8SCA

Rec3 Domain from S. pyogenes Cas9

Summary for 8SCA

• Entry DOI: 10.2210/pdb8sca/pdb
• Descriptor: CRISPR-associated endonuclease Cas9/Csn1, 1,2-ETHANEDIOL (3 entities in total)
• Functional Keywords: crispr cas9, nucleic acid binding protein, rna binding protein
• Biological source: Streptococcus pyogenes
• Total number of polymer chains: 1
• Total formula weight: 26130.97

Authors

D'Ordine, A.M.,Skeens, E.,Lisi, G.P.,Jogl, G. (deposition date: 2023-04-05, release date: 2024-03-13)

Primary citation

Skeens, E.,Sinha, S.,Ahsan, M.,D'Ordine, A.M.,Jogl, G.,Palermo, G.,Lisi, G.P.
High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9.
Sci Adv, 10:eadl1045-eadl1045, 2024

PubMed Abstract: The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

PubMed: 38446895
DOI: 10.1126/sciadv.adl1045

Experimental method

X-RAY DIFFRACTION (1.67 Å)