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

Author(s)

Fernandes, L. G., V ; Hornsby, R. L. ; Nascimento, A. L. T. O. ; Nally, J. E.

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 174, p. 14-pg., 2021-08-01.

Abstract

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain beta 2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates. (AU)

• FAPESP's process: 17/06731-8 - Application of CRISPR-interference for elucidating leptospirosis pathogenesis and development of novel strategies for knockout mutant's obtainment
• Grantee: Luis Guilherme Virgílio Fernandes
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/50981-0 - Search for surface proteins among the genome sequences of Leptospira interrogans: functional and immunological characterization to understanding mechanisms involved in the bacterial pathogenesis
• Grantee: Ana Lucia Tabet Oller do Nascimento
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/20302-8 - CRISPR interference (CRISPRi)-mediated silencing of non-coding RNA (ncRNA) in saprophyte and pathogenic strains of Leptospira
• Grantee: Luis Guilherme Virgílio Fernandes
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor