Cargando…

A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of i...

Descripción completa

Detalles Bibliográficos
Autores principales: Fernandes, Luis G. V., Nascimento, Ana L. T. O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9199861/
https://www.ncbi.nlm.nih.gov/pubmed/35722349
http://dx.doi.org/10.3389/fmicb.2022.915382
_version_ 1784727937903230976
author Fernandes, Luis G. V.
Nascimento, Ana L. T. O.
author_facet Fernandes, Luis G. V.
Nascimento, Ana L. T. O.
author_sort Fernandes, Luis G. V.
collection PubMed
description Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.
format Online
Article
Text
id pubmed-9199861
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-91998612022-06-16 A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems Fernandes, Luis G. V. Nascimento, Ana L. T. O. Front Microbiol Microbiology Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira. Frontiers Media S.A. 2022-05-26 /pmc/articles/PMC9199861/ /pubmed/35722349 http://dx.doi.org/10.3389/fmicb.2022.915382 Text en Copyright © 2022 Fernandes and Nascimento. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Fernandes, Luis G. V.
Nascimento, Ana L. T. O.
A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems
title A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems
title_full A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems
title_fullStr A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems
title_full_unstemmed A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems
title_short A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems
title_sort novel breakthrough in leptospira spp. mutagenesis: knockout by combination of crispr/cas9 and non-homologous end-joining systems
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9199861/
https://www.ncbi.nlm.nih.gov/pubmed/35722349
http://dx.doi.org/10.3389/fmicb.2022.915382
work_keys_str_mv AT fernandesluisgv anovelbreakthroughinleptospirasppmutagenesisknockoutbycombinationofcrisprcas9andnonhomologousendjoiningsystems
AT nascimentoanalto anovelbreakthroughinleptospirasppmutagenesisknockoutbycombinationofcrisprcas9andnonhomologousendjoiningsystems
AT fernandesluisgv novelbreakthroughinleptospirasppmutagenesisknockoutbycombinationofcrisprcas9andnonhomologousendjoiningsystems
AT nascimentoanalto novelbreakthroughinleptospirasppmutagenesisknockoutbycombinationofcrisprcas9andnonhomologousendjoiningsystems