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<p>RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ—also referred to as Cas12n—is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.</p>

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Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年6月5日

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  • Satoshi N. Omura,
  • Lauren E. Alfonse,
  • Alexa Ornstein,
  • Hayato Morinaga,
  • Hisato Hirano,
  • Yuzuru Itoh,
  • Gabrielle Munoz,
  • Anthony J. Garrity,
  • Gregory R. Hoffman,
  • Tia DiTommaso,
  • Winston X. Yan,
  • David R. Cheng,
  • David A. Scott,
  • Zachary Maben,
  • Osamu Nureki

摘要

RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ—also referred to as Cas12n—is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.