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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.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-016-0514-7) contains supplementary material, which is available to authorized users.

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