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CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942
Open accessAbstract
BACKGROUND: Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells. RESULTS: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome. Co-expression of dCas9 and sgRNA conferred effective and stable suppression of EYFP production at efficiencies exceeding 99%, without impairing cell growth. We next integrated the dCas9 and sgRNA targeting endogenous genes essential for glycogen accumulation (glgc) and succinate conversion to fumarate (sdhA and sdhB). Transcription levels of glgc, sdhA and sdhB were effectively suppressed with efficiencies depending on the sgRNA binding site. Targeted suppression of glgc reduced the expression to 6.2%, attenuated the glycogen accumulation to 4.8% and significantly enhanced the succinate titer. Targeting sdhA or sdhB also effectively downregulated the gene expression and enhanced the succinate titer ≈12.5-fold to ≈0.58-0.63 mg/L. CONCLUSIONS: These data demonstrated that CRISPRi-mediated gene suppression allowed for re-directing the cellular carbon flow, thus paving a new avenue to rationally fine-tune the metabolic pathways in PCC 7942 for the production of biotechnological products.
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“This approach provides a valuable means for studying gene function and fine-tuning of metabolic pathways without permanent genomic alterations [119,120].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [120]
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Metadata
- Authors
- Huang, C.-H., Shen, C. R., Li, H., Sung, L.-Y., Wu, M.-Y., & Hu, Y.-C.
- Affiliations
- National Tsing Hua University (TW)
- Corresponding author
- Hu, Yu-Chen
- Venue
- Microbial Cell Factories
- Published
- 2016-11-15
- Volume
- 15
- Issue
- 1
- Pages
- 196-196
- ISSN
- 1475-2859 (ISSN-L)
- PMC
- PMC5111286
- OpenAlex
- W2549981312
- Cited by
- 164
- References
- 53
- Citations by year
- 2026: 10 · 2025: 11 · 2024: 14 · 2023: 15 · 2022: 20 · 2021: 23 · 2020: 21 · 2019: 23 · 2018: 17 · 2017: 10
- FWCI
- 8.66
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Microbial Cell Factories (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Ministry of Science and Technology (MOST 103B7028J2, 104-2622-8-007-001, 105-2622-8-007-009)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
- Keywords
- CRISPR interference, Synechococcus elongatus PCC 7942, cyanobacteria, dCas9, single guide RNA, gene expression inhibition, succinate production, glycogen accumulation, SDHA, SDHB, metabolic pathway engineering, chromosomal integration
- MeSH terms
- Gene Expression Regulation, Bacterial, Succinic Acid, Synechococcus, Metabolic Engineering, Clustered Regularly Interspaced Short Palindromic Repeats
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