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High-specificity gene point mutation detection by PAM-free Cas12a system with double-stranded substrate
Zhujun Liu1, Jinjin Wang2, Zhengguang Yang2
1Department of Pharmacy, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430014, China; Key Laboratory for Molecular Diagnosis of Hubei Province, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430014, China; School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
A new CRISPR/Cas12a system bypasses protospacer adjacent motif (PAM) requirements for enhanced gene point mutation detection. This PAM-free system significantly improves accuracy and sensitivity for clinical diagnostics.
Area of Science:
- Molecular Biology
- Genetic Engineering
- Biotechnology
Background:
- CRISPR/Cas12a is promising for nucleic acid detection.
- Protospacer adjacent motif (PAM) dependence limits Cas12a applications in gene mutation analysis.
- Few known mutation sites naturally contain PAM sequences.
Purpose of the Study:
- To develop a PAM-free Cas12a system for gene point mutation analysis.
- To overcome the sequence limitations of traditional Cas12a systems.
- To provide a high-performance tool for clinical gene point mutation detection.
Main Methods:
- Developed a PAM-free Cas12a system utilizing double-stranded substrate positioning-unwinding (dsPU-Cas12a).
- Engineered "bubble" structures to release single-stranded target DNA as a toehold.
- Employed auxiliary strands to induce DNA unwinding and facilitate R-loop formation.
Main Results:
- Achieved an ultra-low limit of detection of 0.013% for gene point mutations.
- Demonstrated excellent linearity over a mutation abundance range of 0-10%.
- Successfully detected JAK2 V617F mutations in patient blood samples with high accuracy.
Conclusions:
- The dsPU-Cas12a system effectively overcomes PAM sequence limitations.
- This PAM-free strategy offers a simple, universal, and high-performance tool for clinical gene point mutation detection.
- The system shows robust feasibility and accuracy for real-world diagnostic applications.
