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

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. 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. In conclusion, 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 typemore » genetic background.« less
Authors:
 [1] ;  [1] ;  [2] ;  [2] ; ORCiD logo [1]
  1. Washington Univ., St. Louis, MO (United States)
  2. Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States)
Publication Date:
Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 15; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Research Org:
Washington Univ., St. Louis, MO (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Cyanobacteria; Synechococcus; CRISPR; Cas9; Genome modification
OSTI Identifier:
1375815

Wendt, Kristen E., Ungerer, Justin, Cobb, Ryan E., Zhao, Huimin, and Pakrasi, Himadri B.. CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. United States: N. p., Web. doi:10.1186/s12934-016-0514-7.
Wendt, Kristen E., Ungerer, Justin, Cobb, Ryan E., Zhao, Huimin, & Pakrasi, Himadri B.. CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. United States. doi:10.1186/s12934-016-0514-7.
Wendt, Kristen E., Ungerer, Justin, Cobb, Ryan E., Zhao, Huimin, and Pakrasi, Himadri B.. 2016. "CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973". United States. doi:10.1186/s12934-016-0514-7. https://www.osti.gov/servlets/purl/1375815.
@article{osti_1375815,
title = {CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973},
author = {Wendt, Kristen E. and Ungerer, Justin and Cobb, Ryan E. and Zhao, Huimin and Pakrasi, Himadri B.},
abstractNote = {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. 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. In conclusion, 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.},
doi = {10.1186/s12934-016-0514-7},
journal = {Microbial Cell Factories},
number = 1,
volume = 15,
place = {United States},
year = {2016},
month = {6}
}

Works referenced in this record:

Fatty acid production in genetically modified cyanobacteria
journal, April 2011
  • Liu, X.; Sheng, J.; Curtiss III, R.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 17, p. 6899-6904
  • DOI: 10.1073/pnas.1103014108

Photosynthetic conversion of carbon dioxide to ethylene by the recombinant cyanobacterium, Synechococcus sp. PCC 7942, which harbors a gene for the ethylene-forming enzyme of Pseudomonas syringae
journal, January 1997
  • Sakai, Miho; Ogawa, Takahira; Matsuoka, Masayoshi
  • Journal of Fermentation and Bioengineering, Vol. 84, Issue 5, p. 434-443
  • DOI: 10.1016/S0922-338X(97)82004-1

Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism
journal, January 2010

RNA-guided genetic silencing systems in bacteria and archaea
journal, February 2012
  • Wiedenheft, Blake; Sternberg, Samuel H.; Doudna, Jennifer A.
  • Nature, Vol. 482, Issue 7385, p. 331-338
  • DOI: 10.1038/nature10886

Multiplex Genome Engineering Using CRISPR/Cas Systems
journal, January 2013

CRISPR-Cas Systems in Bacteria and Archaea: Versatile Small RNAs for Adaptive Defense and Regulation
journal, December 2011

RNA-guided editing of bacterial genomes using CRISPR-Cas systems
journal, January 2013
  • Jiang, Wenyan; Bikard, David; Cox, David
  • Nature Biotechnology, Vol. 31, Issue 3, p. 233-239
  • DOI: 10.1038/nbt.2508