A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis
| Autor: | Jin Qi, Mei-Yi Yan, Yi-Cheng Sun, Si-Shang Li, Xiaopeng Guo, Xin-Yuan Ding |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Molecular Biology and Physiology
Tuberculosis DNA End-Joining Repair Bacterial Toxins Computational biology Genome Models Biological Microbiology Mycobacterium tuberculosis 03 medical and health sciences Genome editing Virology crispr-cas system medicine CRISPR genome editing Clustered Regularly Interspaced Short Palindromic Repeats Mycobacterium marinum 030304 developmental biology mycobacterium tuberculosis Gene Editing 0303 health sciences biology 030306 microbiology Mycobacterium smegmatis biology.organism_classification medicine.disease QR1-502 nonhomologous end joining Non-homologous end joining Rec A Recombinases mycobacterium marinum CRISPR-Cas Systems mycobacterium smegmatis Genome Bacterial Protein Binding RNA Guide Kinetoplastida Research Article |
| Zdroj: | mBio, Vol 11, Iss 1, p e02364-19 (2020) mBio mBio, Vol 11, Iss 1 (2020) |
| ISSN: | 2150-7511 |
| Popis: | 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. 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. |
| Databáze: | OpenAIRE |
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