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

Authors

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

Published in

Nature structural & molecular biology. Jun 24, 2026. Epub Jun 24, 2026.

Abstract

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.

PMID:
42342973
Bibliographic data and abstract were imported from PubMed on 25 Jun 2026.

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