Efficient prime editing in two-cell mouse embryos using PEmbryo

Here, we show that prime editing, when deployed during a permissive stage of early development and in the presence of an engineered inhibitor of DNA mismatch repair, enables highly efficient editing of mouse embryos with few on-target byproducts. While temporary inhibition of mismatch repair increased nonspecific, off-target indels, as revealed by whole genome sequencing, these primarily -1 bp deletions were found in genomic regions of low sequence complexity and did not result in obvious phenotypic changes nor viability issues in mice. Moreover, on-target editing was of sufficient efficiency and purity to allow us, in proof-of-principle experiments, to directly characterize founder mice for a recessive phenotype without outcrossing. Our findings thus establish PEmbryo as a promising technique for rapidly phenotyping genetic variants and for accelerated germline engineering in mice.