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Cell. Infect. Microbiol."],"abstract":"<jats:p>Until 2015, loss-of-function studies to elucidate protein function in <jats:italic>Leishmania<\/jats:italic> relied on gene disruption through homologous recombination. Then, the CRISPR\/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR\/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to <jats:italic>Leishmania<\/jats:italic> for episomal expression or integration in the \u03b2-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, <jats:italic>Leishmania<\/jats:italic> (<jats:italic>Viannia<\/jats:italic>) <jats:italic>braziliensis<\/jats:italic> is the most frequent etiological agent of tegumentary leishmaniasis. The <jats:italic>L. braziliensis<\/jats:italic> \u03b2-tubulin locus presents significant sequence divergence in comparison with <jats:italic>Leishmania major<\/jats:italic>, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the <jats:italic>L. major<\/jats:italic> \u03b2-tubulin sequences, present in the pTB007, were replaced by a <jats:italic>Leishmania<\/jats:italic> (<jats:italic>Viannia<\/jats:italic>) \u03b2-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the <jats:italic>L. braziliensis<\/jats:italic> M2903 genome, and <jats:italic>in silico<\/jats:italic> predictions suggest that this can also be achieved in other <jats:italic>Viannia<\/jats:italic> species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the <jats:italic>PF16<\/jats:italic> gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in <jats:italic>L. braziliensis<\/jats:italic> and provided an important tool for the study of the biology of this parasite.<\/jats:p>","DOI":"10.3389\/fcimb.2021.772311","type":"journal-article","created":{"date-parts":[[2021,11,10]],"date-time":"2021-11-10T06:40:43Z","timestamp":1636526443000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":22,"title":["Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase"],"prefix":"10.3389","volume":"11","author":[{"given":"Caroline R.","family":"Espada","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"Jr.","given":"Jos\u00e9 Carlos","family":"Quilles","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreia","family":"Albuquerque-Wendt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario C.","family":"Cruz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tom","family":"Beneke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucas B.","family":"Lorenzon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eva","family":"Gluenz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angela K.","family":"Cruz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia R. 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