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Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira
JoVE Journal
Genética
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JoVE Journal Genética
Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

DOI:
10.3791/62631-v

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14:49 min

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August 14, 2021

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, , ,
1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service,United States Department of Agriculture, 2Laboratório de Desenvolvimento de Vacinas,Instituto Butantan

Capítulos

  • 00:04Introduction
  • 02:56Protospacer Definition and Plasmid Construction
  • 04:24Leptospira Transformation by Conjugation
  • 09:35Colony Selection and Transconjugant Growth and Validation
  • 13:01Representative Results
  • 13:51Conclusions

Summary

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Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

Tags

CRISPR InterferenceCRISPRiGene SilencingLeptospiraPathogenic SpeciesHornsby-Alt-Nally MediaLeptospirosisGenetic ToolsDCas9Single-guide RNAIn Vitro ResearchVaccine Development

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