Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C

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dc.contributorLEDV - Laboratório de Desenvolvimento de Vacinaspt_BR
dc.contributor.authorFernandes, Luis Guilherme Vírgíliopt_BR
dc.contributor.authorHornsby, R. L.pt_BR
dc.contributor.authorNascimento, Ana Lúcia Tabet Oller dopt_BR
dc.contributor.authorNally, J. E.pt_BR
dc.date.accessioned2021-01-26T17:48:47Z-
dc.date.available2021-01-26T17:48:47Z-
dc.date.issued2021pt_BR
dc.identifier.citationFernandes LGV, Hornsby R.L., Nascimento ALTO, Nally J.E.. Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C. Commun. Biol.. 2021 Jan;11:1768. doi:10.1038/s41598-021-81400-7.pt_BR
dc.identifier.urihttps://repositorio.butantan.gov.br/handle/butantan/3479-
dc.description.abstractLeptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.pt_BR
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)pt_BR
dc.description.sponsorshipNational Animal Disease Centerpt_BR
dc.format.extent1768pt_BR
dc.language.isoEnglishpt_BR
dc.relation.ispartofCommunications Biologypt_BR
dc.rightsOpen Accesspt_BR
dc.titleGenetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °Cpt_BR
dc.typeArticlept_BR
dc.identifier.doi10.1038/s41598-021-81400-7pt_BR
dc.identifier.urlhttps://doi.org/10.1038/s41598-021-81400-7pt_BR
dc.contributor.externalAgricultural Research Service (ARS)pt_BR
dc.contributor.externalNational Animal Disease Centerpt_BR
dc.contributor.externalUnited States Department of Agriculture (USDA)pt_BR
dc.identifier.citationvolume11pt_BR
dc.relation.ispartofabbreviatedCommun Biolpt_BR
dc.identifier.citationabntv. 11, 1768, jan. 2021pt_BR
dc.identifier.citationvancouver2021 Jan;11:1768pt_BR
dc.contributor.butantanFernandes, Luis Guilherme Vírgílio|:Aluno|:LEDV - Laboratório de Desenvolvimento de Vacinas|:PrimeiroAutor:|Autor de correspondênciapt_BR
dc.contributor.butantanNascimento, Ana Lúcia Tabet Oller do|:Pesquisador|:LEDV - Laboratório de Desenvolvimento de Vacinaspt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2014/50981-0pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2017/06731-8pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2019/20302-8pt_BR
dc.identifier.bvsccBR78.1pt_BR
dc.identifier.bvsdbIBProdpt_BR
dc.description.dbindexedYespt_BR
item.openairetypeArticle-
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item.grantfulltextopen-
item.languageiso639-1English-
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