CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Multiplex nucleic acid detection methods often require expensive equipment and numerous reagents.
• Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems show promise for point-of-care (POC) nucleic acid detection.
• Achieving multiplexed detection with CRISPR/Cas systems remains a significant challenge.

Purpose of the Study:

• To develop an affordable and instrument-free method for multiplex nucleic acid detection.
• To integrate CRISPR-Cas12a trans-cleavage activity with reverse dot blot (RDB) for enhanced biosensing.
• To create a versatile platform for parallel detection of multiple nucleic acid targets.

Main Methods:

• CRISPR-RDB integrates CRISPR-Cas12a trans-cleavage with commercial RDB techniques.
• Different Cas12a-crRNA complexes are used to identify multiple targets in a single sample.
• Colorimetric signals are generated on a nylon membrane with specific oligonucleotide probes.

Main Results:

• A four-channel CRISPR-RDB system successfully detected influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2 simultaneously.
• The method demonstrated versatility by adapting to detect human papillomavirus, reducing detection time significantly compared to PCR-RDB.
• A user-friendly microchip system and smartphone app were developed for convenient use and signal interpretation.

Conclusions:

• CRISPR-RDB offers a sensitive, specific, and cost-effective solution for multiplex nucleic acid detection.
• The developed system is instrument-free, easy to use, and provides visual results.
• CRISPR-RDB is a promising technology for multiplexed biosensing and on-site diagnostics.