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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2020-02-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mBio.02364-19 |
| _version_ | 1818406319992864768 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T09:10:04Z |
| format | Article |
| id | doaj.art-d5efd17612704f668563a19bea374a8f |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-14T09:10:04Z |
| publishDate | 2020-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-d5efd17612704f668563a19bea374a8f2022-12-21T23:08:35ZengAmerican Society for MicrobiologymBio2150-75112020-02-0111110.1128/mBio.02364-19A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>Mei-Yi Yan0Si-Shang Li1Xin-Yuan Ding2Xiao-Peng Guo3Qi Jin4Yi-Cheng Sun5MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaMOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaMOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaMOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaMOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaMOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaABSTRACT 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.https://journals.asm.org/doi/10.1128/mBio.02364-19CRISPR-Cas systemMycobacterium marinumMycobacterium smegmatisMycobacterium tuberculosisgenome editingnonhomologous end joining |
| spellingShingle | 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> mBio CRISPR-Cas system Mycobacterium marinum Mycobacterium smegmatis Mycobacterium tuberculosis genome editing nonhomologous end joining |
| title | 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_short | 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 |
| topic | CRISPR-Cas system Mycobacterium marinum Mycobacterium smegmatis Mycobacterium tuberculosis genome editing nonhomologous end joining |
| url | https://journals.asm.org/doi/10.1128/mBio.02364-19 |
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