Plug-and-Play Photo-Initiated CRISPR-Cas12a One-Pot Nucleic Acid Detection via Universal Repeat RNA Acylation...

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• CRISPR technology requires precise spatiotemporal control for gene editing and diagnostics.
• Current regulatory methods are often sequence-specific, labor-intensive, and difficult to generalize.

Purpose of the Study:

• To develop a generalized, light-inducible CRISPR system for enhanced molecular diagnostics and gene editing.
• To create a streamlined, one-pot assay with improved sensitivity and accuracy.

Main Methods:

• Acylation of the split CRISPR RNA (crRNA) repeat region with photolabile groups, enabling light-activated control.
• Integration of the photo-initiated CRISPR-Cas12a system with recombinase polymerase amplification into a one-pot format (POIROTv2).

Main Results:

• POIROTv2 demonstrated a 100-fold increase in sensitivity compared to conventional Cas12a assays.
• The system achieved analytical performance comparable to two-step assays in a streamlined, one-pot format.
• Clinical validation showed diagnostic accuracy statistically indistinguishable from qPCR for HCMV and EBV detection.

Conclusions:

• The developed photo-initiated CRISPR-Cas12a system (POIROTv2) offers a robust, sensitive, and generalized platform for molecular diagnostics.
• This strategy provides spatiotemporal control over CRISPR activity, with broad applications in infectious disease diagnostics and gene editing.