Analytical chemistryOct 23, 2026

Using Split Cas12a Design for Highly Specific Detection of DNA Strands Without a PAM Sequence

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Oct 24, 2026

Abstract

The split-duplex architecture enhances specificity for detecting PAM-less DNA sequences.

The DNA activator is divided into PAM-proximal (Pp) and PAM-distal (Pd) fragments to separate the PAM requirement from target sequence interrogation.
Robust activation of Cas12a requires both the PAM-containing Pp duplex and the Pd duplex.
PAM-mediated protein-DNA contacts are suggested to stabilize the complex necessary for effective target capture.
Weakening the hybridization between the guide and Pp increased the reliance on PAM stabilization and enhanced selectivity.
This approach allows for improved resolution of single-nucleotide polymorphisms in PAM-less DNA strands.

Simplified

CRISPR-Cas12a is a programmable RNA-guided endonuclease whose activation by double-stranded DNA normally depends on the recognition of an adjacent protospacer-adjacent motif (PAM), which constrains target selection in diagnostic applications. Here, we divide the DNA activator into PAM-proximal (Pp) and PAM-distal (Pd) duplexes, spatially separating the PAM requirement from the interrogation of a PAM-less target sequence. FRET, electrophoretic mobility shift, fluorescence polarization, and functional assays indicate that either fragment can associate with Cas12a, but that robust productive activation requires the PAM-containing Pp duplex together with the Pd duplex. Our data further support a model in which PAM-mediated protein-DNA contacts stabilize an assembly-competent complex and facilitate binding of the PAM-less Pd target capture. Weakening hybridization between the guide and Pp with a chimeric DNA/RNA increased the dependence on PAM-mediated stabilization and improved structural selectivity in the Pp region and single-nucleotide specificity in the Pd region. The resulting split-duplex architecture provides an ultraspecific platform for resolving single-nucleotide polymorphisms in the PAM-less DNA strand.

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Using Split Cas12a Design for Highly Specific Detection of DNA Strands Without a PAM Sequence

Abstract

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