# ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models

## TL;DR

This paper introduces ABE9-SpRY, a new gene editing tool that improves precision in creating mouse models with specific mutations, reducing unwanted side effects.

## Contribution

The novel fusion of ABE9 with a PAM-flexible SpRY Cas9 nickase enhances editing efficiency and reduces off-target effects in mouse embryos.

## Key findings

- ABE9-SpRY achieves up to 96% editing efficiency for A-to-G mutations in mouse embryos.
- Fewer off-target events are observed with ABE9-SpRY compared to ABE8e-SpRY in mouse embryos and R-loop assays.
- ABE9-SpRY improves product purity in mouse embryos and human induced pluripotent stem cells.

## 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.

The online version contains supplementary material available at 10.1038/s41598-026-40642-z.