA structure and regulate the trans-cleavage activity of Cas13a, which has enriched the CRISPR-Cas13a sensing toolbox and will greatly expand its application scope.
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