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TitleStructural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.
Journal, issue, pagesNat Struct Mol Biol, Vol. 33, Issue 7, Page 1062-1074, Year 2026
Publish dateJun 24, 2026
AuthorsMin Duan / Bing Meng / Lei Zhou / Lijie Wu / Xiaohan Tong / Dongchao Huang / Hao Yin / Zhi-Jie Liu / Ying Zhang /
PubMed AbstractThe 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 ...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 NCNNN and NRNNA (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.
External linksNat Struct Mol Biol / PubMed:42342973
MethodsEM (single particle)
Resolution2.41 - 3.15 Å
Structure data

65811

9wac

EMDB-65811, PDB-9wac:
Cryo-EM structure of AtCas9-sgRNA-underwound DNA (TTGA) ternary complex
Method: EM (single particle) / Resolution: 2.89 Å

65812

9wad

EMDB-65812, PDB-9wad:
Cryo-EM structure of AtCas9-sgRNA-B-form DNA ternary complex
Method: EM (single particle) / Resolution: 3.15 Å

67605

21dz

EMDB-67605, PDB-21dz:
Cryo-EM structure of AtCas9-sgRNA-underwound DNA (CATA PAM) ternary complex
Method: EM (single particle) / Resolution: 2.41 Å

67606

21ea

EMDB-67606, PDB-21ea:
Cryo-EM structure of AtCas9-sgRNA-underwound DNA (TATA PAM) ternary complex
Method: EM (single particle) / Resolution: 2.65 Å

Source
  • Alicyclobacillus tengchongensis (bacteria)
  • alicyclobacillus tolerans (bacteria)
KeywordsRNA BINDING PROTEIN/RNA/DNA / Complex / Endonuclease / Immunity / RNA BINDING PROTEIN-RNA-DNA complex

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