Your browser doesn't support javascript.
loading
Structural basis for mismatch surveillance by CRISPR-Cas9.
Bravo, Jack P K; Liu, Mu-Sen; Hibshman, Grace N; Dangerfield, Tyler L; Jung, Kyungseok; McCool, Ryan S; Johnson, Kenneth A; Taylor, David W.
Affiliation
  • Bravo JPK; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
  • Liu MS; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
  • Hibshman GN; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
  • Dangerfield TL; Interdisciplinary Life Sciences Graduate Programs, University of Texas at Austin, Austin, TX, USA.
  • Jung K; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
  • McCool RS; Interdisciplinary Life Sciences Graduate Programs, University of Texas at Austin, Austin, TX, USA.
  • Johnson KA; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
  • Taylor DW; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
Nature ; 603(7900): 343-347, 2022 03.
Article in En | MEDLINE | ID: mdl-35236982
CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1-4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5-7. Although Cas9 variants with greater discrimination against mismatches have been designed8-10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. 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.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: RNA, Guide, Kinetoplastida / DNA Mismatch Repair / CRISPR-Cas Systems / Gene Editing Type of study: Screening_studies Language: En Journal: Nature Year: 2022 Document type: Article Affiliation country: United States Country of publication: United kingdom

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: RNA, Guide, Kinetoplastida / DNA Mismatch Repair / CRISPR-Cas Systems / Gene Editing Type of study: Screening_studies Language: En Journal: Nature Year: 2022 Document type: Article Affiliation country: United States Country of publication: United kingdom