CONVERT: Dynamic crRNA Reconfiguration for Universal One-Pot CRISPR/Cas12a-Based Nucleic Acid Detection.

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Current one-pot CRISPR diagnostics face challenges in controlling hybridization and achieving complete enzymatic silencing, limiting their robustness.
• Existing methods often require extensive modifications or exogenous blockers, increasing complexity and cost.

Purpose of the Study:

• To develop a universal CRISPR diagnostic platform that overcomes limitations of current one-pot systems.
• To introduce a novel strategy using dynamic crRNA reconfiguration for enhanced CRISPR diagnostics.

Main Methods:

• Developed CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology) platform.
• Utilized dynamic crRNA reconfiguration with photocleavable linkers and truncated crRNA (tcrRNA) for Cas12a inhibition and activation.
• Implemented irreversible enzyme sequestration for Cas12a inhibition during recombinase polymerase amplification (RPA).

Main Results:

• Achieved complete Cas12a inhibition during RPA through engineered crRNA acting as a universal suppressor.
• Demonstrated attomolar sensitivity (1 aM) and 100% diagnostic concordance with qPCR for *Enterocytozoon bieneusi* detection.
• Showcased absolute specificity against related pathogens and contamination-resistant operation via spatiotemporal control.

Conclusions:

• CONVERT platform offers a generalized framework for next-generation molecular diagnostics by decoupling CRISPR regulation from sequence-context constraints.
• Dynamic crRNA reconfiguration provides a robust, universal, and cost-effective approach for CRISPR-based detection.
• The system eliminates the need for exogenous blockers or chemical modifications, simplifying workflow and reducing optimization time.