Breaking the PAM Restriction: A Universal Double Stranded DNA Detection Method Based on the Sticky End-Mediated...

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR/Cas12a systems offer precise nucleic acid detection but face limitations due to Protospacer Adjacent Motif (PAM) requirements and complex sticky-end methods.
• Existing methods hinder stable and portable applications for CRISPR/Cas12a-based diagnostics.

Purpose of the Study:

• To develop a universal, PAM-free CRISPR/Cas12a detection platform.
• To enhance CRISPR/Cas12a activity and simplify detection methods for molecular diagnostics.

Main Methods:

• Integrated sticky-end-mediated CRISPR/Cas12a with recombinase polymerase amplification (RPA).
• Incorporated NlaIII recognition sites into RPA primers for uniform sticky end generation.
• Compared sticky-end dsDNA with PAM-containing flat-end dsDNA for Cas12a activity.

Main Results:

• Achieved a detection limit of 40 aM for nucleic acids.
• Successfully identified KRAS G12C mutations at 0.1% frequency.
• Demonstrated enhanced Cas12a activity with sticky-end dsDNA compared to PAM-dependent methods.
• Preliminary one-tube strategy streamlined operations and reduced contamination.

Conclusions:

• Established a simple, sensitive, and universal PAM-free CRISPR/Cas12a detection platform.
• Integrated isothermal amplification with standardized sticky ends for broad diagnostic applications.
• The platform shows significant potential for molecular diagnostics and clinical translation.