7OXA

Target-bound SpCas9 complex with AAVS1 chimeric RNA-DNA guide

7OXA の概要

• エントリーDOI: 10.2210/pdb7oxa/pdb
• 関連するPDBエントリー: 7OX7 7OX8 7OX9
• 分子名称: chimeric RNA-DNA guide, CRISPR-associated endonuclease Cas9/Csn1, AAVS1 target DNA strand, ... (7 entities in total)
• 機能のキーワード: cas9, crispr, chrdna, aavs1, nuclease, hydrolase
• 由来する生物種: Streptococcus pyogenes serotype M1; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 197790.62

構造登録者

Donohoue, P.,Pacesa, M.,Lau, E.,Vidal, B.,Irby, M.J.,Nyer, D.B.,Rotstein, T.,Banh, L.,Toh, M.T.,Gibson, J.,Kohrs, B.,Baek, K.,Owen, A.L.G.,Slorach, E.M.,van Overbeek, M.,Fuller, C.K.,May, A.P.,Jinek, M.,Cameron, P. (登録日: 2021-06-22, 公開日: 2021-09-15, 最終更新日: 2024-01-31)

主引用文献

Donohoue, P.D.,Pacesa, M.,Lau, E.,Vidal, B.,Irby, M.J.,Nyer, D.B.,Rotstein, T.,Banh, L.,Toh, M.S.,Gibson, J.,Kohrs, B.,Baek, K.,Owen, A.L.G.,Slorach, E.M.,van Overbeek, M.,Fuller, C.K.,May, A.P.,Jinek, M.,Cameron, P.
Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.
Mol.Cell, 81:3637-3649.e5, 2021

PubMed Abstract: The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

PubMed: 34478654
DOI: 10.1016/j.molcel.2021.07.035

実験手法

X-RAY DIFFRACTION (2.15 Å)