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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 gene mutation detection. This PAM-free system significantly enhances the ability to identify point mutations, including in clinical samples.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
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 have adjacent PAM sequences.
Purpose of the Study:
- To develop a protospacer adjacent motif (PAM)-free CRISPR/Cas12a system.
- To overcome the sequence limitations of Cas12a for gene point mutation detection.
- To enhance the sensitivity and applicability of Cas12a in clinical diagnostics.
Main Methods:
- Developed a PAM-free Cas12a system utilizing double-stranded substrate positioning-unwinding (dsPU-Cas12a).
- Engineered 'bubble' structures and utilized auxiliary strands to facilitate R-loop formation.
- Optimized the dsPU-Cas12a system for enhanced DNA unwinding and target recognition.
Main Results:
- Achieved an ultra-low limit of detection of 0.013% for gene point mutations.
- Demonstrated excellent linearity (0-10% mutation abundance).
- Successfully detected JAK2 V617F mutations in patient blood samples with high accuracy.
Conclusions:
- The dsPU-Cas12a system effectively overcomes the sequence limitations of traditional Cas12a.
- This PAM-free strategy provides a high-performance, universal tool for clinical gene point mutation detection.
- The system shows robust feasibility and accuracy for diagnosing conditions like myeloproliferative neoplasms.
