CRISPR/Cas-mediated "one to more" lighting-up nucleic acid detection using aggregation-induced emission luminogens

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Chemical Biology

Background:

• CRISPR diagnostics offer potential but face limitations in signal transduction, sensitivity, and stability.
• Current methods often rely on trans-cleavage of single-stranded nucleic acid fluorescent reporters, hindering efficiency.

Purpose of the Study:

• To develop an enhanced CRISPR diagnostic platform, CrisprAIE, with improved signal transduction and sensitivity.
• To integrate CRISPR/Cas reactions with aggregation-induced emission luminogens (AIEgens) for a novel "one to more" fluorescence lighting-up strategy.

Main Methods:

• Developed CrisprAIE by combining CRISPR/Cas systems with AIEgens incorporated into double-stranded DNA reporters (Q-dsDNA/AIEgens-Q).
• Utilized the trans-cleavage activity of Cas proteins to trigger AIEgen fluorescence.
• Integrated the system with spherical nucleic acid-modified AIEgens (SNA/AIEgens) and a portable cellphone-based readout device.

Main Results:

• CrisprAIE demonstrated superior performance in detecting norovirus and SARS-CoV-2 nucleic acids without amplification.
• The enhanced CrisprAIE system showed 80-fold and 270-fold improvements in sensitivity using Q-dsDNA/AIEgen-Q and SNA/AIEgen reporters, respectively.
• Achieved significant sensitivity enhancements compared to conventional CRISPR diagnostics.

Conclusions:

• CrisprAIE offers a robust and highly sensitive platform for clinical nucleic acid detection.
• The developed system can be extended as a universal signal generation strategy to improve existing CRISPR-based diagnostics.
• This approach holds promise for advancing rapid and sensitive molecular diagnostic tools.