Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

• DOI: 10.1128/msystems.00180-20
• Publication Date: 2020-08-10T13:24:50Z
• Authors: Katlyn Borgers; Kristof Vandewalle; Annelies Van Hecke; Gitte Michielsen; Evelyn Plets; Loes van Schie; Sandrine Vanmarcke; Laurent Schindfessel; Nele Festjens; Nico Callewaert
• Keywords: RECOMBINASE; EXPRESSION; REPAIR; transposon mutagenesis; 0301 basic medicine; MUTAGENESIS; EFFICIENT TRANSGENESIS; characterized transposon library; optimized Himar1 transposon; TUBERCULOSIS; Microbiology; unmarked mutants; QR1-502; I-SCEI MEGANUCLEASE; 3. Good health; 03 medical and health sciences; GENE DELETIONS; Mycobacterium tuberculosis complex; Medicine and Health Sciences; Research Article

Mutant resources are essential to improve our understanding of the biology slow-growing mycobacteria, which include causative agents tuberculosis in various species, including humans. The generation deletion mutants mycobacteria a gene-by-gene approach order make genome-wide ordered mutant is still laborious and costly approach, despite recent development improved methods. On other hand, transposon mutagenesis combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows creation large archived Mycobacterium insertion libraries. However, such contain selection marker genes risk polar gene effects, undesired both for research use these as live attenuated vaccines. In this paper, derivative Himar1 described clean, markerless knockouts from By incorporating FRT sites FlpE/FRT-mediated recombination I-SceI ISceIM-based removal, we enable two thoroughly experimentally validated possibilities create unmarked marked mutants. highly efficient but leaves an scar genome, whereas I-SceI-mediated can without any heterologous DNA genome. combined CP-CSeq optimized was applied BCG Danish 1331 vaccine strain (WHO reference 07/270), creating largest ordered, characterized resource member complex (18,432 clones, mutating 83% nonessential M. homologues), be easily generated.IMPORTANCE While speeding up many fields (e.g., yeast, plant, Caenorhabditis elegans), collections elusive mycobacterial research. We developed methods generate time- cost-effective manner newly engineered efficiently generated. Our library WHO bovis targets all made publicly available via BCCM/ITM Mycobacteria Collection. This will speed drug resistance development) paves way similar strains complex. stretch full collection now much shorter, just 17% remaining targeted using approaches, effective have recently also been described.