Nature Biotechnology ( IF 33.1 ) Pub Date : 2024-09-05 , DOI: 10.1038/s41587-024-02370-5
Chloe B Fishman 1 , Kate D Crawford 1, 2 , Santi Bhattarai-Kline 1, 3 , Darshini Poola 1, 4 , Karen Zhang 1, 2 , Alejandro González-Delgado 1 , Matías Rojas-Montero 1 , Seth L Shipman 1, 5, 6
Bacteriophage genome editing can enhance the efficacy of phages to eliminate pathogenic bacteria in patients and in the environment. However, current methods for editing phage genomes require laborious screening, counterselection or in vitro construction of modified genomes. Here, we present a scalable approach that uses modified bacterial retrons called recombitrons to generate recombineering donor DNA paired with single-stranded binding and annealing proteins for integration into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. The approach also supports larger insertions and deletions, which can be combined with simultaneous counterselection for >99% efficiency. Moreover, we show that the process is continuous, with more edits accumulating the longer the phage is cultured with the host, and multiplexable. We install up to five distinct mutations on a single lambda phage genome without counterselection in only a few hours of hands-on time and identify a residue-level epistatic interaction in the T7 gp17 tail fiber.
中文翻译:
使用 recombitron 进行连续多重噬菌体基因组编辑
噬菌体基因组编辑可以增强噬菌体消除患者和环境中病原菌的功效。然而,目前编辑噬菌体基因组的方法需要费力的筛选、反选或体外构建修饰的基因组。在这里,我们提出了一种可扩展的方法,该方法使用称为 recombitrons 的修饰细菌逆转录素生成重组供体 DNA,并与单链结合和退火蛋白配对,以整合到噬菌体基因组中。该系统可以在多个噬菌体中有效地产生基因组修饰,而无需反向选择。该方法还支持更大的插入和缺失,可以与同时反向选择相结合,以实现 >99% 的效率。此外,我们表明该过程是连续的,噬菌体与宿主一起培养的时间越长,积累的编辑就越多,并且是可多重的。我们只需几个小时的动手操作时间,即可在单个 lambda 噬菌体基因组上安装多达五个不同的突变,无需反选,并鉴定出 T7 gp17 尾纤维中的残基水平上位相互作用。