Kinetics and Evolution-guided Design of High Fidelity CRISPR-Cas Endonucleases

Physics-driven, PLM-assisted atomistic simulations identify kinetic determinants of R-loop formation and enable high-fidelity SpyCas9 and FnCas12a variants with 4.49-fold and 6.69-fold increases over wild-type.

• Why it matters: High-fidelity CRISPR variants are needed for precise in vitro DNA detection and safer in vivo gene-editing, but de novo design and directed evolution face costly trials and limited success.
• What they did: The team used atomistic molecular dynamics with automated path searching to extract transition-state structures for R-loop conformational changes linked to off-target recognition and cleavage, then used PLM filtering on fidelity-diminishing residues.
• The result: The resulting designs produced high-fidelity SpyCas9 and FnCas12a variants, validated with only a few wet-lab tests using cellular genome-editing and ribonucleoprotein cleavage assays.

AI summary of the abstract · may contain errors · not medical advice · read the paper.