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<p>The CRISPR–Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C <i>Alicyclobacillus tengchongensis</i> Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for N<sub>4</sub>CNNN and N<sub>4</sub>RNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.</p>

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Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology

  • 期刊论文

  • 发表日期：

    2026年6月24日

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  • Min Duan,
  • Bing Meng,
  • Lei Zhou,
  • Lijie Wu,
  • Xiaohan Tong,
  • Dongchao Huang,
  • Hao Yin,
  • Zhi-Jie Liu,
  • Ying Zhang

摘要

The CRISPR–Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for N4CNNN and N4RNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.