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Cited by 232CRISPR and Genetic EngineeringMicrobial Metabolic Engineering and BioproductionBacterial Genetics and Biotechnology

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

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Wendt, Kristen E., Ungerer, Justin, Cobb, Ryan E., Zhao, Huimin, Pakrasi, Himadri B.·2016·Microbial Cell Factories
DOI 10.1186/s12934-016-0514-7PMC PMC4917971
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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.

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Crossref · 10.1186/s12934-016-0514-7

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Fig. 1 Plasmids were generated using pCRISPomyces-2 backbone to engineer the ∆nblA line. a The nblA deletion plasmid including cas9 and b the nblA editing plasmid excluding cas9 are depicted
Fig. 1 Plasmids were generated using pCRISPomyces-2 backbone to engineer the ∆nblA line. a The nblA deletion plasmid including cas9 and b the nblA editing plasmid excluding cas9 are depictedCC BY · PubMed Central
Fig. 2 Synechococcus 2973 exconjugants do not exhibit characteristic bleaching under nitrogen deprivation conditions. Triparental mating was used to introduce the ∆nblA CRISPR/Cas9 plasmid into Synechococcus 2973. Exconjugants were patched onto selective media and then transferred to liquid cultures in standard and nitrogen deprivation conditions
Fig. 2 Synechococcus 2973 exconjugants do not exhibit characteristic bleaching under nitrogen deprivation conditions. Triparental mating was used to introduce the ∆nblA CRISPR/Cas9 plasmid into Synechococcus 2973. Exconjugants were patched onto selective media and then transferred to liquid cultures in standard and nitrogen deprivation conditionsCC BY · PubMed Central
Fig. 3 Double homologous recombination was used to generate the markerless deletion. a Schematic of the double homologous recombination event that results in deletion of nblA from the chromosome is shown. Black arrows indicate primers, yellow rectangles indicate homology arms, and the blue arrow represents the nblA gene. b PCR was used to confirm the deletion of nblA. Colony PCR using Primer A and Primer B of mutant Synechococcus 2973 yielded a product that is 180 base pairs lower in molecular weight than the band produced by wild type culture
Fig. 3 Double homologous recombination was used to generate the markerless deletion. a Schematic of the double homologous recombination event that results in deletion of nblA from the chromosome is shown. Black arrows indicate primers, yellow rectangles indicate homology arms, and the blue arrow represents the nblA gene. b PCR was used to confirm the deletion of nblA. Colony PCR using Primer A and Primer B of mutant Synechococcus 2973 yielded a product that is 180 base pairs lower in molecular weight than the band produced by wild type cultureCC BY · PubMed Central
Fig. 4 The cas9 gene was present in early patches, but absent once curing had occurred. Primers were designed to sit on the pCRISPomyces-2 backbone, outside cas9, yielding a 4.252 kb product if the plasmid was present
Fig. 4 The cas9 gene was present in early patches, but absent once curing had occurred. Primers were designed to sit on the pCRISPomyces-2 backbone, outside cas9, yielding a 4.252 kb product if the plasmid was presentCC BY · PubMed Central
Fig. 5 Curing of the CRISPR ∆nblA editing plasmid during consecutive serial patching. Top plate exhibits the growth of nine exconjugants on nonselective medium, and bottom plate shows the absence of growth on media containing apramycin. By patch ten, no growth is seen on the antibiotic-containing plate
Fig. 5 Curing of the CRISPR ∆nblA editing plasmid during consecutive serial patching. Top plate exhibits the growth of nine exconjugants on nonselective medium, and bottom plate shows the absence of growth on media containing apramycin. By patch ten, no growth is seen on the antibiotic-containing plateCC BY · PubMed Central

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Metadata

Authors
Wendt, K. E., Ungerer, J., Cobb, R. E., Zhao, H., & Pakrasi, H. B.
Affiliations
  • Washington University in St. Louis (US)
  • University of Illinois Urbana-Champaign (US)
Corresponding author
Pakrasi, Himadri B.
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2016-06-23
Volume
15
Issue
1
Pages
115-115
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/s12934-016-0514-7
PMC
PMC4917971
OpenAlex
W2470889463
Cited by
232
References
38
Citations by year
2026: 15 · 2025: 15 · 2024: 24 · 2023: 17 · 2022: 29 · 2021: 23 · 2020: 22 · 2019: 34 · 2018: 31 · 2017: 16 · 2016: 6
FWCI
15.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
  • Office of Science
  • National Institute of General Medical Sciences (GM077596)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
CRISPR/Cas9, cyanobacteria, genome editing, conjugative transfer
MeSH terms
Endonucleases, Genetic Techniques, Mutagenesis, Synechococcus, Clustered Regularly Interspaced Short Palindromic Repeats

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