Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host’s single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.

高效的基因组编辑方法对于生物技术和基础研究至关重要。同源重组 (HR) 是最通用的基因组编辑方法，但依赖宿主 RecA 介导途径的技术效率低下且费力。噬菌体编码的单链 DNA 退火蛋白 (SSAP) 可将 HR 提高到内源水平以上 1,000 倍。但是，它们的功能并不广泛。使用大肠杆菌、乳酸乳球菌、耻垢分枝杆菌、鼠李糖乳杆菌和新月柄杆菌，我们调查了 SSAP 的有限可移植性。我们发现这些蛋白质特异性识别宿主单链 DNA 结合蛋白 (SSB) 的 C 末端尾部，并且只有在与该宿主域保持相容性的情况下才可在物种之间移植。此外，我们发现将 SSAP 与 SSB 共表达可以显着提高基因组编辑效率，在某些物种中即使没有宿主兼容性也能实现 SSAP 功能。最后，我们发现高效 HR 远远超过了常用随机诱变方法的突变能力，产生了通过顺序核苷酸转换无法获得的特殊表型。