{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T03:00:51Z","timestamp":1787626851754,"version":"build-2736575974"},"reference-count":94,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2025,12,21]],"date-time":"2025-12-21T00:00:00Z","timestamp":1766275200000},"content-version":"vor","delay-in-days":20,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,12,1]],"date-time":"2025-12-01T00:00:00Z","timestamp":1764547200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["MicrobiologyOpen"],"published-print":{"date-parts":[[2025,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                    (\n                    <jats:italic>Mtb<\/jats:italic>\n                    ), the etiological agent of tuberculosis, is one of the most challenging pathogens due to its complex physiology, diverse clinical manifestations, and growing multidrug resistance. The global rise of drug\u2010resistant\n                    <jats:italic>Mtb<\/jats:italic>\n                    strains has prompted the search for innovative genetic and molecular strategies to accelerate drug discovery and vaccine development. Progress in\n                    <jats:italic>Mtb<\/jats:italic>\n                    research has long been hindered by its slow replication rate and impermeable cell envelope, which limit the efficacy of genetic manipulation. This review outlines methodological advances that have transformed the study of\n                    <jats:italic>Mtb<\/jats:italic>\n                    pathogenesis and drug resistance mechanisms. Traditional homologous recombination\u2013based approaches, including allelic exchange and specialized transduction, laid the groundwork for targeted mutagenesis but were limited by low efficiency. The advent of phage\u2010derived recombineering systems, such as the Che9c RecET, has substantially improved the precision and throughput of genetic modification. Hybrid systems such as ORBIT, which combines oligonucleotide\u2010mediated recombineering with Bxb1 integrase, have further enabled rapid and versatile genome engineering across mycobacterial species. Parallel developments in conditional gene expression systems (e.g., the use of TetR\/Pip\u2010based promoters) have facilitated the functional analysis of essential genes and the validation of novel drug targets. The advent of CRISPR\u2013Cas technologies has represented a paradigm shift, by enabling programmable, high\u2010fidelity gene regulation and functional genomics even in slow\u2010growing mycobacteria. Together, these genetic innovations are transforming\n                    <jats:italic>Mtb<\/jats:italic>\n                    research by accelerating drug discovery and vaccine design, and shedding light on host\u2013pathogen interactions.\n                  <\/jats:p>","DOI":"10.1002\/mbo3.70206","type":"journal-article","created":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T05:26:51Z","timestamp":1766381211000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Uncovering Insights Into the Biology of\n                    <i>Mycobacterium tuberculosis<\/i>\n                    Using Genetic Tools"],"prefix":"10.1002","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7065-1501","authenticated-orcid":false,"given":"Alessandro","family":"Stamilla","sequence":"first","affiliation":[{"name":"Dipartimento di Biologia e Biotecnologie \u201cLazzaro Spallanzani\u201d Universit\u00e0 di Pavia Pavia 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