5x2h
From Proteopedia
Crystal structure of Campylobacter jejuni Cas9 in complex with sgRNA and target DNA (AGAAACA PAM)
Structural highlights
Function[CAS9_CAMJE] 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 in Cas9:gRNAs mixing experiments with N.meningitidis strain Z2491 and P.multocoda strain Pm70.[HAMAP-Rule:MF_01480][1] Publication Abstract from PubMedThe RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.,Yamada M, Watanabe Y, Gootenberg JS, Hirano H, Ran FA, Nakane T, Ishitani R, Zhang F, Nishimasu H, Nureki O Mol Cell. 2017 Mar 16;65(6):1109-1121.e3. doi: 10.1016/j.molcel.2017.02.007. PMID:28306506[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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Categories: Camje | Large Structures | Hirano, H | Ishitani, R | Nakane, T | Nishimasu, H | Nureki, O | Watanabe, Y | Yamada, M | Complex | Crispr-cas9 | Dna | Hydrolase-rna-dna complex | Nuclease | Rna
