7s4u
Cryo-EM structure of Cas9 in complex with 12-14MM DNA substrate, 5 minute time-point
Structural highlights
FunctionCAS9_STRP1 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) (Probable). 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. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.[1] [2] Publication Abstract from PubMedCRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage(1-4), and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood(5-7). Although Cas9 variants with greater discrimination against mismatches have been designed(8-10), these suffer from substantially reduced rates of on-target DNA cleavage(5,11). Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants. Structural basis for mismatch surveillance by CRISPR-Cas9.,Bravo JPK, Liu MS, Hibshman GN, Dangerfield TL, Jung K, McCool RS, Johnson KA, Taylor DW Nature. 2022 Mar;603(7900):343-347. doi: 10.1038/s41586-022-04470-1. Epub 2022 , Mar 2. PMID:35236982[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
| ||||||||||||||||||
