Listeria Phages Induce Cas9 Degradation to Protect Lysogenic Genomes

• Published in: Cell host & microbe 2020-07, Vol.28 (1), p.31-40.e9
• Main Authors: Osuna, Beatriz A., Karambelkar, Shweta, Mahendra, Caroline, Christie, Kathleen A., Garcia, Bianca, Davidson, Alan R., Kleinstiver, Benjamin P., Kilcher, Samuel, Bondy-Denomy, Joseph
• Format: Article
• Language: English
• Subjects: anti-CRISPR; bacteriophage; Cas9; CRISPR-Cas; Listeria; lysogen; prophage
• ISSN: 1931-3128; 1934-6069
• DOI: 10.1016/j.chom.2020.04.001

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse “anti-CRISPR” proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1’s pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome. [Display omitted] •Listeria phages use independent Cas9 inhibitors to support lytic and lysogenic states•AcrIIA1 induces degradation of Cas9 during lysogeny, but is ineffective in lytic growth•AcrIIA1 binds directly to Apo or sgRNA-loaded Cas9 via the HNH domain•AcrIIA1 has a broad spectrum of inactivation, inhibiting many Listeria Cas9 orthologs Bacteriophages inactivate CRISPR-Cas immunity by encoding “anti-CRISPR” proteins. Osuna et al. reveal that a protein commonly encoded by Listeria phages, AcrIIA1, directly binds to the Cas9 HNH domain and stimulates its degradation to stabilize the lysogenic state, while the phages use an independent Acr protein for lytic replication.