6rjd
Cryo-EM structure of St1Cas9-sgRNA-tDNA59-ntPAM complex.Cryo-EM structure of St1Cas9-sgRNA-tDNA59-ntPAM complex.
Structural highlights
FunctionCAS9A_STRTD CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). Cas9 recognizes the protospacer adjacent motif (PAM) in the CRISPR repeat sequences to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. PAM recognition is also required for catalytic activity (By similarity). Cuts target DNA when Cas9 and gRNAs are mixed.[HAMAP-Rule:MF_01480][1] Publication Abstract from PubMedIn 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. 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. 2019 Sep 26. pii: S1097-2765(19)30697-5. doi:, 10.1016/j.molcel.2019.09.012. PMID:31604602[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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