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

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

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.

Electronic supplementary material

The online version of this article (10.1186/s12934-017-0815-5) contains supplementary material, which is available to authorized users.

Related collections

Most cited references 43

Record: found
Abstract: found
Article: not found

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.

Alexis Komor, Ahmed H Badran, David R. Liu (2017)

The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.

0 comments Cited 389 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

CRISPR/Cpf1-mediated DNA-free plant genome editing

Hyeran Kim, Sang-Tae Kim, Jahee Ryu … (2017)

Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1–crRNA complex is an effective DNA-free genome-editing tool for plant genome editing.

0 comments Cited 208 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

Niclas Bengtsson, John Hall, Guy Odom … (2017)

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx 4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.

0 comments Cited 165 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Jiao Liu: liu_j@tib.cas.cn

Yu Wang: wang_y@tib.cas.cn

Yujiao Lu: lu_yj@tib.cas.cn

Ping Zheng: zheng_p@tib.cas.cn

Jibin Sun: sun_jb@tib.cas.cn

Yanhe Ma: ma_yh@tib.cas.cn

Journal

Journal ID (nlm-ta): Microb Cell Fact

Journal ID (iso-abbrev): Microb. Cell Fact

Title: Microbial Cell Factories

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2859

Publication date (Electronic): 16 November 2017

Publication date PMC-release: 16 November 2017

Publication date Collection: 2017

Volume: 16

Electronic Location Identifier: 205

Affiliations

[1 ]ISNI 0000000119573309, GRID grid.9227.e, Key Laboratory of Systems Microbial Biotechnology, , Chinese Academy of Sciences, ; Tianjin, 300308 People’s Republic of China

[2 ]ISNI 0000000119573309, GRID grid.9227.e, Tianjin Institute of Industrial Biotechnology, , Chinese Academy of Sciences, ; Tianjin, 300308 People’s Republic of China

Article

Publisher ID: 815

DOI: 10.1186/s12934-017-0815-5

PMC ID: 5693361

PubMed ID: 29145843

SO-VID: 88bbc851-4e5e-4931-920f-64f45930c96c

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 29 June 2017

Date accepted : 8 November 2017

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;

Award ID: 31700044

Award ID: 31370829

Award Recipient : Yu Wang Jibin Sun

Funded by: Key Research Program of the Chinese Academy of Sciences

Award ID: ZDRW-ZS-2016-2

Award Recipient : Ping Zheng

Funded by: Special Support Plan for Talents Development and High-level Innovation and Entrepreneurship Team of the Tianjin Municipal City

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: crispr/cas9,genome editing,corynebacterium glutamicum,plasmid-borne templates,gene deletion/insertion,single-nucleotide editing,double-locus editing

Data availability:

ScienceOpen disciplines: Biotechnology

Keywords: crispr/cas9, genome editing, corynebacterium glutamicum, plasmid-borne templates, gene deletion/insertion, single-nucleotide editing, double-locus editing

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

Read this article at

Abstract

Background

Results

Conclusions

Electronic supplementary material

Related collections

CRISPR/Cas9 editing in human blood

Most cited references 43

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.

CRISPR/Cpf1-mediated DNA-free plant genome editing

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

Author and article information

Contributors

Journal

Affiliations

Article

History

Funding

Categories

Custom metadata

Comments

Comment on this article

Similar content 808

Cited by 47

Most referenced authors 1,071