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

International audienceLeptospirosis 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

Landau Analog Levels for Dipoles in the Noncommutative Space and Phase Space

In the present contribution we investigate the Landau analog energy quantization for neutral particles, that possesses a nonzero permanent magnetic and electric dipole moments, in the presence of an homogeneous electric and magnetic external fields in the context of the noncommutative quantum mechanics. Also, we analyze the Landau--Aharonov--Casher and Landau--He--McKellar--Wilkens quantization due to noncommutative quantum dynamics of magnetic and electric dipoles in the presence of an external electric and magnetic fields and the energy spectrum and the eigenfunctions are obtained. Furthermore, we have analyzed Landau quantization analogs in the noncommutative phase space, and we obtain also the energy spectrum and the eigenfunctions in this context.Comment: 20 pages, references adde