{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T21:30:41Z","timestamp":1792704641289,"version":"4.5.0"},"reference-count":76,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2018,1,10]],"date-time":"2018-01-10T00:00:00Z","timestamp":1515542400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2018,1,10]],"date-time":"2018-01-10T00:00:00Z","timestamp":1515542400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The sexually-transmitted parasite\n                    <jats:italic>Trichomonas vaginalis<\/jats:italic>\n                    infects ~1\/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying\n                    <jats:italic>T. vaginalis<\/jats:italic>\n                    pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single\n                    <jats:italic>T. vaginalis<\/jats:italic>\n                    cell. We then created DNA constructs required to express several components essential to drive CRISPR\/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR\/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR\/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the\n                    <jats:italic>T. vaginalis<\/jats:italic>\n                    genome. This first report of the use of the CRISPR\/Cas9 system in\n                    <jats:italic>T. vaginalis<\/jats:italic>\n                    greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.\n                  <\/jats:p>","DOI":"10.1038\/s41598-017-18442-3","type":"journal-article","created":{"date-parts":[[2018,1,4]],"date-time":"2018-01-04T05:57:19Z","timestamp":1515045439000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":54,"title":["CRISPR\/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis"],"prefix":"10.1038","volume":"8","author":[{"given":"Brian D.","family":"Janssen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi-Pei","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brenda M.","family":"Molgora","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4460-7221","authenticated-orcid":false,"given":"Shuqi E.","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Augusto","family":"Simoes-Barbosa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia J.","family":"Johnson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2018,1,10]]},"reference":[{"key":"18442_CR1","doi-asserted-by":"crossref","unstructured":"Rowley RTaN, F. Global incidence and prevalence of selected curable sexually transmitted infections-2008. 20 (World Health Organization, 2012).","DOI":"10.1016\/S0968-8080(12)40660-7"},{"key":"18442_CR2","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1097\/QCO.0000000000000128","volume":"28","author":"RP Hirt","year":"2015","unstructured":"Hirt, R. P. & Sherrard, J. Trichomonas vaginalis origins, molecular pathobiology and clinical considerations. Curr Opin Infect Dis 28, 72\u201379, https:\/\/doi.org\/10.1097\/QCO.0000000000000128 (2015).","journal-title":"Curr Opin Infect Dis"},{"key":"18442_CR3","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1136\/sti.2003.005124","volume":"80","author":"H Swygard","year":"2004","unstructured":"Swygard, H., Sena, A. C., Hobbs, M. M. & Cohen, M. S. Trichomoniasis: clinical manifestations, diagnosis and management. Sex Transm Infect 80, 91\u201395 (2004).","journal-title":"Sex Transm Infect"},{"key":"18442_CR4","doi-asserted-by":"publisher","first-page":"939","DOI":"10.3201\/eid1806.111590","volume":"18","author":"RD Kirkcaldy","year":"2012","unstructured":"Kirkcaldy, R. D. et al. Trichomonas vaginalis antimicrobial drug resistance in 6 US cities, STD Surveillance Network, 2009\u20132010. Emerg Infect Dis 18, 939\u2013943, https:\/\/doi.org\/10.3201\/eid1806.111590 (2012).","journal-title":"Emerg Infect Dis"},{"key":"18442_CR5","doi-asserted-by":"publisher","first-page":"4209","DOI":"10.1128\/AAC.00814-06","volume":"50","author":"JR Schwebke","year":"2006","unstructured":"Schwebke, J. R. & Barrientes, F. J. Prevalence of Trichomonas vaginalis isolates with resistance to metronidazole and tinidazole. Antimicrob Agents Chemother 50, 4209\u20134210, https:\/\/doi.org\/10.1128\/AAC.00814-06 (2006).","journal-title":"Antimicrob Agents Chemother"},{"key":"18442_CR6","doi-asserted-by":"crossref","first-page":"3004","DOI":"10.1128\/JCM.38.8.3004-3009.2000","volume":"38","author":"LJ Snipes","year":"2000","unstructured":"Snipes, L. J. et al. Molecular epidemiology of metronidazole resistance in a population of Trichomonas vaginalis clinical isolates. J Clin Microbiol 38, 3004\u20133009 (2000).","journal-title":"J Clin Microbiol"},{"key":"18442_CR7","doi-asserted-by":"publisher","first-page":"334","DOI":"10.1071\/SH09011","volume":"6","author":"JA Upcroft","year":"2009","unstructured":"Upcroft, J. A. et al. Metronidazole resistance in Trichomonas vaginalis from highland women in Papua New Guinea. Sex Health 6, 334\u2013338, https:\/\/doi.org\/10.1071\/SH09011 (2009).","journal-title":"Sex Health"},{"key":"18442_CR8","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/j.pt.2012.10.004","volume":"29","author":"MD Conrad","year":"2013","unstructured":"Conrad, M. D., Bradic, M., Warring, S. D., Gorman, A. W. & Carlton, J. M. Getting trichy: tools and approaches to interrogating Trichomonas vaginalis in a post-genome world. Trends Parasitol 29, 17\u201325, https:\/\/doi.org\/10.1016\/j.pt.2012.10.004 (2013).","journal-title":"Trends Parasitol"},{"key":"18442_CR9","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1016\/B978-0-12-391429-3.00006-X","volume":"77","author":"RP Hirt","year":"2011","unstructured":"Hirt, R. P. et al. Trichomonas vaginalis pathobiology new insights from the genome sequence. Adv Parasitol 77, 87\u2013140, https:\/\/doi.org\/10.1016\/B978-0-12-391429-3.00006-X (2011).","journal-title":"Adv Parasitol"},{"key":"18442_CR10","doi-asserted-by":"publisher","first-page":"177","DOI":"10.1016\/j.molbiopara.2012.03.003","volume":"183","author":"D Leitsch","year":"2012","unstructured":"Leitsch, D., Drinic, M., Kolarich, D. & Duchene, M. Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis. Mol Biochem Parasitol 183, 177\u2013183, https:\/\/doi.org\/10.1016\/j.molbiopara.2012.03.003 (2012).","journal-title":"Mol Biochem Parasitol"},{"key":"18442_CR11","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1111\/mmi.12455","volume":"91","author":"D Leitsch","year":"2014","unstructured":"Leitsch, D., Janssen, B. D., Kolarich, D., Johnson, P. J. & Duchene, M. Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance. 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