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TitleStructural basis for Cas9-directed spacer acquisition in type II-A CRISPR-Cas systems.
Journal, issue, pagesMol Cell, Vol. 86, Issue 5, Page 805-816.e4, Year 2026
Publish dateMar 5, 2026
AuthorsZhaoxing Li / Yutao Li / Jianping Kong / Qianqian Wu / Pingping Huang / Yu Zhang / Wanqian Wu / Meirong Chen / Yongfeng Liu / HanFeng Lin / Liqiu Hou / Gongyu Liu / Ting Zeng / Yutong He / Chunyi Hu / Zhenhuang Yang / Meiling Lu / Min Luo / Yibei Xiao /
PubMed AbstractCRISPR-Cas systems confer prokaryotic immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer-adjacent motif (PAM) recognition and coordinate ...CRISPR-Cas systems confer prokaryotic immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer-adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-electron microscopy (cryo-EM) structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo state (Cas9₂-Csn2₈-Cas1₈-Cas2₄) is a resting complex, while DNA binding forms a prespacer-catching complex threading DNA through Csn2's channel, enabling Cas9 to interrogate the PAM sequence while sliding along the DNA. Cas9 and Csn2 jointly define a 30-bp DNA segment matching the prespacer length. Cas9 dissociation triggers structural reconfiguration of the Csn2-Cas1-Cas2 assembly. This exposes the PAM-proximal DNA, allowing Cas1-Cas2 to bind the exposed site for subsequent prespacer processing and directional integration. These findings reveal how Cas9, Csn2, and Cas1-Cas2 couple PAM recognition with prespacer selection, ensuring fidelity during adaptation.
External linksMol Cell / PubMed:41702404
MethodsEM (single particle)
Resolution2.96 - 3.9 Å
Structure data

65106

9vj8

EMDB-65106, PDB-9vj8:
Type II-A CRISPR integrase complex, apo form
Method: EM (single particle) / Resolution: 3.9 Å

65107

9vj9

EMDB-65107: Raw consensus map of Type II-A CRISPR integrase prespacer catching complex, State I
PDB-9vj9: Type I-A CRISPR integrase prespacer catching complex, State I
Method: EM (single particle) / Resolution: 2.96 Å

65108

9vja

EMDB-65108, PDB-9vja:
Type I-A CRISPR integrase prespacer catching complex, State II
Method: EM (single particle) / Resolution: 3.01 Å

65109

9vjb

EMDB-65109, PDB-9vjb:
Type II-A CRISPR integrase pre-integration complex
Method: EM (single particle) / Resolution: 3.76 Å

Chemicals

ChemComp-CA:
Unknown entry

Source
  • enterococcus faecalis (bacteria)
  • synthetic construct (others)
KeywordsDNA BINDING PROTEIN / CRISPR / Cas9 / Cas1 / Cas2 / Csn2 / DNA BINDING PROTEIN/DNA/RNA / Csn2 DNA binding protein / DNA BINDING PROTEIN-DNA-RNA complex / DNA BINDING PROTEIN/DNA / DNA BINDING PROTEIN-DNA complex

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