Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C

Abstract Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira...

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Autores principales:	L. G. V. Fernandes, R. L. Hornsby, A. L. T. O. Nascimento, J. E. Nally
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c8860077aafe4cccbb4637a9656bf816
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spelling	oai:doaj.org-article:c8860077aafe4cccbb4637a9656bf8162021-12-02T15:23:47ZGenetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C10.1038/s41598-021-81400-72045-2322https://doaj.org/article/c8860077aafe4cccbb4637a9656bf8162021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81400-7https://doaj.org/toc/2045-2322Abstract Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira 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 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes 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 L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. 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 Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.L. G. V. FernandesR. L. HornsbyA. L. T. O. NascimentoJ. E. NallyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q L. G. V. Fernandes R. L. Hornsby A. L. T. O. Nascimento J. E. Nally Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C
description	Abstract Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira 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 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes 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 L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. 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 Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.
format	article
author	L. G. V. Fernandes R. L. Hornsby A. L. T. O. Nascimento J. E. Nally
author_facet	L. G. V. Fernandes R. L. Hornsby A. L. T. O. Nascimento J. E. Nally
author_sort	L. G. V. Fernandes
title	Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C
title_short	Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C
title_full	Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C
title_fullStr	Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C
title_full_unstemmed	Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C
title_sort	genetic manipulation of pathogenic leptospira: crispr interference (crispri)-mediated gene silencing and rapid mutant recovery at 37 °c
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/c8860077aafe4cccbb4637a9656bf816
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Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C

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