Temperature-Responsive Competitive Inhibition of CRISPR-Cas9

• Published in: Molecular cell 2019-02, Vol.73 (3), p.601-610.e5
• Main Authors: Jiang, Fuguo, Liu, Jun-Jie, Osuna, Beatriz A., Xu, Michael, Berry, Joel D., Rauch, Benjamin J., Nogales, Eva, Bondy-Denomy, Joseph, Doudna, Jennifer A.
• Format: Article
• Language: English
• Subjects: AcrIIA2; AcrllA2; anti-CRISPR; bacteriophage; BASIC BIOLOGICAL SCIENCES; Binding, Competitive; Cas9; Cas9 inhibitors; CRISPR-Associated Protein 9 - antagonists & inhibitors; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Associated Protein 9 - ultrastructure; CRISPR-Cas; CRISPR-Cas Systems; CRISPR-Cas9; Cryoelectron Microscopy; DNA - genetics; DNA - metabolism; DNA - ultrastructure; Escherichia coli - enzymology; Escherichia coli - genetics; Gene Editing - methods; Gene Expression Regulation, Bacterial; genome editing; Models, Molecular; Nucleic Acid Conformation; PAM blockage; Protein Binding; Protein Conformation; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - virology; Pseudomonas Phages - genetics; Pseudomonas Phages - metabolism; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; RNA, Guide, CRISPR-Cas Systems - ultrastructure; Structure-Activity Relationship; Temperature; temperature-dependent inhibition; Viral Proteins - genetics; Viral Proteins - metabolism; Viral Proteins - ultrastructure
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2018.11.016

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory “anti-CRISPR” (Acr) proteins, including six inhibitors (AcrIIA1–AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally. [Display omitted] •Atomic cryo-EM structure of AcrIIA2- or AcrIIA2b-bound SpyCas9-sgRNA complex•AcrIIA2 inhibitor family shows a convergent Cas9 inhibition mechanism with AcrIIA4•AcrIIA2 exhibits a strong temperature-dependent anti-CRISPR activity Jiang et al. report cryo-EM structures of type II-A anti-CRISPRs (AcrIIA2 and its homolog AcrIIA2b) bound to S. pyogenes Cas9, revealing a convergent inhibition mechanism between AcrIIA2 and AcrIIA4. The temperature-dependent differences between AcrIIA2 and AcrIIA2b provide an interesting condition-dependent variable that could be exploited for developing Cas9-based tools.