Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for ‘programmable addition via site-specific targeting elements’, a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells.

将基因靶向整合到哺乳动物基因组中的方法存在可编程性低、效率低或特异性低的问题。在这里，我们证明噬菌体辅助的持续进化增强了引物编辑辅助的位点特异性整合酶基因编辑（PASSIGE），它将引物编辑的可编程性与重组酶的能力结合起来，以精确整合超过10 kilobase的大DNA货物。进化和工程化的 Bxb1 重组酶变体（evoBxb1 和 eeBxb1）在具有预装重组酶着陆位点的人类细胞系中介导高达 60% 的供体整合（野生型 Bxb1 的 3.2 倍）。在安全港和治疗相关位点的单次转染实验中，使用 eeBxb1 进行 PASSIGE 的平均靶向基因整合效率为 23%（野生型 Bxb1 的 4.2 倍）。值得注意的是，原代人成纤维细胞中多个位点的整合效率超过 30%。带有 evoBxb1 或 eeBxb1 的 PASSIGE 的性能平均分别比 PASTE（“通过位点特异性靶向元件进行可编程添加”，一种使用与重组酶融合的 Prime 编辑器的方法）性能高出 9.1 倍和 16 倍。具有不断进化的重组酶的 PASSIGE 是一种在哺乳动物细胞中靶向整合基因的异常有效的方法。