美国加州大学旧金山分校Seth L. Shipman小组开发出使用recombitron的连续多重噬菌体基因组编辑。这一研究成果于2024年9月5日在线发表在国际学术期刊《自然—生物技术》上。

研究人员提出了一种可扩展的方法，使用称为recombitron的改造细菌反转录子来生成与单链结合和退火蛋白配对的重组体供体DNA，以便整合到噬菌体基因组中。该系统可以高效地在多个噬菌体中创建基因组修改，而无需对抗选择。该方法还支持更大的插入和删除，可以与同时进行的对抗选择相结合，实现超过99%的效率。

此外，研究人员还展示了该过程是连续的，噬菌体与宿主培养时间越长，编辑数量越多，并且可以进行多重编辑。研究人员在仅几个小时的操作时间内在单个λ噬菌体基因组上安装了多达五个不同的突变，无需对抗选择，并在T7 gp17尾部纤维中识别出残基水平的表观遗传相互作用。

据介绍，噬菌体基因组编辑可以增强噬菌体在患者和环境中清除致病细菌的效果。然而，目前的噬菌体基因组编辑方法需要繁琐的筛选、对抗选择或体外构建修改的基因组。

附：英文原文

Title: Continuous multiplexed phage genome editing using recombitrons

Author: Fishman, Chloe B., Crawford, Kate D., Bhattarai-Kline, Santi, Poola, Darshini, Zhang, Karen, Gonzlez-Delgado, Alejandro, Rojas-Montero, Matas, Shipman, Seth L.

Issue&Volume: 2024-09-05

Abstract: 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. Phage genomes are readily engineered using retron recombineering.

DOI: 10.1038/s41587-024-02370-5

Source: https://www.nature.com/articles/s41587-024-02370-5