Abstract
Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR) system is emerging as a next-generation nucleic acid detection technology, offering versatility, convenience and rapid detection. However, CRISPR methods are significantly limited by the protospacer adjacent motif (PAM) sequence, and achieving a one-pot reaction for detecting single nucleotide variations (SNVs) within a short time still remains challenging. Here, we developed a comprehensive method for screening PAM sequences, which significantly expands the CRISPR detection scope. Additionally, we also proposed a one-pot CRISPR method, termed "SIMPLE", capable of identifying SNVs within 30 min. We applied the SIMPLE method to the clinical diagnostics of drug-resistant bacteria and the screening of cancer hotspot mutations. The SIMPLE method successfully detected drug-resistant bacteria mediated by canonical PAM TTN sequence with a sensitivity of 10 copies per reaction and achieved 100% consistency with next-generation sequencing results. Furthermore, the SIMPLE method proved effective in detecting hotspot mutations in cancer, even at a low mutation rate of 1% in the presence of high background interference mediated by non-canonical PAM ATN sequence. Therefore, the SIMPLE method not only expands the CRISPR detection scope but also offers a one-pot reaction with high specificity for SNVs identification, making it a promising tool for next-generation molecular diagnostics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43657-025-00240-x.