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

Article · Liu J, Wang Y, Lu Y, Zheng P, Sun J, Ma Y

- Archive id: doi:10.1186/s12934-017-0815-5
- Published: 2017-11-16
- Venue: Microbial cell factories
- DOI: https://doi.org/10.1186/s12934-017-0815-5
- PMID: 29145843
- PMCID: PMC5693361
- Citations: 95
- Source URL: https://doi.org/10.1186/s12934-017-0815-5
- PDF: https://europepmc.org/articles/PMC5693361/pdf
- Provenance: europepmc

## 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.

_Abstract reproduced under CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)._

Topics: [CRISPR & Gene-Editing Therapies](https://space-kitty.com/topics/gene-editing-therapies)

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