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<p>CRISPR technology has profoundly transformed both genome editing and the next generation of nucleic acid detection. In this study, we characterized a Cas12a ortholog, Gs12-9 (EsoCas12a), which recognizes a broad PAM motif (NYYN, where Y denotes C or T) and maintains robust <i>cis</i>- and <i>trans</i>-cleavage activity across a wide temperature range (16–60 ℃), outperforming established Cas12a variants such as LbCas12a. Leveraging Gs12-9, we established a rapid, highly sensitive, and temperature-tolerant nucleic acid detection platform that enables precise genotyping without stringent thermal control. By integrating recombinase polymerase amplification (RPA) with CRISPR/Gs12-9-mediated detection, we accurately discriminated myostatin (<i>MSTN</i>) knockout pigs from wild-type individuals, achieving a detection limit of 40 copies per reaction with 100% accuracy. While the <i>MSTN</i> knockout (KO) pig served as a proof-of-concept model in this work, the versatility of Gs12-9 supports its broader application in CRISPR-based diagnostics, including pathogen screening and genetic variant detection.</p>

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A rapid and highly sensitive CRISPR-Cas12a ortholog-assisted assay for genotyping of myostatin knockout pigs

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月29日

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  • Yuan Wang,
  • Hui Yang,
  • Wenhua Zhang,
  • Dagang Tao,
  • Suyu Shi,
  • Sheng Li,
  • Xiao Wu,
  • Yunlong Ma,
  • Jinxue Ruan,
  • Lu Jing,
  • Kang Ma,
  • Xinyun Li,
  • Xiaosong Han,
  • Xuewen Xu,
  • Shengsong Xie

摘要

CRISPR technology has profoundly transformed both genome editing and the next generation of nucleic acid detection. In this study, we characterized a Cas12a ortholog, Gs12-9 (EsoCas12a), which recognizes a broad PAM motif (NYYN, where Y denotes C or T) and maintains robust cis- and trans-cleavage activity across a wide temperature range (16–60 ℃), outperforming established Cas12a variants such as LbCas12a. Leveraging Gs12-9, we established a rapid, highly sensitive, and temperature-tolerant nucleic acid detection platform that enables precise genotyping without stringent thermal control. By integrating recombinase polymerase amplification (RPA) with CRISPR/Gs12-9-mediated detection, we accurately discriminated myostatin (MSTN) knockout pigs from wild-type individuals, achieving a detection limit of 40 copies per reaction with 100% accuracy. While the MSTN knockout (KO) pig served as a proof-of-concept model in this work, the versatility of Gs12-9 supports its broader application in CRISPR-based diagnostics, including pathogen screening and genetic variant detection.