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Here, we developed a second-generation genetic toolbox based on leucine auxotrophy that enables antibiotic-free positive selection. A \u0394\n                      <jats:italic toggle=\"yes\">leuB<\/jats:italic>\n                      mutant, lacking the gene encoding isopropyl malate dehydrogenase in the leucine biosynthesis pathway, was generated by targeted gene deletion. This mutant requires exogenous leucine for growth and can be complemented by plasmid-borne\n                      <jats:italic toggle=\"yes\">leuB<\/jats:italic>\n                      , establishing a robust auxotrophy-based selection system. To support genetic manipulation, we constructed a suite of second-generation vectors, including a multicopy replicative vector (pRep-amp-\n                      <jats:italic toggle=\"yes\">leuB<\/jats:italic>\n                      ), a single-copy integrative vector (pInt-amp-\n                      <jats:italic toggle=\"yes\">leuB<\/jats:italic>\n                      ), and a suicide vector (pSuc-amp-\n                      <jats:italic toggle=\"yes\">leuB<\/jats:italic>\n                      ) for allelic replacement. These vectors enable gene overexpression, complementation, and targeted gene deletion, respectively, without reliance on aminoglycoside resistance markers. Using this system, we demonstrate efficient transformation and functional complementation of the \u0394\n                      <jats:italic toggle=\"yes\">leuB<\/jats:italic>\n                      mutant, achieving high transformation efficiencies and near-zero background growth under leucine selection. In contrast to antibiotic-based systems, this approach eliminates nonspecific background and avoids activation of stress response pathways, such as the\n                      <jats:italic toggle=\"yes\">whiB7<\/jats:italic>\n                      regulon. Overall, this auxotrophy-based toolbox provides a versatile platform for precise genetic manipulation in\n                      <jats:italic toggle=\"yes\">M. abscessus<\/jats:italic>\n                      , improving selection stringency and enabling antibiotic-free functional genomics approaches, as demonstrated by deleting\n                      <jats:italic toggle=\"yes\">aac(2<\/jats:italic>\n                      \u2032),\n                      <jats:italic toggle=\"yes\">eis2,<\/jats:italic>\n                      and a 19-kb fragment of the\n                      <jats:italic toggle=\"yes\">gpl<\/jats:italic>\n                      locus.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      The development of a leucine auxotroph-based genetic system for\n                      <jats:italic toggle=\"yes\">Mycobacterium abscessus<\/jats:italic>\n                      addresses critical challenges in mycobacterial genetics. By avoiding established antibiotic resistance markers, this approach reduces selective pressure for antibiotic-resistant transformants, supports antibiotic stewardship, and minimizes costly disposal from laboratory waste. It also avoids unintended activation of\n                      <jats:italic toggle=\"yes\">whiB7<\/jats:italic>\n                      , a master regulator of approximately 100 genes, particularly those involved in antibiotic stress responses, thereby improving the accuracy of phenotypic drug susceptibility testing. The versatile genetic toolbox developed here, including novel replicative, integrative, and suicide plasmids, provides precise control over functional studies, overexpression, complementation, and gene deletion. It significantly reduces dependency on antibiotics for genetic manipulation, aligning with the goals of sustainable research and offering new opportunities for studying this clinically significant pathogen. This approach represents a critical advance in microbial genetics, enhancing our capacity to explore the molecular basis of pathogenesis and drug resistance in\n                      <jats:italic toggle=\"yes\">M. abscessus<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00712-26","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T13:02:00Z","timestamp":1784206920000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Leucine auxotrophic marker for genetic manipulation of\n                    <i>Mycobacterium abscessus<\/i>"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-8297-461X","authenticated-orcid":true,"given":"Tizian","family":"Griesser","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02crff812","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Medical Microbiology, University of Zurich","place":["Zurich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8305-3316","authenticated-orcid":true,"given":"Petra","family":"Selchow","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02crff812","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Medical Microbiology, University of Zurich","place":["Zurich, Switzerland"]},{"id":[{"id":"https:\/\/ror.org\/02crff812","id-type":"ROR","asserted-by":"publisher"}],"name":"National Reference Laboratory for Mycobacteria, University of Zurich","place":["Zurich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1581-0682","authenticated-orcid":true,"given":"Peter","family":"Sander","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02crff812","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Medical Microbiology, University of Zurich","place":["Zurich, Switzerland"]},{"id":[{"id":"https:\/\/ror.org\/02crff812","id-type":"ROR","asserted-by":"publisher"}],"name":"National Reference Laboratory for Mycobacteria, University of Zurich","place":["Zurich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1098\/rsob.160185"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/s44259-024-00060-5"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1177\/23247096251334229"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1002\/9780471729259.mc10d01s18"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-020-0331-1"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.02179"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkr578"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms7030090"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkae288"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1099\/ijsem.0.001376"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01879-19"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.chest.2021.07.035"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1513\/AnnalsATS.202003-222CME"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/S2666-5247(21)00173-7"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.633510"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.02508-19"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkw466"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkx284"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkr209"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1099\/13500872-140-1-133"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.01914-08"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01347-17"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","unstructured":"Corley JM Congel JH Haist KC Ochoa AE Malcolm KC Janssen WJ Nick JA Hisert KB. 2025. 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