Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but are generally specific for a narrow subset of Cas nucleases that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits diverse Cas9 sub-types in vitro and human cells. Single-molecule fluorescence imaging reveals that AcrIIA11 interferes with the first steps of target search by reducing S. aureus Cas9’s diffusion on non-specific DNA. DNA cleavage is inhibited because the AcrIIA11:Cas9 complex is kinetically trapped at PAM-rich decoy sites, preventing Cas9 from reaching its target. This work establishes that DNA trapping can be used to inhibit a broad spectrum of Cas9 orthologs in vitro and during mammalian genome editing.