1. Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira
- Author
-
Silvio Arruda Vasconcellos, Marcos Bryan Heinemann, Ana L. T. O. Nascimento, Luis G. V. Fernandes, Mathieu Picardeau, Linda P. Guaman, Instituto Butantan [São Paulo], Universidad Tecnologica Equinoccial [Quito, Ecuador] (UTE), Universidade de São Paulo (USP), Biologie des Spirochètes / Biology of Spirochetes, Institut Pasteur [Paris], The following Brazilian agencies: FAPESP (grant 2014/50981-0), CNPq (grants 301229/2017-1 and 441449/2014-0) and Fundacao Butantan, financially supported this work, LGVF has fellowship from FAPESP (2017/06731-8). This work was also supported by the Institut Pasteur, Paris, France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., We are deeply in debt to Dr. Albert Leyva for English editing this manuscript., Universidade de São Paulo = University of São Paulo (USP), and Institut Pasteur [Paris] (IP)
- Subjects
0301 basic medicine ,Streptococcus pyogenes ,[SDV]Life Sciences [q-bio] ,lcsh:Medicine ,ENGENHARIA GENÉTICA ,Biology ,Article ,Genome engineering ,03 medical and health sciences ,0302 clinical medicine ,Plasmid ,CRISPR-Associated Protein 9 ,Gene silencing ,CRISPR ,Gene Silencing ,lcsh:Science ,Gene ,Genetics ,Leptospira ,CRISPR interference ,Multidisciplinary ,Cas9 ,lcsh:R ,030104 developmental biology ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Coding strand ,RNA ,lcsh:Q ,CRISPR-Cas Systems ,Genetic Engineering ,030217 neurology & neurosurgery ,RNA, Guide, Kinetoplastida - Abstract
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.
- Published
- 2019
- Full Text
- View/download PDF