7OXA

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

Summary for 7OXA

• Entry DOI: 10.2210/pdb7oxa/pdb
• Related: 7OX7 7OX8 7OX9
• Descriptor: chimeric RNA-DNA guide, CRISPR-associated endonuclease Cas9/Csn1, AAVS1 target DNA strand, ... (7 entities in total)
• Functional Keywords: cas9, crispr, chrdna, aavs1, nuclease, hydrolase
• Biological source: Streptococcus pyogenes serotype M1; More
• Total number of polymer chains: 4
• Total formula weight: 197790.62

Authors

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. (deposition date: 2021-06-22, release date: 2021-09-15, Last modification date: 2024-01-31)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (2.15 Å)