8YE6

Cryo-EM structure of Cas9-sgRNA-A32 complex

Summary for 8YE6

• Entry DOI: 10.2210/pdb8ye6/pdb
• EMDB information: 39190
• Descriptor: A32, CRISPR-associated endonuclease Cas9/Csn1, RNA (92-MER) (3 entities in total)
• Functional Keywords: crispr, complex, immune system/rna, immune system-rna complex
• Biological source: Streptococcus; More
• Total number of polymer chains: 3
• Total formula weight: 205227.51

Authors

Zheng, J.,Zhu, Y.,Huang, Z. (deposition date: 2024-02-22, release date: 2024-09-11)

Primary citation

Zheng, J.,Zhu, Y.,Huang, T.,Gao, W.,He, J.,Huang, Z.
Inhibition mechanisms of CRISPR-Cas9 by AcrIIA25.1 and AcrIIA32.
Sci China Life Sci, 67:1781-1791, 2024

PubMed Abstract: In the ongoing arms race between bacteria and bacteriophages, bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems. Recently, AcrIIA25.1 and AcrIIA32 have been found to effectively inhibit the activity of SpyCas9 both in bacterial and human cells. However, their molecular mechanisms remain elusive. Here, we report the cryo-electron microscopy structures of ternary complexes formed by AcrIIA25.1 and AcrIIA32 bound to SpyCas9-sgRNA. Using structural analysis and biochemical experiments, we revealed that AcrIIA25.1 and AcrIIA32 recognize a novel, previously-unidentified anti-CRISPR binding site on SpyCas9. We found that both AcrIIA25.1 and AcrIIA32 directly interact with the WED domain, where they spatially obstruct conformational changes of the WED and PI domains, thereby inhibiting SpyCas9 from recognizing protospacer adjacent motif (PAM) and unwinding double-stranded DNA. In addition, they may inhibit nuclease activity by blocking the dynamic conformational changes of the SpyCas9 surveillance complex. In summary, our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins, provide new strategies for the modulation of SpyCas9 activity, and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.

PubMed: 38842649
DOI: 10.1007/s11427-024-2607-8

Experimental method

ELECTRON MICROSCOPY (2.95 Å)