A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a hig...

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Autores principales:	Mei-Yi Yan, Si-Shang Li, Xin-Yuan Ding, Xiao-Peng Guo, Qi Jin, Yi-Cheng Sun
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	CRISPR-Cas system Mycobacterium marinum Mycobacterium smegmatis Mycobacterium tuberculosis genome editing nonhomologous end joining Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d5efd17612704f668563a19bea374a8f
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spelling	oai:doaj.org-article:d5efd17612704f668563a19bea374a8f2021-11-15T15:56:58ZA CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>10.1128/mBio.02364-192150-7511https://doaj.org/article/d5efd17612704f668563a19bea374a8f2020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02364-19https://doaj.org/toc/2150-7511ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.Mei-Yi YanSi-Shang LiXin-Yuan DingXiao-Peng GuoQi JinYi-Cheng SunAmerican Society for MicrobiologyarticleCRISPR-Cas systemMycobacterium marinumMycobacterium smegmatisMycobacterium tuberculosisgenome editingnonhomologous end joiningMicrobiologyQR1-502ENmBio, Vol 11, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR-Cas system Mycobacterium marinum Mycobacterium smegmatis Mycobacterium tuberculosis genome editing nonhomologous end joining Microbiology QR1-502
spellingShingle	CRISPR-Cas system Mycobacterium marinum Mycobacterium smegmatis Mycobacterium tuberculosis genome editing nonhomologous end joining Microbiology QR1-502 Mei-Yi Yan Si-Shang Li Xin-Yuan Ding Xiao-Peng Guo Qi Jin Yi-Cheng Sun A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
description	ABSTRACT New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli. IMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis. More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.
format	article
author	Mei-Yi Yan Si-Shang Li Xin-Yuan Ding Xiao-Peng Guo Qi Jin Yi-Cheng Sun
author_facet	Mei-Yi Yan Si-Shang Li Xin-Yuan Ding Xiao-Peng Guo Qi Jin Yi-Cheng Sun
author_sort	Mei-Yi Yan
title	A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_short	A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full	A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_fullStr	A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full_unstemmed	A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_sort	crispr-assisted nonhomologous end-joining strategy for efficient genome editing in <named-content content-type="genus-species">mycobacterium tuberculosis</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/d5efd17612704f668563a19bea374a8f
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A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

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