{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T07:33:45Z","timestamp":1778052825182,"version":"3.51.4"},"reference-count":19,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,7,9]],"date-time":"2025-07-09T00:00:00Z","timestamp":1752019200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82272375"],"award-info":[{"award-number":["82272375"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    The increasing global prevalence of\n                    <jats:italic>Mycobacterium abscessus<\/jats:italic>\n                    infections presents a significant clinical challenge due to the pathogen\u2019s intrinsic resistance to multiple antibiotics and poor treatment outcomes. Despite the necessity of genetic tools for studying its physiology, pathogenesis, and drug resistance, efficient methods for large-fragment deletions remain underdeveloped. Here, we report a CRISPR\/Cas9-based dual-sgRNA system employing\n                    <jats:italic>Streptococcus thermophilus<\/jats:italic>\n                    CRISPR1-Cas9 (Sth1Cas9), enabling efficient large-fragment knockout in\n                    <jats:italic>M. abscessus<\/jats:italic>\n                    with deletion efficiencies exceeding 90% at certain loci and spanning up to 16.7\u202fkb. Furthermore, we systematically optimized the modular arrangement of genetic components in Cas9\/dual-sgRNA expression plasmids and refined their construction workflow, achieving a significant reduction in cassette loss rates while enabling single-step plasmid assembly. Notably, deletion efficiency was position-dependent rather than correlated with target size, suggesting an influence of chromatin structure on editing outcomes. As the first CRISPR\/Cas9-based platform capable of kilobase-scale deletions in\n                    <jats:italic>M. abscessus<\/jats:italic>\n                    , this system advances functional genomics studies and facilitates targeted investigations into virulence and antibiotic resistance mechanisms.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2025.1608274","type":"journal-article","created":{"date-parts":[[2025,7,10]],"date-time":"2025-07-10T05:45:49Z","timestamp":1752126349000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["A CRISPR\/Cas9-based system using dual-sgRNAs for efficient gene deletion in Mycobacterium abscessus"],"prefix":"10.3389","volume":"16","author":[{"given":"Linai","family":"Li","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuxiang","family":"Hu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shengjuan","family":"Bao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Taibing","family":"Deng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qinglan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,7,9]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"106924","DOI":"10.1016\/j.mimet.2024.106924","article-title":"Gene knock-out in Mycobacterium abscessus using Streptococcus thermophilus CRISPR\/Cas","volume":"220","author":"Akter","year":"2024","journal-title":"J. Microbiol. Methods"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"3757","DOI":"10.1111\/febs.15041","article-title":"A guide to Mycobacterium mutagenesis","volume":"286","author":"Borgers","year":"2019","journal-title":"FEBS J."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"633510","DOI":"10.3389\/fmicb.2021.633510","article-title":"Genetic manipulation of non-tuberculosis mycobacteria","volume":"12","author":"Chimukuche","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"712","DOI":"10.1016\/j.cmi.2023.08.035","article-title":"Epidemiology of Mycobacterium abscessus","volume":"30","author":"Cristancho-Rojas","year":"2024","journal-title":"Clin. Microbiol. Infect."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"120","DOI":"10.1016\/j.ijid.2022.10.013","article-title":"Global trends of pulmonary infections with nontuberculous mycobacteria: a systematic review","volume":"125","author":"Dahl","year":"2022","journal-title":"Int. J. Infect. Dis."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"706207","DOI":"10.3389\/fmicb.2021.706207","article-title":"Increased virulence of outer membrane porin mutants of Mycobacterium abscessus","volume":"12","author":"de Moura","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"120","DOI":"10.1016\/j.chest.2017.04.166","article-title":"Microbiological and clinical outcomes of treating non-Mycobacterium avium complex nontuberculous mycobacterial pulmonary disease: a systematic review and meta-analysis","volume":"152","author":"Diel","year":"2017","journal-title":"Chest"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"e000111","DOI":"10.1099\/mgen.0.000111","article-title":"Genomic epidemiology of a national outbreak of post-surgical Mycobacterium abscessus wound infections in Brazil","volume":"3","author":"Everall","year":"2017","journal-title":"Microb. Genom."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"1331","DOI":"10.1128\/aem.01914-08","article-title":"Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools","volume":"75","author":"Medjahed","year":"2009","journal-title":"Appl. Environ. Microbiol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"101983","DOI":"10.1016\/j.tube.2020.101983","article-title":"Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9","volume":"124","author":"Meijers","year":"2020","journal-title":"Tuberculosis"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"e0033523","DOI":"10.1128\/jb.00335-23","article-title":"A dual-plasmid CRISPR\/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus","volume":"206","author":"Neo","year":"2024","journal-title":"J. Bacteriol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"810","DOI":"10.1093\/jac\/dkr578","article-title":"Mycobacterium abscessus: a new antibiotic nightmare","volume":"67","author":"Nessar","year":"2012","journal-title":"J. Antimicrob. Chemother."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"1167","DOI":"10.1046\/j.1365-2958.2003.03662.x","article-title":"Mycobacterial porins\u2014new channel proteins in unique outer membranes","volume":"49","author":"Niederweis","year":"2003","journal-title":"Mol. Microbiol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"16274","DOI":"10.1038\/nmicrobiol.2016.274","article-title":"Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform","volume":"2","author":"Rock","year":"2017","journal-title":"Nat. Microbiol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"1091","DOI":"10.3201\/eid2406.172095","article-title":"Pulmonary infections with nontuberculous mycobacteria, Catalonia, Spain, 1994\u20132014","volume":"24","author":"Santin","year":"2018","journal-title":"Emerg. Infect. Dis."},{"key":"ref16","first-page":"35","article-title":"Phage L5 integrating vectors are present within the mycobacterial cell in an equilibrium between integrated and excised states","volume":"7","author":"Saviola","year":"2009","journal-title":"Cancer Ther."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1016\/s2213-2600(16)30444-1","article-title":"Multidrug-resistant Mycobacterium abscessus threatens patients with cystic fibrosis","volume":"5","author":"van Dorn","year":"2017","journal-title":"Lancet Respir. Med."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"69","DOI":"10.3389\/fcimb.2018.00069","article-title":"A simple and rapid gene disruption strategy in Mycobacterium abscessus: on the design and application of glycopeptidolipid mutants","volume":"8","author":"Viljoen","year":"2018","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"e02364-19","DOI":"10.1128\/mBio.02364-19","article-title":"A CRISPR-assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis","volume":"11","author":"Yan","year":"2020","journal-title":"mBio"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1608274\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,10]],"date-time":"2025-07-10T13:38:56Z","timestamp":1752154736000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1608274\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,9]]},"references-count":19,"alternative-id":["10.3389\/fmicb.2025.1608274"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1608274","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2025.05.28.656731","asserted-by":"object"}]},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,9]]},"article-number":"1608274"}}