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Microb Cell Fact. 2016 Jun 23;15(1):115. doi: 10.1186/s12934-016-0514-7.

CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973.

Author information

1
Department of Biology, Washington University, St. Louis, MO, 63130, USA.
2
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
3
Department of Biology, Washington University, St. Louis, MO, 63130, USA. Pakrasi@wustl.edu.

Abstract

BACKGROUND:

As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions.

RESULTS:

First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain.

CONCLUSIONS:

High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.

KEYWORDS:

CRISPR; Cas9; Cyanobacteria; Genome modification; Synechococcus

PMID:
27339038
PMCID:
PMC4917971
DOI:
10.1186/s12934-016-0514-7
[Indexed for MEDLINE]
Free PMC Article

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