Highly specific detection of single gene mutations using a Cas12a system without PAM that unwinds double-stranded DNA

The PAM-free Cas12a system achieved an ultra-low limit of detection of 0.013% for gene point mutations.

• The developed system utilizes double-stranded substrate positioning-unwinding to facilitate detection.
• Bubble structures formed by unpaired base pairs help release a partial single-stranded target strand.
• Local unwinding of double-stranded DNA is induced by excess auxiliary strands to aid R-loop formation.
• The system demonstrated excellent linearity for mutation abundance ranging from 0-10%.
• It showed robust feasibility and accuracy in detecting the JAK2 V617F mutation in patient blood samples.

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