ArticleScientific reports2019
Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira.
Article in Scientific reports, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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30 citing papers in PubMed, 45 citations in OpenAlex.
- Identification and characterization of small RNAs expressed by Leptospira borgpetersenii serovar Hardjo and their conservation in the genus Leptospira.BMC microbiology · 2026Article
- TheInfection and immunity · 2026Article
- DMEM and EMEM as alternate growth media for pathogenic Leptospira.PLoS neglected tropical diseases · 2026Article
- Transposon Sequencing in Leptospira interrogans.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Gene Knockout in Leptospira spp. by CRISPR/Cas9-NHEJ (Non-Homologous End-Joining) and CRISPR-Prime Editing.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation.Scientific reports · 2025Article
- DMEM and EMEM are suitable surrogate media to mimic host environment and expand leptospiral pathogenesis studies usingbioRxiv : the preprint server for biology · 2025Article
- CRISPR-Cas technologies in neurodegenerative disorders: mechanistic insights, therapeutic potential, and translational challenges.Frontiers in neurology · 2025Review
- Article
- Evolutionary insights into the emergence of virulent Leptospira spirochetes.PLoS pathogens · 2024Article
- Evolutionary insights into the emergence of virulentbioRxiv : the preprint server for biology · 2024Article
- Optimal delivery of RNA interference by viral vectors for cancer therapy.Molecular therapy : the journal of the American Society of Gene Therapy · 2023Review
- LipL41 and LigA/LigB Gene Silencing on a LipL32 KnockoutPathogens (Basel, Switzerland) · 2023Article
- The Arsenal ofAntioxidants (Basel, Switzerland) · 2023Review
- Evaluation ofFrontiers in microbiology · 2023Article
- Advances in CRISPR/Cas-Based Strategies on Zoonosis.Transboundary and emerging diseases · 2023Review
- A Novel Breakthrough inFrontiers in microbiology · 2022Article
- Transcriptional analysis of CRISPR I-B arrays ofFrontiers in microbiology · 2022Article
- CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria.Frontiers in genome editing · 2022Review
- Article
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Authors and funding
6 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
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.
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