{"id":"doi:10.1186/s12934-017-0815-5","kind":"article","title":"Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum","authors":["Liu J","Wang Y","Lu Y","Zheng P","Sun J","Ma Y"],"author_count":6,"year":2017,"published":"2017-11-16","venue":"Microbial cell factories","cited_by":95,"doi":"10.1186/s12934-017-0815-5","arxiv_id":null,"nct_id":null,"source_url":"https://doi.org/10.1186/s12934-017-0815-5","archive_url":"https://space-kitty.com/papers/doi:10.1186/s12934-017-0815-5","updated":null,"pmid":"29145843","pmcid":"PMC5693361","openalex_id":null,"pdf_url":"https://europepmc.org/articles/PMC5693361/pdf","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_license":"CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)","license":"CC-BY","is_open_access":true,"retracted":false,"peer_reviewed":null,"categories":[],"details":null,"provenance":"europepmc","attribution":"Abstract of \"Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum\" by Liu J, Wang Y, Lu Y et al., https://doi.org/10.1186/s12934-017-0815-5, used under its Creative Commons Attribution license (CC-BY); markup removed, otherwise unmodified.","topics":[{"slug":"gene-editing-therapies","name":"CRISPR & Gene-Editing Therapies","domain_slug":"medicine"}],"cited_in_dossiers":[]}