PT - JOURNAL ARTICLE AU - Scott H. Saunders AU - Ayesha M. Ahmed TI - ORBIT for <em>E. coli</em>: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency AID - 10.1101/2023.06.28.546561 DP - 2023 Jan 01 TA - bioRxiv PG - 2023.06.28.546561 4099 - http://biorxiv.org/content/early/2023/07/11/2023.06.28.546561.short 4100 - http://biorxiv.org/content/early/2023/07/11/2023.06.28.546561.full AB - Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput applications. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for E. coli. Our redesigned plasmid toolkit achieved nearly 1000x higher efficiency than λ Red recombination and enabled precise, stable knockouts (&lt;134 kb) and integrations (&lt;11 kb) of various sizes. Additionally, we constructed multi-mutants (double and triple) in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (&gt;30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.Competing Interest StatementThe authors have declared no competing interest.