Disarming of type IF CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9

Egle Kupcinskaite, Marijonas Tutkus, Aurimas Kopūstas, Simonas Ašmontas, Marija Jankunec, Mindaugas Zaremba, Giedre Tamulaitiene, Tomas Sinkunas (see publication in Journal )

Abstract

CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection.