OR09
An enhanced Single-Strand Break-Based Strategy for safer and more efficient Genome Editing
E Anagnostou(1,2) S Haider(1,2) A L Cavallo(3) R Nitsch(4) M Goncalves(5) C Mussolino(1,2)
1:Institute for Transfusion Medicine and Gene Therapy, Medical Center, University of Freiburg, 79106 Freiburg, Germany; 2:Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany; 3:Discovery Cell and Gene Therapy integrated Bioanalysis, R&D Biopharmaceuticals, AstraZeneca, 43183 Mölndal, Sweden; 4:Gene therapy team, Clinical and pharmacological safety sciences, AstraZeneca, 43183 Mölndal, Sweden; 5:Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, 2333 ZC, The Netherlands
Precise genome editing requires recombination between a DNA repair template and chromosomal sequences, typically initiated by DNA double-strand breaks (DSBs). However, DSBs are highly genotoxic and pose significant safety concerns. Emerging technologies such as base and prime editing reduce genotoxic risk by relying on single-strand breaks (SSBs). Despite their promise, base editing has been linked to unintended DSB byproducts, while prime editing remains limited to small insertions or edits. Designer nickases have been explored as low-genotoxic tools for genome editing, yet the inherent inefficiency of SSB-mediated editing has driven efforts to enhance its performance. Strategies such as double nicks or coordinated paired nicks—targeting both the chromosomal site and the repair template—have shown potential for increased editing efficiency. Building on this concept, we developed a novel approach using Cas9 nickase (nCas9) fused to DNA repair pathway effectors. These fusions significantly improve the integration of a GFP expression cassette at a genomic locus via the in trans paired nicks strategy, achieving a 3-fold increase in efficiency compared to unmodified nCas9. Ongoing work involves testing additional nCas9 variants and repair effectors to optimize SSB resolution through homology-directed repair. These DSB-free strategies offer a safer alternative to traditional genome editing, mitigating risks associated with non-homologous end joining (NHEJ)-mediated repair. This approach holds particular promise for clinical applications, especially in targeting coding regions implicated in dominant genetic disorders.