High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9

Skeens, Erin; Sinha, Souvik; Ahsan, Mohd; D’Ordine, Alexandra M.; Jogl, Gerwald; Palermo, Giulia; Lisi, George P.

doi:10.1126/sciadv.adl1045

Title: High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9

Journal Article · 05 March 2024 · Science Advances

DOI: https://doi.org/10.1126/sciadv.adl1045 · OSTI ID:2470269

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Brown University, Providence, RI (United States)
University of California, Riverside, CA (United States)

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.

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Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: National Energy Research Scientific Computing Center (NERSC); National Institute of General Medical Sciences; National Institutes of Health (NIH); National Science Foundation (NSF); San Diego Supercomputer Center; USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0012704

OSTI ID:: 2470269

Journal Information:: Science Advances, Journal Name: Science Advances Journal Issue: 10 Vol. 10; ISSN 2375-2548

Publisher:: AAASCopyright Statement

Country of Publication:: United States

Language:: English

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