Genome editing of phylogenetically distinct bacteria using cross-species retron-mediated recombineering

Advanced genome editing technologies have enabled rapid, flexible rewriting of the Escherichia coli genome, but most have not been tested in other bacterial species. Recombitrons-a genome editing tool created by pairing modified, donor-producing bacterial retrons with single-stranded binding and annealing proteins-have increased the efficiency of recombineering to install flexible, precise edits in the prokaryotic chromosome. Here, to extend their utility outside of E. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, Bacillota and Actinomycetota) and a total of 15 different species. We found that retron recombineering is variable across the species tested but functional in all of them, reaching editing efficiencies >20% in six of them, >40% in three and >90% in two. Efficiencies in the remaining nine species ranged from 0.015% to 7.4%. We also tested the extension of the recombitron architecture, operon and strain modifications in a subset of hosts in which species-specific modifications were required to increase editing rates.