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<p>Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5′ side favor <i>trans</i>-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although <i>trans</i>-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.</p>

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CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月24日

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  • Roser Montagud-Martínez,
  • Raúl Ruiz,
  • Sara Baldanta,
  • Rubén Delicado-Mateo,
  • Guillermo Rodrigo

摘要

Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5′ side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.