A crispr-assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis

Yan，Mei Yi1; Li，Si Shang1; Ding，Xin Yuan1; Guo，Xiao Peng1; Jin，Qi1; Sun，Yi Cheng1,2

2020

10.1128/mBio.02364-19

mBio   影响因子和分区

2161-2129

2150-7511

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.

CRISPR-Cas system Genome editing Mycobacterium marinum Mycobacterium smegmatis Mycobacterium tuberculosis Nonhomologous end joining

SCI

英语

其他

National Natural Science Foundation of China[31870067]

Microbiology

Microbiology

WOS:000518763400117

AMER SOC MICROBIOLOGY

2-s2.0-85078688419

Scopus

1.MOH Key Laboratory of Systems Biology of Pathogens,Institute of Pathogen Biology,Center for Tuberculosis Research,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing,China
2.Sanming Project of Medicine in Shenzhen on Construction of Novel Systematic Network against Tuberculosis,National Clinical Research Center for Infectious Diseases,Shenzhen Third People’s Hospital,Southern University of Science and Technology,Shenzhen,China

Yan，Mei Yi,Li，Si Shang,Ding，Xin Yuan,et al. A crispr-assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis[J]. mBio,2020,11(1).

Yan，Mei Yi,Li，Si Shang,Ding，Xin Yuan,Guo，Xiao Peng,Jin，Qi,&Sun，Yi Cheng.(2020).A crispr-assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis.mBio,11(1).

Yan，Mei Yi,et al."A crispr-assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis".mBio 11.1(2020).