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<p>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&#xa0;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.</p>

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SIMPLE: A One-Pot RPA-Cas12b Method for Single Nucleotide Variations Identification with an Expanded Scope and Clinical Applications

  • 期刊论文

  • 发表日期：

    2025年6月8日

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  • Kaiming Guo,
  • Yongkang Lai,
  • Yiru Han,
  • Linlin Bai,
  • Ting Wang,
  • Shengzhou Wang,
  • Ziqin Zhang,
  • Dianwei Liu,
  • Yanju Chen,
  • Jiulong Zhao,
  • Yanan Pang,
  • Feng Qian,
  • Yanni Mo,
  • Yongming Wang,
  • Rui Wang

摘要

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.