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Updated: Jun 29, 2025
Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
Published on: August 16, 2024
A PAM-Free One-Step Asymmetric RPA and CRISPR/Cas12b Combined Assay (OAR-CRISPR) for Rapid and Ultrasensitive DNA
Lei Yang1, Guanwei Chen1,2, Jian Wu3
1State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, P.R.China, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
A new one-step assay, OAR-CRISPR, combines asymmetric recombinase polymerase amplification (RPA) with CRISPR/Cas12b for rapid, sensitive DNA detection. This PAM-free method simplifies diagnostics and enables naked-eye identification of low DNA concentrations.
Area of Science:
- Molecular Diagnostics
- CRISPR-Cas Systems
- Nucleic Acid Amplification
Background:
- Current molecular diagnostics often combine isothermal amplification with CRISPR/Cas systems for enhanced sensitivity.
- Existing two-step assays are complex and prone to contamination.
- One-step assays face challenges due to inherent incompatibilities and the protospacer adjacent motif (PAM) requirement for Cas12 activation.
Purpose of the Study:
- To develop a novel, PAM-free, one-step assay for CRISPR/Cas12b-based DNA detection.
- To overcome limitations of existing methods, including PAM site dependency and multi-step procedures.
- To improve the speed, simplicity, and sensitivity of molecular diagnostic approaches.
Main Methods:
- Development of a one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR).
- Asymmetric RPA generates single-stranded DNA (ssDNA) for CRISPR RNA binding, bypassing PAM site requirements.
- Integration of ssDNA cleavage by PAM-free crRNA without interfering with RPA amplification.
Main Results:
- The OAR-CRISPR assay achieves sensitivity comparable to quantitative PCR (qPCR) in one-quarter of the time.
- Demonstrates naked-eye detection of as few as 60 copies/μL DNA within 8 minutes.
- Successfully integrates asymmetric RPA into a one-step CRISPR-based detection system.
Conclusions:
- OAR-CRISPR represents a significant advancement in one-step CRISPR/Cas12b-based detection.
- The PAM-free, single-step approach simplifies operations and reduces contamination risks.
- This method broadens the applicability of CRISPR-based detection to diverse DNA targets.
