A universal light-controlled highly sensitive one-pot CRISPR/Cas12a diagnostic based on structure-engineered crRNA.

Area of Science:

• Molecular diagnostics
• Biotechnology
• CRISPR-based detection

Background:

• CRISPR-based nucleic acid detection offers speed and accuracy.
• One-pot formats face limitations in sensitivity and field suitability.

Purpose of the Study:

• To develop a universal light-controlled, high-sensitivity, one-pot CRISPR/Cas12a testing (ULTRAt) platform.
• To overcome sensitivity and enrichment limitations in existing one-pot CRISPR assays.

Main Methods:

• Developed structure-engineered CRISPR RNA (crRNA) scaffolds with photocaged groups.
• Utilized UV irradiation to control Cas12a activity and enable target enrichment.
• Integrated isothermal amplification with CRISPR/Cas12a trans-cleavage.

Main Results:

• Achieved a limit of detection of two copies of monkeypox virus per reaction.
• Demonstrated a 100-fold sensitivity improvement over conventional assays with a 15-min time-to-result.
• Successfully performed single-nucleotide polymorphism discrimination and HPV genotyping, with strong clinical sample concordance.

Conclusions:

• The ULTRAt platform enables rapid, ultra-sensitive, and versatile one-pot detection.
• ULTRAt supports near-patient diagnostics and genotyping applications.
• This technology advances molecular diagnostics with improved sensitivity and field suitability.