{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T13:42:48Z","timestamp":1767274968881,"version":"3.48.0"},"posted":{"date-parts":[[2025,12,17]]},"group-title":"Microbiology","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T00:00:00Z","timestamp":1765929600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,17]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  <jats:italic>Mycobacterium abscessus<\/jats:italic>\n                  (\n                  <jats:italic>Mab<\/jats:italic>\n                  ) is a multidrug-resistant nontuberculous mycobacterium that causes debilitating TB-like pulmonary infections for which effective treatment options are lacking. Poor\n                  <jats:italic>in vivo<\/jats:italic>\n                  drug efficacy may stem from altered vulnerability of drug targets driven by host-specific environmental conditions. To enable validation and prioritization of candidate drug targets\n                  <jats:italic>in vivo<\/jats:italic>\n                  , we exploited CRISPRi (CRi) gene silencing in multiple mouse infection models. Inducible silencing of\n                  <jats:italic>ftsZ<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  , a previously validated target, and three predicted targets (\n                  <jats:italic>leuS<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  ,\n                  <jats:italic>folP<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  ,\n                  <jats:italic>fusA<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  ) confirmed their essentiality\n                  <jats:italic>in vitro<\/jats:italic>\n                  . We then assessed the\n                  <jats:italic>in vivo<\/jats:italic>\n                  vulnerability of these targets in both immunocompetent C57BL\/6N and immunodeficient NSG mice by assessing the impact of CRi silencing on pulmonary mycobacterial burden. In NSG mice, silencing of all four genes led to comparable decreases in\n                  <jats:italic>Mab<\/jats:italic>\n                  burden. However, in C57BL\/6N mice, the degree of\n                  <jats:italic>Mab<\/jats:italic>\n                  clearance varied among targets, suggesting that immune pressure may influence the outcome of CRi-mediated gene silencing. Notably, repression of\n                  <jats:italic>fusA<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  yielded a larger decline in mycobacterial burden in C57BL\/6N mice despite a lower level of gene silencing\n                  <jats:italic>in vitro<\/jats:italic>\n                  , consistent with enhanced vulnerability of this target. Overall, this study demonstrated that\n                  <jats:italic>ftsZ<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  ,\n                  <jats:italic>leuS<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  ,\n                  <jats:italic>folP<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  , and\n                  <jats:italic>fusA<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>Mab<\/jats:italic>\n                  <\/jats:sub>\n                  are essential for\n                  <jats:italic>Mab<\/jats:italic>\n                  growth\n                  <jats:italic>in vitro<\/jats:italic>\n                  and, for the first time, validated their vulnerability to inhibition by CRi during infection. These data also identified potential context-dependent target vulnerabilities, which could inform the prioritization of bacterial drug targets and accelerate the development of effective therapeutics for\n                  <jats:italic>Mab<\/jats:italic>\n                  infections.\n                <\/jats:p>","DOI":"10.64898\/2025.12.16.694790","type":"posted-content","created":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T11:24:02Z","timestamp":1766057042000},"source":"Crossref","is-referenced-by-count":0,"title":["CRISPRi-mediated validation of candidate\n                  <i>Mycobacterium abscessus<\/i>\n                  drug targets during host infection"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2196-3397","authenticated-orcid":false,"given":"Rashmi","family":"Gupta","sequence":"first","affiliation":[{"name":"Division of Immunity and Pathogenesis, College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida, 6900 Lake Nona Blvd, FL 32827, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Breven S.","family":"Simcox","sequence":"additional","affiliation":[{"name":"Division of Immunity and Pathogenesis, College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida, 6900 Lake Nona Blvd, FL 32827, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9838-3238","authenticated-orcid":false,"given":"Kyle H.","family":"Rohde","sequence":"additional","affiliation":[{"name":"Division of Immunity and Pathogenesis, College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida, 6900 Lake Nona Blvd, FL 32827, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026010105410547000_2025.12.16.694790v1.1","doi-asserted-by":"publisher","DOI":"10.1097\/QCO.0000000000000104"},{"key":"2026010105410547000_2025.12.16.694790v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/S0272-2712(02)00018-5"},{"key":"2026010105410547000_2025.12.16.694790v1.3","doi-asserted-by":"publisher","DOI":"10.1164\/rccm.200604-571ST"},{"key":"2026010105410547000_2025.12.16.694790v1.4","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciq237"},{"key":"2026010105410547000_2025.12.16.694790v1.5","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.02448-14"},{"key":"2026010105410547000_2025.12.16.694790v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/j.drup.2012.04.001"},{"key":"2026010105410547000_2025.12.16.694790v1.7","doi-asserted-by":"publisher","DOI":"10.1513\/AnnalsATS.201804-236OC"},{"key":"2026010105410547000_2025.12.16.694790v1.8","doi-asserted-by":"crossref","unstructured":"Dal Molin M , Gut M , Rominski A , Haldimann K , Becker K , Sander P. 2018. Molecular Mechanisms of Intrinsic Streptomycin Resistance in Mycobacterium abscessus. Antimicrob Agents Chemother 62.","DOI":"10.1128\/AAC.01427-17"},{"key":"2026010105410547000_2025.12.16.694790v1.9","doi-asserted-by":"crossref","unstructured":"Hurst-Hess K , Rudra P , Ghosh P. 2017. Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance. Antimicrob Agents Chemother 61.","DOI":"10.1128\/AAC.01347-17"},{"key":"2026010105410547000_2025.12.16.694790v1.10","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01275-08"},{"key":"2026010105410547000_2025.12.16.694790v1.11","doi-asserted-by":"crossref","unstructured":"Rudra P , Hurst-Hess K , Lappierre P , Ghosh P. 2018. High Levels of Intrinsic Tetracycline Resistance in Mycobacterium abscessus Are Conferred by a Tetracycline-Modifying Monooxygenase. 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