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

作者：Jinlong Ai, Jinhai Deng, Jingjing Hu, Xingxiang Pu, Tongyan Yuan, Yanling Teng, Han Li, Bojie Chen, Jinlian Du, Ling Jiang, Xiaoyan Chen, Erhu Xiong, Ronghua Yang · 发表于：JACS Au · 年份：2025 · DOI：10.1021/jacsau.5c00011 · 被引用次数：27 · 研究领域：CRISPR and Genetic Engineering、Innovation and Socioeconomic Development、Genetics, Aging, and Longevity in Model Organisms

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.