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Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic <jats:italic>Leptospira<\/jats:italic> continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6\u00a0weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the <jats:italic>Streptococcus pyogenes<\/jats:italic> CRISPR\/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic <jats:italic>L. interrogans<\/jats:italic>. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37\u00a0\u00b0C in 3% CO<jats:sub>2<\/jats:sub> within 8\u00a0days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic <jats:italic>Leptospira<\/jats:italic> serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.<\/jats:p>","DOI":"10.1038\/s41598-021-81400-7","type":"journal-article","created":{"date-parts":[[2021,1,19]],"date-time":"2021-01-19T11:09:05Z","timestamp":1611054545000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":46,"title":["Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37\u00a0\u00b0C"],"prefix":"10.1038","volume":"11","author":[{"given":"L. G. 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