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Phosphorothioate Modification-Regulated One-Pot CRISPR Assay for Arbovirus Detection
Songkuan Zhuang1,2, Jie Li1, Zhoubin Fang1,3
1Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518060, China.
ACS Infectious Diseases
|March 23, 2026
Summary
A new psHOLMES method simplifies Chikungunya virus (CHIKV) diagnostics using CRISPR technology. This rapid, one-pot system offers high accuracy for detecting CHIKV RNA in clinical samples within 30 minutes.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Arthropod-borne viruses like Chikungunya virus (CHIKV) necessitate rapid and accurate diagnostic tools for effective epidemic control.
- CRISPR diagnostic (CRISPR-Dx) technology presents a promising avenue, but optimizing one-pot systems requires balancing amplification and Cas cleavage.
- Current methods often involve complex fine-tuning of Cas cis-cleavage activity.
Purpose of the Study:
- To develop a simplified, one-pot, two-step CRISPR-Dx system for efficient Chikungunya virus RNA detection.
- To introduce a novel method utilizing photocleavable partially phosphorothioate-modified DNA (ppPS-DNA) to regulate Cas12a activity.
- To enable rapid development of sensitive and accurate diagnostic tools for clinical applications.
Main Methods:
- Developed the psHOLMES method employing ppPS-DNA to control Cas12a activity in a one-pot, two-step process.
- Cas12a activity was inhibited during isothermal amplification by ppPS-DNA binding.
- Cas12a was reactivated post-amplification via UV-mediated photolysis of ppPS-DNA, initiating trans-cleavage detection.
Main Results:
- psHOLMES achieved attomolar sensitivity for CHIKV RNA detection.
- Demonstrated zero cross-reactivity with other related arboviruses.
- Achieved 100% accuracy (50/50) in clinical samples, detecting CHIKV within 30 minutes.
Conclusions:
- The psHOLMES method provides a simple and rapid approach for developing one-pot CRISPR-Dx systems.
- Eliminates the need for traditional fine-tuning of Cas cis-cleavage activity, accelerating diagnostic development.
- Offers a highly sensitive, accurate, and rapid diagnostic solution for Chikungunya virus, suitable for clinical settings.
