biostudies-literatureCarusillo AGerman Research FoundationEuropean Union's Horizon 2020University of FreiburgMedical Informatics Funding Scheme4660-4673https://www.ebi.ac.uk/biostudies/studies/S-EPMC10201422biostudies-literatureUnknown51(9)Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the Nucleic acids researchA novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.PMC102014227652693861/3-1EkoEstMed–FKZ 01ZZ2015Chmielewski KOMussolino CKlermund JSchafer RTurk DCarusillo AMosti LCornu TIRhiel MHaider SAndrieux GCathomen TfalseA novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the 2023-01-01T00:00:00Z2023 May2026-05-28T17:51:18.988Z2025-04-04T14:30:00.915ZS-EPMC102014223707019210.1093/nar/gkad255