Abstract

Rapid, specific, and instrument-independent detection of bacterial pathogens and antibiotic resistance genes remains a major challenge in clinical diagnostics. Recombinase polymerase amplification (RPA) integrated with CRISPR/Cas12a is a promising molecular diagnostic platform, but its performance is fundamentally limited by the strict requirement for protospacer adjacent motifs (PAM) to activate Cas12a. To address this constraint, we developed a P AM- E ngineered A basic-site probe-assisted R PA–CRISPR (PEAR) assay that introduces a structurally defined artificial PAM sequence directly during the RPA process. The system incorporates an Endonuclease IV (Nfo)-cleavable probe containing an abasic site (d’Spacer) and an embedded PAM motif. Upon target-specific probe hybridization and Nfo-mediated cleavage, the PAM sequence is precisely integrated into the newly synthesized amplicon, generating PAM-containing double-stranded DNA that efficiently triggers Cas12a trans-cleavage activity. This design enables PAM-independent target adaptability while maintaining high analytical sensitivity. Using plasmid-derived templates, PEAR successfully detected the Klebsiella pneumoniae species-specific khe gene and the carbapenem resistance gene bla KPC-2 , achieving a detection limit of 1 copy/μL with 100% analytical specificity. Evaluation with 30 clinical samples yielded diagnostic accuracies of 93.3% for khe and 87.7% for bla KPC-2 , demonstrating practical clinical applicability. Overall, PEAR represents a sensitive, specific, and potentially universal platform for CRISPR/Cas12a-mediated nucleic acid detection, providing a generalizable strategy to eliminate PAM dependence and expand translational applications in rapid pathogen detection and antimicrobial resistance surveillance.