Fishman Chloe B, Crawford Kate D, Bhattarai-Kline Santi, Zhang Karen, González-Delgado Alejandro, Shipman Seth L
Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.
Graduate Program in Bioengineering, University of California, San Francisco and Berkeley, CA, USA.
bioRxiv. 2023 Mar 25:2023.03.24.534024. doi: 10.1101/2023.03.24.534024.
Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply. Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or construction of modified genomes. These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation. Here, we present a scalable approach for engineering phage genomes using recombitrons: modified bacterial retrons that generate recombineering donor DNA paired with single stranded binding and annealing proteins to integrate those donors into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. Moreover, the process is continuous, with edits accumulating in the phage genome the longer the phage is cultured with the host, and multiplexable, with different editing hosts contributing distinct mutations along the genome of a phage in a mixed culture. In lambda phage, as an example, recombitrons yield single-base substitutions at up to 99% efficiency and up to 5 distinct mutations installed on a single phage genome, all without counterselection and only a few hours of hands-on time.
噬菌体能够自然塑造细菌群落，可被用作一种生物技术，以帮助清除我们体内和食物供应中的致病细菌。噬菌体基因组编辑是构建更有效噬菌体技术的关键工具。然而，传统上编辑噬菌体基因组的效率很低，需要进行费力的筛选、反选择或构建修饰基因组。这些要求限制了噬菌体修饰的类型和通量，进而限制了我们的知识和创新潜力。在这里，我们提出了一种使用重组子工程化噬菌体基因组的可扩展方法：经过修饰的细菌反转录子，其产生重组工程供体DNA，并与单链结合和退火蛋白配对，以将这些供体整合到噬菌体基因组中。该系统可以在多个噬菌体中高效地进行基因组修饰，而无需反选择。此外，该过程是连续的，噬菌体与宿主培养的时间越长，编辑在噬菌体基因组中积累得就越多，并且该过程是可多重化的，在混合培养中，不同的编辑宿主会在噬菌体基因组上产生不同的突变。例如，在λ噬菌体中，重组子产生单碱基替换的效率高达99%，并且在单个噬菌体基因组上可安装多达5种不同的突变，所有这些都无需反选择，且只需几个小时的实际操作时间。
