title: CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973.
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.
authors.affiliation: Department of Biology, Washington University, St. Louis, MO, 63130, USA.
authors.forename: Kristen E
authors.initials: KE
authors.lastname: Wendt
authors.affiliation: Department of Biology, Washington University, St. Louis, MO, 63130, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Ungerer
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Cobb
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Biology, Washington University, St. Louis, MO, 63130, USA. Pakrasi@wustl.edu.
authors.forename: Himadri B
authors.identifier: http://orcid.org/0000-0001-8240-2123
authors.initials: HB
authors.lastname: Pakrasi
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism; D005821:Genetic Techniques; D016296:Mutagenesis; D046940:Synechococcus / Q000235:genetics* / Q000254:growth & development
chemical_list: D004720:Endonucleases
keywords: CRISPR; Cas9; Cyanobacteria; Genome modification; Synechococcus