Application of CRISPR interference (CRISPRi) for gene silencing in pathogenic species of Leptospira

Authors: FERNANDES, LUIS - Butantan Institute; Hornsby, Richard; NASCIMENTO, ALTO - Butantan Institute; Nally, Jarlath

Submitted to: The Journal of Visualized Experiments (JoVE)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/2021
Publication Date: 8/14/2021
Citation: Fernandes, L.G., Hornsby, R.L., Nascimento, A., Nally, J.E. 2021. Application of CRISPR interference (CRISPRi) for gene silencing in pathogenic species of Leptospira. The Journal of Visualized Experiments (JoVE). 174. Article e62631. https://doi.org/10.3791/62631.
DOI: https://doi.org/10.3791/62631

Interpretive Summary: Our group has developed a novel methodology to silence the expression of specific genes within the genome of a unique group of bacteria that belong to the genus Leptospira. These bacteria cause leptospirosis, a global and zoonotic disease that affects animals as well as people. However, very little is known about how this group of bacteria cause disease. The ability to silence specific genes allows us to perform functional genomics and investigate exactly how leptospires cause disease. This manuscript describes the methods used to silence gene expression in leptospires.

Technical Abstract: Here, the application of the CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Mutant colonies of Leptospira are transformed by conjugation with plasmids expressing dCas9 (“dead” Cas9, with no nuclease activity) and single-guide RNA (sgRNA), which contains a 20-nt long sequence at its 5’ responsible for base pairing to the desired genomic target. Methods to validate gene silencing and evaluate mutant phenotype are also presented.