Application of CRISPR-interference for elucidating leptospirosis pathogenesis and development of novel strategies for knockout mutant's obtainment

Abstract

Leptospirosis is a zoonosis of global importance recently included in the List of Neglected Tropical Diseases. In urban areas, rodents play the role of the main reservoirs of the disease by allowing the colonization of leptospires in proximal tubes and excreting them alive via urine. According to the World Health Organization, the number of annual clinical cases worldwide remains underestimated. A better understanding of the pathogenesis of the bacteria is crucial for the development of an effective vaccine. The generation of Leptospira mutants is a key step in the elucidation of virulence factors and the basic biology of the bacterium. During the development of the Postdoctoral Project (2017/06731-8 and BEPE 2019/20302-8), the CRISPR-interference tool for epissomal and complete gene silencing in saprophytic and pathogenic strains was developed, and for the first time in the literature, species besides L. interrogans were manipulated. The CRISPRi system requires the constitutive expression of dCas9 ("dead" Cas9 protein) and a guide RNA (sgRNA), which directs the protein to the targets of interest, blocking transcription. This tool has been shown to be extremely useful in vitro and in vivo for elucidation of the role of Leptospira spp. proteins. In this context, our group is working with numerous potential genes for silencing by this technique, and with this additional period, it is intended to apply and to disseminate the CRISPRi technique in our laboratory, as well as to explore these potential targets. It is also intended to develop new plasmids for genetic knockout, through the use of the enzyme Cas9 and co-expression of Mycobacterium tuberculosis proteins that act in the repair of genomic breakdown induced by the enzyme.