A novel breakthrough in Leptospira spp. mutagenesis: knockout by combination of CRISPR/Cas9 and non-homologous end-joining systems

Full metadata record
DC FieldValueLanguage
dc.contributorLab. de Desenvolvimento de Vacinaspt_BR
dc.contributor.authorFernandes, Luis Guilherme Vírgíliopt_BR
dc.contributor.authorNascimento, Ana Lúcia Tabet Ollerpt_BR
dc.date.accessioned2022-06-21T14:48:29Z-
dc.date.available2022-06-21T14:48:29Z-
dc.date.issued2022pt_BR
dc.identifier.citationFernandes LGV, Nascimento ALTO. A novel breakthrough in Leptospira spp. mutagenesis: knockout by combination of CRISPR/Cas9 and non-homologous end-joining systems. Front. Microbiol. 2022 May;13:915382. doi:10.3389/fmicb.2022.915382.pt_BR
dc.identifier.urihttps://repositorio.butantan.gov.br/handle/butantan/4395-
dc.description.abstractLeptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.pt_BR
dc.description.sponsorship(FAPESP) Fundação de Amparo à Pesquisa do Estado de São Paulopt_BR
dc.description.sponsorship(CNPq) Conselho Nacional de Desenvolvimento Científico e Tecnológicopt_BR
dc.language.isoEnglishpt_BR
dc.relation.ispartofFrontiers in Microbiologypt_BR
dc.rightsOpen accesspt_BR
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/pt_BR
dc.titleA novel breakthrough in Leptospira spp. mutagenesis: knockout by combination of CRISPR/Cas9 and non-homologous end-joining systemspt_BR
dc.typeArticlept_BR
dc.rights.licenseCC BYpt_BR
dc.identifier.doi10.3389/fmicb.2022.915382pt_BR
dc.identifier.urlhttps://doi.org/10.3389/fmicb.2022.915382pt_BR
dc.contributor.external(USP) Universidade de São Paulopt_BR
dc.identifier.citationvolume13pt_BR
dc.identifier.citationissue915382pt_BR
dc.subject.keywordLeptospira interroganspt_BR
dc.subject.keywordleptospirosispt_BR
dc.subject.keywordCRISPR/Cas9pt_BR
dc.subject.keywordmycobacterium tuberculosispt_BR
dc.subject.keywordmycobacterium smegmatispt_BR
dc.subject.keywordnon-homologous end-joiningpt_BR
dc.subject.keywordknockout mutantspt_BR
dc.subject.keywordLeptospira biflexapt_BR
dc.relation.ispartofabbreviatedFront. Microbiolpt_BR
dc.identifier.citationabntv. 13, 915382, maio. 2022pt_BR
dc.identifier.citationvancouver2022 May;13:915382pt_BR
dc.contributor.butantanFernandes, Luis Guilherme Vírgílio|:Desvinculado|:Lab. de Desenvolvimento de Vacinas|:PrimeiroAutor:Autor de correspondênciapt_BR
dc.contributor.butantanNascimento, Ana Lúcia Tabet Oller do|:Pesquisador|:Lab. de Desenvolvimento de Vacinaspt_BR
dc.sponsorship.butantan(FAPESP) Fundação de Amparo à Pesquisa do Estado de São Paulo¦¦2019/17488-2pt_BR
dc.sponsorship.butantan(FAPESP) Fundação de Amparo à Pesquisa do Estado de São Paulo¦¦2017/06731-8pt_BR
dc.sponsorship.butantan(CNPq) Conselho Nacional de Desenvolvimento Científico e Tecnológico¦¦301229/2017-1pt_BR
dc.identifier.bvsccBR78.1pt_BR
dc.identifier.bvsdbIBProdpt_BR
dc.description.dbindexedYespt_BR
item.fulltextCom Texto completo-
item.languageiso639-1English-
item.grantfulltextopen-
item.openairetypeArticle-
crisitem.author.dept#PLACEHOLDER_PARENT_METADATA_VALUE#-
crisitem.author.dept#PLACEHOLDER_PARENT_METADATA_VALUE#-
crisitem.author.orcid#PLACEHOLDER_PARENT_METADATA_VALUE#-
crisitem.author.orcid0000-0003-4851-0870-
crisitem.author.parentorg#PLACEHOLDER_PARENT_METADATA_VALUE#-
crisitem.journal.journalissn#PLACEHOLDER_PARENT_METADATA_VALUE#-
crisitem.journal.journaleissn#PLACEHOLDER_PARENT_METADATA_VALUE#-
Appears in Collections:Artigos de periódicos


Files in This Item:

fmicb-13-915382.pdf
Description:
Size: 4.21 MB
Format: Adobe PDF
View/Open
Show simple item record

This item is licensed under a Creative Commons License Creative Commons