Abstract
Point mutations cause many genetic disorders, but modelling them in organisms is technically challenging. Creating mouse models that mimic these mutations is crucial for establishing a causal relationship between mutations and disease phenotype, thereby supporting the development of therapeutic strategies. Adenine base editors (ABEs) can correct single-nucleotide variants (SNVs) in disease modelling without double-stranded breaks (DSBs) or donor DNA, achieving higher product purity than traditional Cas9 methods. Earlier ABE techniques faced issues like limited targetability, bystander editing, and off-target effects. By combining two editor advancements, we introduced and tested ABE9-SpRY, an improved ABE variant fused with a PAM-flexible SpRY-Cas9 nickase. Our results show that ABE9-SpRY effectively generates three out of four targeted A-to-G mutations in mouse embryos, achieving desired editing efficiencies of up to 96% in individual adult founder mice. Furthermore, we observe fewer off-target events at predicted DNA sites in mouse embryos and in an orthogonal R-loop assay compared with ABE8e-SpRY. ABE9-SpRY also enhances product purity in mouse embryos under pooled sgRNA injections and, as a proof-of-concept, at a single endogenous locus in human induced pluripotent stem cells (hiPSCs), relative to ABE8e-SpRY. Our findings support ABE9-SpRY’s precision at the loci tested and PAM-flexible versatility. Although performance remains sequence-dependent, these data support ABE9-SpRY as a PAM-flexible tool for generating precise point-mutation models where bystander editing is a concern.
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Data availability
Oligonucleotide and sgRNA sequences used in this study are listed in Supplementary Tables 2, 3, 5, and 6. All raw NGS sequencing data generated in this study were in the NCBI Sequence Read Archive (SRA) database as a BioProject with the accession number PRJNA1291991. Plasmids encoding pCMV_ABE9-SpRY (/#242976) and pEF1α_ABE9-SpRY (#242977) are available at Addgene. Any other data and reagents will be made available upon reasonable request.
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Acknowledgements
Sayari Bhunia is a member of HBIGS, the Heidelberg Biosciences International Graduate School. We thank the entire Freichel lab for their constructive feedback on the manuscript. We thank Maren Schneider for help with cloning pEF1α_ABE9-SpRY and XMAS-TREE sgRNAs. We thank the whole team from the Interfakultäre Biomedizinische Forschungseinrichtung (IBF) at Heidelberg University for their expert technical assistance. We thank Angela Wirth for writing the animal proposal. While preparing this work, the authors used AI-assisted technologies (Grammarly and ChatGPT) to improve readability and language. We acknowledge financial support from the Heidelberg University Publikationsfonds for the open-access publication fee.
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Open Access funding enabled and organized by Projekt DEAL. This research was funded by the German Research foundation (DFG) through the Collaborative Research Centres CRC1550 (FKZ 464424253, projects B09 and S01), CRC1328 (335447717, project A21), DFG project FR 1638/5-1 (531047917), the DZHK (German Centre for Cardiovascular Research) and the BMBF (German Ministry of Education and Research); and The Health + Life Science Alliance Heidelberg Mannheim.
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J.K.O. and A.C. designed the study and developed the methodology. J.K.O. and A.C. curated the data and performed the formal analysis. J.K.O., S.B., B.H., V.K., S.D. and F.Z. performed the investigation. J.K.O., S.B. and A.C. conducted validation. J.K.O. and A.C. visualised the data. J.K.O. and A.C. wrote the original draft of the manuscript. J.K.O., S.B., V.K., M.F. and A.C. reviewed and edited the manuscript. M.F. acquired funding and provided resources. A.C. supervised the work.
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Alex Cornean is a co-founder and shareholder of DataHarmony Ltd, a company that provides gene editing-related services and software solutions.
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Ong, J.K., Bhunia, S., Hilbert, B. et al. ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40642-z
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DOI: https://doi.org/10.1038/s41598-026-40642-z
