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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 β-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.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira