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Cryo-EM structures of multicomponent complexes CRISPR single guide RNA, target DNA, Cas9 and the dimeric AcrIIA6 phage protein

The CRISPR/Cas9 bacterial system (in fact a true bacterial “immune response” system) attracted a large interest over the past few years thanks to its versatility for the production of genome editing tools. In nature, the transcripts of CRISPR DNA sequences that accumulate during phage infections in bacteria, can drive the bacterial endonuclease Cas9 to specific sequences within the DNA of infecting bacteriophages. Such specific targeting allows double strand cleavage of invading DNA, thus halting the viral infection.  Conversely, in response to such defense apparatus, phages have evolved protein systems that allow blocking the Cas9 nuclease activity through various mechanisms.      

One such system (AcrIIA6) has been presented in a recent single particle cryo EM analysis developed through a collaboration between Aix-Marseille University (Architecture et Fonction des Macromolécules Biologiques, CNRS, Luminy Campus, France), Laval University (Département de biochimie, de microbiologie, et de bio-informatique, Quebec, Canada), and the cryo-EM lab at the University of Milano (Dept. Biosciences). This study presents four cryo-EM structures (in the 3.0 – 3.3 Å resolution range) that encompass in multicomponent complexes CRISPR single guide RNA, target DNA, Cas9 (from Streptococcus thermophilus) and the dimeric AcrIIA6 phage protein (2 x 183residues). The cryo EM analyses show that the homodimeric AcrIIA6 protein can bind one or two Cas9 complexes at the same time (see Figure), and that the nuclease inhibitory mechanism relies on an allosteric effect whereby binding of the inhibitor protein alters the structural and dynamic properties of a Cas9 region involved in binding of the protospacer adjacent motif (PAM) sequence presented by the target DNA stretch. More details on the cryo-EM and associated biochemical analyses re reported in Fuchsbauer, Swuec et al. (2019) Molecular Cell, 76, 1-16.

Figure caption: The Cas9 (light blue)/sgRNA(green)/target DNA(yellow)/AcrIIA6 dimer(purple) complex in its monomeric and dimeric assemblies. Non all Cas9 domains are fully resolved, as previously observed, due to conformational disorder.