An Efficient CRISPR/Cas Cooperative Shearing Platform for Clinical Diagnostics Applications.

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• The CRISPR/Cas system is a versatile genome editing tool with broad applications.
• Single Cas-CRISPR RNA (crRNA) duplexes face limitations in recognition and shearing efficiency due to target sequence folding.
• Efficient and rapid nucleic acid detection remains a challenge in molecular diagnostics.

Purpose of the Study:

• To develop an improved CRISPR/Cas system for enhanced genome editing and nucleic acid detection.
• To overcome the limitations of traditional CRISPR/Cas systems in target recognition and shearing efficiency.
• To establish a sensitive and rapid platform for unamplified nucleic acid electrochemical assays.

Main Methods:

• Development of a CRISPR/Cas cooperative shearing (CRISPR-CS) system utilizing two crRNA duplexes.
• Simultaneous recognition of different target sites by two CRISPR/Cas-crRNA duplexes.
• Electrochemical detection of cleaved methylene blue-ssDNA reporters.

Main Results:

• The CRISPR-CS system demonstrated increased recognition possibility and shearing efficiency compared to traditional CRISPR/Cas systems.
• Achieved an unamplified nucleic acid electrochemical assay in under 5 minutes with a detection limit of 9.5×10^-20 M.
• The platform accurately detected monkeypox, human papilloma virus, and amyotrophic lateral sclerosis with up to 98.1% accuracy.

Conclusions:

• The CRISPR-CS system significantly enhances CRISPR/Cas-mediated shearing efficiency and recognition.
• This cooperative shearing approach enables highly sensitive and rapid unamplified nucleic acid electrochemical detection.
• The CRISPR-CS platform shows great potential for accurate and efficient molecular diagnostics of various diseases.