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Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b
Jiaqi Wang1, Xiaofang Ye1, Yuanfang Liu1
1Guangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, Guangdong, China.
Nature Communications
|July 10, 2025
Summary
A novel split single guide RNA (sgRNA) strategy enhances CRISPR-Cas12b diagnostics. This adaptable system allows precise nucleic acid detection, including microRNAs, with improved regulation and simplified protocols for clinical applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Nucleic Acid Detection
Background:
- CRISPR-Cas12b systems offer advanced molecular diagnostics.
- Current limitations include reliance on long single guide RNAs (sgRNAs) >100 nt, hindering precise control.
- Need for adaptable and regulatable CRISPR systems for diverse applications.
Purpose of the Study:
- To develop a split sgRNA strategy for CRISPR-Cas12b to enable precise regulation and broader applications.
- To create a versatile Cas12b system using universal components and a customizable Spacer.
- To introduce dynamic control mechanisms for Cas12b activity.
Main Methods:
- Designed a split sgRNA system for Cas12b with universal components and a replaceable Spacer.
- Utilized glyoxal labeling and photo-cleavable linkers for dynamic Cas12b activity modulation.
- Validated detection of Epstein-Barr virus in clinical samples and microRNAs without amplification.
Main Results:
- The split sgRNA strategy allows simple target detection by replacing the Spacer.
- Glyoxal labeling and photo-cleavable linkers provide temperature- and UV-mediated dynamic regulation.
- Achieved sensitive detection of Epstein-Barr virus in plasma, comparable to qPCR.
- Demonstrated direct microRNA detection without reverse transcription or amplification.
- Consistent results with RT-qPCR in clinical samples (healthy individuals, colorectal cancer patients).
Conclusions:
- The split sgRNA strategy significantly enhances CRISPR-Cas12b systems for molecular diagnostics.
- This approach offers a simplified, highly adaptable, and precisely regulated method for clinical nucleic acid analysis.
- The system shows promise for direct detection of biomarkers like microRNAs.
