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TitleStructural basis for inhibition of SpyCas9 by the anti-CRISPR protein AcrIIA26.
Journal, issue, pagesBiochem J, Year 2026
Publish dateJan 19, 2026
AuthorsScott Bailey / Iris Zheng / Brian Learn /
PubMed AbstractCRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this ...CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 Å resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-sgRNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central β-sheet flanked by two α-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the PAM duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple Acrs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications.
External linksBiochem J / PubMed:41553775
MethodsEM (single particle)
Resolution2.98 Å
Structure data

74542

9zq6

EMDB-74542, PDB-9zq6:
Structure of SpyCas9 in complex with the anti-CRISPR protein AcrIIA26
Method: EM (single particle) / Resolution: 2.98 Å

Source
  • streptococcus pyogenes m1 gas (bacteria)
  • streptococcus (bacteria)
KeywordsIMMUNE SYSTEM / CRISPR / Cas9 / Acr

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