Dual-target CRISPR-Cas12 diagnostics based on asymmetrically chemical-modified DNA probe

Area of Science:

• Molecular Biology
• Biotechnology
• Nucleic Acid Detection

Background:

• CRISPR-based nucleic acid detection offers rapid, precise, and cost-effective target identification.
• Current limitations exist in achieving simultaneous multi-target detection within a single reaction tube.
• Further research is needed to overcome these challenges in multiplexed detection.

Purpose of the Study:

• To develop a novel nucleic acid detection module based on the CRISPR-Cas12i system.
• To engineer an effective dual-target nucleic acid detection platform by combining Cas12i and AapCas12b nucleases.
• To demonstrate the platform's capability for simultaneous detection of multiple nucleic acid targets.

Main Methods:

• Utilized the CRISPR-Cas12i system for nucleic acid detection.
• Investigated the trans-cleavage preferences of Cas12i and AapCas12b with modified DNA probes.
• Combined Cas12i and AapCas12b nucleases for dual-target detection in a single tube.
• Integrated loop-mediated isothermal amplification (LAMP) for enhanced sensitivity.

Main Results:

• Identified opposite trans-cleavage preferences between Cas12i and AapCas12b for specific DNA probes.
• Developed a dual-target detection platform by combining these two Cas12 nucleases.
• Demonstrated high specificity and sensitivity in detecting human papillomavirus (HPV) 16/18 and Influenza A virus (FluA)/Respiratory syncytial virus.
• Achieved high detection rates in clinical samples when combined with LAMP (18/18 FluA and 18/18 GAPDH).

Conclusions:

• The developed CRISPR-Cas12i based dual-target detection platform effectively overcomes limitations in multiplexed nucleic acid detection.
• The differential cleavage preferences of Cas12i and AapCas12b enable simultaneous detection of multiple targets in one tube.
• This platform shows significant potential for broad applications in infectious disease diagnostics and environmental monitoring.