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Immunol."],"abstract":"<jats:p>Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from <jats:italic>Escherichia coli<\/jats:italic>, induces improved protection against <jats:italic>Mycobacterium tuberculosis<\/jats:italic> (<jats:italic>Mtb<\/jats:italic>) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR\/Cas9 to generate unmarked mutations in the <jats:italic>lysA<\/jats:italic> gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying <jats:italic>lysA<\/jats:italic> and <jats:italic>ltak63<\/jats:italic> gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCG\u0394-LTAK63) at comparable levels to the original construct. The intranasal challenge with <jats:italic>Mtb<\/jats:italic> confirmed the superior protection induced by rBCG\u0394-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCG\u0394-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR\/Cas9 in the tuberculosis vaccine development field.<\/jats:p>","DOI":"10.3389\/fimmu.2022.867195","type":"journal-article","created":{"date-parts":[[2022,3,30]],"date-time":"2022-03-30T13:23:09Z","timestamp":1648646589000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":13,"title":["CRISPR\/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate"],"prefix":"10.3389","volume":"13","author":[{"given":"Luana","family":"Moraes","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monalisa Martins","family":"Trentini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dimitrios","family":"Fousteris","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silas 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