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ABSTRACT Advanced genome editing technologies have enabled rapid and flexible rewriting of theEscherichia coligenome, benefiting fundamental biology and biomanufacturing. Unfortunately, some of the most useful technologies to advance genome editing inE. colihave not yet been ported into other bacterial species. For instance, the addition of bacterial retrons to the genome editing toolbox has increased the efficiency of recombineering inE. coliby enabling sustained, abundant production of ssDNA recombineering donors by reverse transcription that install flexible, precise edits in the prokaryotic chromosome. To extend the utility of this technology beyondE. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, BacillotaandActinomycetota) and a total of 15 different species. We found that retron recombineering is functional in all species tested, reaching editing efficiencies above 20% in six of them, above 40% in three of them, and above 90% in two of them. We also tested the extension of the recombitron architecture optimizations and strain backgrounds in a subset of hosts to additionally increase editing rates. The broad recombitron survey carried out in this study forms the basis for widespread use of retron-derived technologies through the whole Bacteria domain.more » « lessFree, publicly-accessible full text available June 17, 2026
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