Molecular mechanism of CRISPR-SpyCas9 inhibition by AcrIIA26

In the ongoing arms race between bacteria and phages, CRISPR-Cas systems and anti-CRISPR proteins (Acrs) have evolved to counteract one another. Recently, AcrIIA26 from Streptococcus sp. was identified as a modulator of SpyCas9 activity, although its molecular mechanism remains unclear. Here, we present the cryo-EM structure of the SpyCas9-sgRNA-AcrIIA26 complex, revealing a two-domain architecture. The 5A domain of AcrIIA26 binds the PI and WED domains of Cas9, while the 4A domain contacts the REC2 domain. This dual interaction sterically blocks target DNA binding and prevents the conformational changes required for cleavage. Notably, AcrIIA26's sgRNA-independent binding to Cas9 allows the modulation of gene editing across an extended temporal window. Our study elucidates the molecular mechanism of AcrIIA26 and provides novel strategies for precise SpyCas9 regulation.