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Updated: Aug 3, 2025
Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
Published on: August 16, 2024
CRISPR-Cas12-Based Diagnostic Applications in Infectious and Zoonotic Diseases
Linxian Li1, Shiyuan Li2, Dayong Gu1
1Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, China.
Abstract:
Rapid detection of infectious and zoonotic diseases is very important for pathogen identification and infection control. Molecular diagnostic assays are well-known for high accuracy and sensitivity; however, conventional methods such as real-time PCR may require professional instruments and operations, preventing their wide applications in scenarios including animal quarantine. The recently developed CRISPR diagnostic (CRISPR-Dx) methods, employing the trans-cleavage activities of either Cas12 (e.g., HOLMES) or Cas13 (e.g., SHERLOCK), have shown great potential in rapid and convenient nucleic acid detection. Guided by specially designed CRISPR RNA (crRNA), Cas12 binds target DNA sequences and trans-cleaves ssDNA reporters, generating detectable signals, while Cas13 recognizes target ssRNA and trans-cleaves ssRNA reporters. To achieve high detection sensitivity, both HOLMES and SHERLOCK systems can be combined with pre-amplification procedures including both PCR and isothermal amplifications. Here, we present the employment of the HOLMESv2 method for convenient detection of the infectious and zoonotic diseases. Specifically, target nucleic acid is first amplified by LAMP or RT-LAMP, and the products are then detected by the thermophilic Cas12b. In addition, Cas12b reaction can be combined with LAMP amplification to achieve one-pot reaction systems. In this chapter, we provide a step-by-step description of the HOLMESv2-mediated rapid and sensitive detection of Japanese encephalitis virus (JEV), an RNA pathogen as an example.
Insights
Rapid detection of infectious diseases is crucial. The HOLMESv2 CRISPR diagnostic method, combined with LAMP amplification, offers a sensitive and convenient tool for pathogen identification, exemplified by Japanese encephalitis virus detection.
Area of Science:
- Molecular diagnostics
- Biotechnology
- Infectious disease research
Background:
- Accurate and rapid detection of infectious and zoonotic diseases is vital for pathogen identification and infection control.
- Conventional molecular assays like real-time PCR require specialized equipment, limiting their use in field settings such as animal quarantine.
- CRISPR-based diagnostic (CRISPR-Dx) methods, utilizing Cas12 or Cas13 enzymes, offer promising alternatives for rapid and convenient nucleic acid detection.
Purpose of the Study:
- To present the application of the HOLMESv2 method for the convenient and sensitive detection of infectious and zoonotic diseases.
- To demonstrate a streamlined diagnostic workflow combining pre-amplification and CRISPR-based detection.
- To provide a detailed protocol for HOLMESv2-mediated detection using Japanese encephalitis virus (JEV) as an example.
Main Methods:
- Utilized the HOLMESv2 CRISPR diagnostic system employing the thermophilic Cas12b enzyme.
- Integrated Loop-mediated Isothermal Amplification (LAMP) or Reverse Transcription LAMP (RT-LAMP) for target nucleic acid amplification.
- Developed a one-pot reaction system combining LAMP amplification and Cas12b detection for enhanced convenience.
Main Results:
- Successfully employed HOLMESv2 for the rapid and sensitive detection of target nucleic acids.
- Demonstrated the feasibility of combining LAMP/RT-LAMP with Cas12b detection for sensitive results.
- Showcased a one-pot reaction system integrating amplification and detection for simplified workflow.
Conclusions:
- The HOLMESv2 method, coupled with LAMP/RT-LAMP, provides a rapid, sensitive, and convenient approach for infectious and zoonotic disease detection.
- This CRISPR-Dx system holds significant potential for applications in resource-limited settings and field diagnostics.
- The presented protocol offers a valuable tool for the timely identification of pathogens like Japanese encephalitis virus.
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