Precise generation of bystander-free mouse models with ABE9-SpRY.

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, with significantly fewer off-target effects than ABE8e-SpRY, achieving desired editing efficiencies of up to 96% in individual adult founder mice.ABE9-SpRY also enhances product purity in mouse embryos and human induced pluripotent stem cells (hiPSCs) compared to ABE8e-SpRY. Our findings showcase ABE9-SpRYs precision and versatility, highlighting it as a powerful tool for accurate in vivo point mutation modelling.