Development and Optimization of CRISPR/Cas9-Assisted Recombineering in Escherichia albertii

Escherichia albertii is a zoonotic pathogen frequently misidentified as diarrheagenic E. coli due to shared phenotypic and genetic traits, yet functional genomic studies in this species have been limited by the inefficiency of traditional genetic tools. To address this, we developed a bipartite CRISPR/Cas9-assisted lambda red (𝛌-Red) recombineering system for efficient, markerless genome editing in E. albertii . As proof of principle, we targeted the nonessential lacZ gene for negative selection by using Cas9 to generate lethal double stranded breaks in unedited cells. Using a recombinogenic single-stranded DNA (ssDNA) oligonucleotide to introduce a premature stop codon and an NheI restriction site, we achieved a recombination efficiency of 53%. Extending the induction time of the 𝛌-Red recombinase genes enhanced recombineering efficiency to 80%. This optimized CRISPR-assisted platform, the first reported application of its kind in E. albertii, enables the rapid generation of scarless mutations and provides a robust tool for the systematic analysis of E. albertii virulence factors and regulatory networks.