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Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

Fuchsbauer O, Swuec P, Zimberger C, Amigues B, Levesque S, Agudelo D, Duringer A, Chaves-Sanjuan A, Spinelli S, Rousseau GM, Velimirovic M, Bolognesi M, Roussel A, Cambillau C, Moineau S, Doyon Y, Goulet A
Mol Cell 76 922-937.e7 (2019)
Related entries: 6rja, 6rjd, 6rjg

Cited: 26 times
EuropePMC logo PMID: 31604602

Abstract

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

Reviews citing this publication (10)

  1. Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies. Marino ND, Pinilla-Redondo R, Csörgő B, Bondy-Denomy J. Nat Methods 17 471-479 (2020)
  2. Controlling and enhancing CRISPR systems. Shivram H, Cress BF, Knott GJ, Doudna JA. Nat Chem Biol 17 10-19 (2021)
  3. Structures and Strategies of Anti-CRISPR-Mediated Immune Suppression. Wiegand T, Karambelkar S, Bondy-Denomy J, Wiedenheft B. Annu Rev Microbiol 74 21-37 (2020)
  4. Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins. Jia N, Patel DJ. Nat Rev Mol Cell Biol 22 563-579 (2021)
  5. How low can we go? Structure determination of small biological complexes using single-particle cryo-EM. Wu M, Lander GC. Curr Opin Struct Biol 64 9-16 (2020)
  6. Type II anti-CRISPR proteins as a new tool for synthetic biology. Zhang Y, Marchisio MA. RNA Biol 18 1085-1098 (2021)
  7. A comprehensive appraisal of mechanism of anti-CRISPR proteins: an advanced genome editor to amend the CRISPR gene editing. Choudhary N, Tandi D, Verma RK, Yadav VK, Dhingra N, Ghosh T, Choudhary M, Gaur RK, Abdellatif MH, Gacem A, Eltayeb LB, Alqahtani MS, Yadav KK, Jeon BH. Front Plant Sci 14 1164461 (2023)
  8. Mechanisms regulating the CRISPR-Cas systems. Zakrzewska M, Burmistrz M. Front Microbiol 14 1060337 (2023)
  9. Inhibitors of bacterial immune systems: discovery, mechanisms and applications. Mayo-Muñoz D, Pinilla-Redondo R, Camara-Wilpert S, Birkholz N, Fineran PC. Nat Rev Genet 25 237-254 (2024)
  10. The Many (Inter)faces of Anti-CRISPRs: Modulation of CRISPR-Cas Structure and Dynamics by Mechanistically Diverse Inhibitors. Belato HB, Lisi GP. Biomolecules 13 264 (2023)

Articles citing this publication (16)



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