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<p>CRISPR–Cas12a is a versatile RNA-guided nuclease that has rapidly gained prominence for its dual functionality in genome editing and nucleic acid detection. In this Review, we discuss the structural, biochemical and mechanistic features of Cas12a that underpin its autonomous processing of the guide RNA and indiscriminate cleavage of single-stranded DNA, which enable Cas12a applications ranging from gene therapy to rapid diagnostics. We discuss key allosteric regulators and functional modules that orchestrate Cas12a activity, focusing on the core regulatory structural elements that control maturation of the guide RNA, target specificity, and both <i>cis</i>-cleavage and <i>trans</i>-cleavage activities, including the determinants of off-target cleavage. We provide a comparative analysis of Cas12a and the widely used Cas9, which further illuminates the distinctive attributes of Cas12a, and discuss recent advances in the characterization of its orthologues and in the development of engineered variants that expand its capabilities. Collectively, we present a comprehensive understanding of Cas12a and its increasing impact on biotechnology, therapeutics and molecular diagnostics.</p>

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Molecular mechanisms and biotechnology applications of CRISPR–Cas12a

  • 期刊论文

  • 发表日期：

    2026年4月27日

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  • Aakash Saha,
  • Rodrigo Fregoso Ocampo,
  • Jacquelyn T. Wright,
  • David W. Taylor,
  • Giulia Palermo

摘要

CRISPR–Cas12a is a versatile RNA-guided nuclease that has rapidly gained prominence for its dual functionality in genome editing and nucleic acid detection. In this Review, we discuss the structural, biochemical and mechanistic features of Cas12a that underpin its autonomous processing of the guide RNA and indiscriminate cleavage of single-stranded DNA, which enable Cas12a applications ranging from gene therapy to rapid diagnostics. We discuss key allosteric regulators and functional modules that orchestrate Cas12a activity, focusing on the core regulatory structural elements that control maturation of the guide RNA, target specificity, and both cis-cleavage and trans-cleavage activities, including the determinants of off-target cleavage. We provide a comparative analysis of Cas12a and the widely used Cas9, which further illuminates the distinctive attributes of Cas12a, and discuss recent advances in the characterization of its orthologues and in the development of engineered variants that expand its capabilities. Collectively, we present a comprehensive understanding of Cas12a and its increasing impact on biotechnology, therapeutics and molecular diagnostics.