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Microb Cell Fact. 2017 Nov 16;16(1):205. doi: 10.1186/s12934-017-0815-5.

Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum.

Liu J1,2, Wang Y1,2, Lu Y1,2, Zheng P3,4, Sun J5,6, Ma Y2.

Author information

1
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.
2
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China.
3
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. zheng_p@tib.cas.cn.
4
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. zheng_p@tib.cas.cn.
5
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. sun_jb@tib.cas.cn.
6
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. sun_jb@tib.cas.cn.

Abstract

BACKGROUND:

Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum.

RESULTS:

Herein, we developed a versatile CRISPR/Cas9 genome editing toolbox for C. glutamicum. Cas9 and gRNA expression cassettes were reconstituted to combat Cas9 toxicity and facilitate effective termination of gRNA transcription. Co-transformation of Cas9 and gRNA expression plasmids was exploited to overcome high-frequency mutation of cas9, allowing not only highly efficient gene deletion and insertion with plasmid-borne editing templates (efficiencies up to 60.0 and 62.5%, respectively) but also simple and time-saving operation. Furthermore, CRISPR/Cas9-mediated ssDNA recombineering was developed to precisely introduce small modifications and single-nucleotide changes into the genome of C. glutamicum with efficiencies over 80.0%. Notably, double-locus editing was also achieved in C. glutamicum. This toolbox works well in several C. glutamicum strains including the widely-used strains ATCC 13032 and ATCC 13869.

CONCLUSIONS:

In this study, we developed a CRISPR/Cas9 toolbox that could facilitate markerless gene deletion, gene insertion, precise base editing, and double-locus editing in C. glutamicum. The CRISPR/Cas9 toolbox holds promise for accelerating the engineering of C. glutamicum and advancing its application in the production of biochemicals and biofuels.

KEYWORDS:

CRISPR/Cas9; Corynebacterium glutamicum; Double-locus editing; Gene deletion/insertion; Genome editing; Plasmid-borne templates; Single-nucleotide editing

PMID:
29145843
PMCID:
PMC5693361
DOI:
10.1186/s12934-017-0815-5
[Indexed for MEDLINE]
Free PMC Article

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