Compact bacterial recombination complexes drive efficient kilobase-scale knock-in in mammalian cells

Abstract Efficient homologous recombination, homology-directed repair (HDR), remains a major hurdle for precise genome editing in mammalian cells, particularly for kilobase-scale insertions. Bacterial recombineering proteins, such as RecE and RecT, offer potential solutions, but their activity in eukaryotic systems has been largely uncharacterized. Here, we identify Escherichia coli RecE (EcRecE) as a potent enhancer of HDR in mammalian cells. Targeted recruitment of EcRecE via CRISPR/Cas9 significantly increased HDR efficiency at multiple genomic loci across different cellular contexts, including human embryonic stem cells, achieving a 3–6-fold enhancement in the integration efficiency of kilobase-scale sequences. Furthermore, in combination with RecT and a catalytically inactive Cas9 (dCas9), applying functional domain engineering, we developed a dCas9-miniRecTE editor that enhances large-fragment integration without introducing double-strand breaks in human cells and primary mouse neurons, achieving ∼20% kilobase-scale knock-in efficiency. These results establish EcRecE as a versatile tool for improving precision genome engineering, with potential applications in therapeutic gene editing.