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

Abstract

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:

Wendt, Kristen E.; Ungerer, Justin; Cobb, Ryan E.; Zhao, Huimin; Pakrasi, Himadri B.

• Search DOE PAGES for author "Pakrasi, Himadri B."
• Search DOE PAGES for ORCID "0000-0001-8240-2123"

Publication Date:

Thu Jun 23 00:00:00 EDT 2016

Research Org.:

Washington Univ., St. Louis, MO (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1618804

Alternate Identifier(s):

OSTI ID: 1375815

Resource Type:

Published Article

Journal Name:

Microbial Cell Factories

Additional Journal Information:

Journal Name: Microbial Cell Factories Journal Volume: 15 Journal Issue: 1; Journal ID: ISSN 1475-2859

Publisher:

Springer Science + Business Media

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Cyanobacteria; Synechococcus; CRISPR; Cas9; Genome modification