ORBIT: a new paradigm for geneticengineering of mycobacterial chromosomes

Abstract

Current methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic \"targeting oligonucleotide\" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a \"payload plasmid\" that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes in M. tuberculosis and M. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target- or modification-specific double-stranded DNA recombination substrates.