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Abstract Most CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent approach quantifies analyte concentration by sizing λ DNA reporters via gel electrophoresis, providing a simple solution for label-free detection. Here, we report an alternative strategy for label-free CRISPR-Cas12a, which relies on Cas12atrans-nicking induced supercoil relaxation of dsDNA plasmid reporters to generate a robust and ratiometric readout. The ratiometric CRISPR (rCRISPR) measures the relative percentage of supercoiled plasmid DNA to the relaxed circular DNA by gel electrophoresis for more accurate target concentration quantification. This simple method is two orders of magnitude more sensitive than the typical fluorescent reporter. This self-referenced strategy solves the potential application limitations of previously demonstrated DNA sizing-based CRISPR-Dx without compromising the sensitivity. Finally, we demonstrated the applicability of rCRISPR for detecting various model DNA targets such as HPV 16 and real AAV samples, highlighting its feasibility for point-of-care CRISPR-Dx applications.
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A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double‐Stranded λ DNA Reporter
Abstract CRISPR‐based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR‐Cas12a‐based sensing platform to detect ssDNA targets by sizing double‐stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massivetrans‐nuclease activity of Cas12a toward λ DNA is due to the presence of single‐stranded looped structures along the λ DNA sequence. In addition, we observed a strong binding affinity between Cas12a and λ DNA, which further promotes thetrans‐cleavage activity and helps achieve sub‐picomolar detection sensitivity, ≈100 times more sensitive than the fluorescent counterpart. The concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as CRISPR reporters.
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- Award ID(s):
- 1944167
- PAR ID:
- 10443474
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 61
- Issue:
- 50
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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