PAM-Independent CRISPR-Cas12a System for Specific Assays of Single Nucleotide Variants

The CRISPR-Cas12a system has been extensively utilized in nucleic acid detection owing to its remarkable sensitivity and specificity. Nonetheless, its strict dependency on the presence of a protospacer adjacent motif (PAM) within double-stranded DNA (dsDNA) introduces considerable limitations, thereby constraining its applicability, flexibility, and broader accessibility in molecular diagnostics. Here, we communicate a universal, robust, and high-fidelity method for a PAM-independent nucleic acid assay based on the CRISPR-Cas12a system, named TRACER (mutant target-recognized PAM-independent CRISPR-Cas12a enzyme reporting system). TRACER can effectively distinguish target nucleic acids at concentrations as low as 0.5 aM, thereby enabling it to identify the presence of a 0.1% single nucleotide variant (SNV)-included mutant-type gene in heterozygotes. Thus, TRACER exhibits comparable sensitivity, specificity, and accuracy to Sanger sequencing in analyzing the SNV-related clinical tumor samples. Overall, TRACER introduces a brand-new perspective for SNV assays by eliminating the dependency on PAM sites and significantly expands the application range of the CRISPR-Cas12a system, thus holding immense potential for clinical diagnostics, biomedical research, and drug discovery.