7QQR

SpCas9 bound to AAVS1 off-target5 DNA substrate

Summary for 7QQR

• Entry DOI: 10.2210/pdb7qqr/pdb
• Related: 7OX9
• Descriptor: AAVS1 sgRNA, CRISPR-associated endonuclease Cas9/Csn1, AAVS1 off-target5 target strand, ... (8 entities in total)
• Functional Keywords: crispr, cas9, off-target, ternary complex, hydrolase
• Biological source: Streptococcus pyogenes; More
• Total number of polymer chains: 4
• Total formula weight: 198919.15

Authors

Pacesa, M.,Jinek, M. (deposition date: 2022-01-10, release date: 2022-10-26, Last modification date: 2024-01-31)

Primary citation

Pacesa, M.,Lin, C.H.,Clery, A.,Saha, A.,Arantes, P.R.,Bargsten, K.,Irby, M.J.,Allain, F.H.,Palermo, G.,Cameron, P.,Donohoue, P.D.,Jinek, M.
Structural basis for Cas9 off-target activity.
Cell, 185:4067-4081.e21, 2022

PubMed Abstract: The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

PubMed: 36306733
DOI: 10.1016/j.cell.2022.09.026

Experimental method

X-RAY DIFFRACTION (2.75 Å)