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Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira.
Fernandes, L G V; Guaman, L P; Vasconcellos, S A; Heinemann, Marcos B; Picardeau, M; Nascimento, A L T O.
Afiliación
  • Fernandes LGV; Laboratório Especial de Desenvolvimento de Vacinas, Instituto Butantan, Avenida Vital Brasil, 1500, 05503-900, Sao Paulo, SP, Brazil. luis.fernandes@butantan.gov.br.
  • Guaman LP; Universidad Tecnológica Equinoccial, Centro de Investigación Biomédica, Facultad de Ciencias de la Salud Eugenio Espejo, Avenida Mariscal Sucre y Mariana de Jesús. Campus Occidental, 170105, Quito, Ecuador.
  • Vasconcellos SA; Laboratório de Zoonoses Bacterianas do VPS, Faculdade de Medicina Veterinária e Zootecnia, USP, Avenida Prof. Dr. Orlando Marques de Paiva, 87, 05508-270, Sao Paulo, SP, Brazil.
  • Heinemann MB; Laboratório de Zoonoses Bacterianas do VPS, Faculdade de Medicina Veterinária e Zootecnia, USP, Avenida Prof. Dr. Orlando Marques de Paiva, 87, 05508-270, Sao Paulo, SP, Brazil.
  • Picardeau M; Institut Pasteur, Biology of Spirochetes Unit, 25 rue du Dr Roux, 75723, Paris, France.
  • Nascimento ALTO; Laboratório Especial de Desenvolvimento de Vacinas, Instituto Butantan, Avenida Vital Brasil, 1500, 05503-900, Sao Paulo, SP, Brazil. ana.nascimento@butantan.gov.br.
Sci Rep ; 9(1): 1839, 2019 02 12.
Article en En | MEDLINE | ID: mdl-30755626
Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Streptococcus pyogenes / Ingeniería Genética / ARN Guía de Kinetoplastida / Leptospira Idioma: En Revista: Sci Rep Año: 2019 Tipo del documento: Article País de afiliación: Brasil Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Streptococcus pyogenes / Ingeniería Genética / ARN Guía de Kinetoplastida / Leptospira Idioma: En Revista: Sci Rep Año: 2019 Tipo del documento: Article País de afiliación: Brasil Pais de publicación: Reino Unido