Combined Use of Engineered PfAGO and LbCas12a for Nucleic Acid Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• The COVID-19 pandemic highlighted the need for advanced nucleic acid detection methods.
• Existing technologies often lack the required speed, sensitivity, or accessibility for widespread use.

Purpose of the Study:

• To develop and validate NAEPLS (Nucleic Acid Detection Based on Engineered PfAgo and LbCas12a with Split crRNA), a novel diagnostic platform.
• To achieve ultrasensitive and specific detection of SARS-CoV-2 RNA for improved diagnostics.

Main Methods:

• Integration of engineered Pyrococcus furiosus PfAgo (mPfAgo) with CRISPR-Cas12a system.
• Utilized mPfAgo's RNA-cleaving ability and LbCas12a's split crRNA compatibility.
• Combined with Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) for enhanced sensitivity.

Main Results:

• NAEPLS detected SARS-CoV-2 RNA with a limit of detection (LOD) of 10 copies/mL when coupled with RT-RPA.
• Demonstrated single-nucleotide specificity, distinguishing key mutations like D614G and S371P.
• Achieved 100% concordance with RT-qPCR in clinical samples (Ct 20-39), including low viral loads (Ct > 35).

Conclusions:

• NAEPLS represents a transformative approach to nucleic acid diagnostics, merging PfAgo precision with CRISPR amplification.
• The platform's compatibility with lateral flow strips enables rapid point-of-care testing (POCT), especially in resource-limited areas.
• This technology offers high-performance, field-deployable pathogen detection capabilities.