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Cited by 164Microbial Metabolic Engineering and BioproductionCRISPR and Genetic EngineeringAlgal biology and biofuel production

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CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

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Huang, Chun-Hung, Shen, Claire R., Li, Hung, Sung, Li-Yu, Wu, Meng-Ying, Hu, Yu-Chen·2016·Microbial Cell Factories
DOI 10.1186/s12934-016-0595-3PMC PMC5111286
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Abstract

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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Fig. 1 Establishment of CRISPRi in PCC 7942. a Map of pSdCas9-CY’ harboring CmR, dCas9 under Psmt and eyfp under PconII, which was flanked by homology arms targeting the NSI site. Trrnb, transcriptional terminator. Psmt consists of the smtA promoter and the smtB repressor. b Map of psgRNA harboring the cassette expressing KmR and sgRNA under the PJ23119 promoter, which was flanked by NSII-targeting homology arms. The sgRNAs were designed to target no sequence on the PCC 7942 chromosome (Φ) or the eyfp cassette at the non-template strand of promoter (P1) or coding regions near the transcription start site (NT1 and NT2). The numbers indicate the position relative to the transcription start site. c Confocal micrographs of cells. d Flow cytometry analysis data. pSdCas9-CY’ was first transformed into PCC 7942 for cassette integration into NSI site, re-streaked, then individual psgRNA was transformed into the recombinant cells for integration into NSII site. The transformants colonies were transferred to shake flasks and cultured to OD730 = 1–1.5 in BG-11 medium containing appropriate antibiotics
Fig. 1 Establishment of CRISPRi in PCC 7942. a Map of pSdCas9-CY’ harboring CmR, dCas9 under Psmt and eyfp under PconII, which was flanked by homology arms targeting the NSI site. Trrnb, transcriptional terminator. Psmt consists of the smtA promoter and the smtB repressor. b Map of psgRNA harboring the cassette expressing KmR and sgRNA under the PJ23119 promoter, which was flanked by NSII-targeting homology arms. The sgRNAs were designed to target no sequence on the PCC 7942 chromosome (Φ) or the eyfp cassette at the non-template strand of promoter (P1) or coding regions near the transcription start site (NT1 and NT2). The numbers indicate the position relative to the transcription start site. c Confocal micrographs of cells. d Flow cytometry analysis data. pSdCas9-CY’ was first transformed into PCC 7942 for cassette integration into NSI site, re-streaked, then individual psgRNA was transformed into the recombinant cells for integration into NSII site. The transformants colonies were transferred to shake flasks and cultured to OD730 = 1–1.5 in BG-11 medium containing appropriate antibioticsCC BY · PubMed Central
Fig. 2 Effect of CRISPRi on PCC 7942 growth and persistent transgene repression. a Mean fluorescence intensity (FI) profiles. b Growth curves. The recombinant cells were cultured in 40 ml BG-11 medium containing Km/Cm. Wild-type (WT) cells were cultured in 40 ml BG-11 without antibiotics. The cells were cultured for 21 days, during which cells were sampled for OD730 or flow cytometry analysis
Fig. 2 Effect of CRISPRi on PCC 7942 growth and persistent transgene repression. a Mean fluorescence intensity (FI) profiles. b Growth curves. The recombinant cells were cultured in 40 ml BG-11 medium containing Km/Cm. Wild-type (WT) cells were cultured in 40 ml BG-11 without antibiotics. The cells were cultured for 21 days, during which cells were sampled for OD730 or flow cytometry analysisCC BY · PubMed Central
Fig. 3 Schematic illustration of plasmids used for CRISPRi-mediated suppression of glgc, sdhA and sdhB. Φ indicates the scramble sgRNA. Glgc1, glgc2, sdhA1, sdhA2, sdhB1 and sdhB2 are the sgRNA targeting different regions of endogenous genes. pSdCas9 expressed dCas9 under Psmt
Fig. 3 Schematic illustration of plasmids used for CRISPRi-mediated suppression of glgc, sdhA and sdhB. Φ indicates the scramble sgRNA. Glgc1, glgc2, sdhA1, sdhA2, sdhB1 and sdhB2 are the sgRNA targeting different regions of endogenous genes. pSdCas9 expressed dCas9 under PsmtCC BY · PubMed Central
Fig. 4 Effective suppression of endogenous genes by CRISPRi. a glgc expression. b sdhA expression. c sdhB expression. PCC 7942 cells were transformed with pSdCas9 first for integration into NSI site, and were transformed again with individual psgRNA plasmids for integration into NSII site. The KmR/CmR colonies were transferred to 40 ml BG-11 medium containing Km/Cm and cultured to OD730 = 0.7–1.3, and 5 ml cells were sampled for qRT-PCR analysis. The expression levels were normalized to those in the WT cells
Fig. 4 Effective suppression of endogenous genes by CRISPRi. a glgc expression. b sdhA expression. c sdhB expression. PCC 7942 cells were transformed with pSdCas9 first for integration into NSI site, and were transformed again with individual psgRNA plasmids for integration into NSII site. The KmR/CmR colonies were transferred to 40 ml BG-11 medium containing Km/Cm and cultured to OD730 = 0.7–1.3, and 5 ml cells were sampled for qRT-PCR analysis. The expression levels were normalized to those in the WT cellsCC BY · PubMed Central
Fig. 5 Effects of CRISPRi-mediated suppression on glycogen accumulation and succinate production. a Glycogen titer (µg/OD730). b Succinate titer (mg/l). For glycogen analysis, KmR/CmR cells were cultured to OD730 ≈ 0.7–1.3, centrifuged and cultured in shake flasks containing 40 ml nitrogen-deplete (0×N) BG-11 for 2 days and subjected to glycogen analysis. To analyze succinate titer, PCC 7942 cells cultured in shake flasks to stationary phase (OD73 ≈ 2.0) were centrifuged, and resuspended in 20 ml nitrate-free BG-11 medium. After centrifugation, the cells were resuspended in 40 ml nitrate-deplete (0×N) BG-11 medium containing Km/Cm and cultured in shake flasks. The WT cells were cultured in the nitrate-deplete (0×N) BG-11 or BG-11 (nitrate-replete) (1×N) medium
Fig. 5 Effects of CRISPRi-mediated suppression on glycogen accumulation and succinate production. a Glycogen titer (µg/OD730). b Succinate titer (mg/l). For glycogen analysis, KmR/CmR cells were cultured to OD730 ≈ 0.7–1.3, centrifuged and cultured in shake flasks containing 40 ml nitrogen-deplete (0×N) BG-11 for 2 days and subjected to glycogen analysis. To analyze succinate titer, PCC 7942 cells cultured in shake flasks to stationary phase (OD73 ≈ 2.0) were centrifuged, and resuspended in 20 ml nitrate-free BG-11 medium. After centrifugation, the cells were resuspended in 40 ml nitrate-deplete (0×N) BG-11 medium containing Km/Cm and cultured in shake flasks. The WT cells were cultured in the nitrate-deplete (0×N) BG-11 or BG-11 (nitrate-replete) (1×N) mediumCC BY · PubMed Central

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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
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2016-11-15
Volume
15
Issue
1
Pages
196-196
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/s12934-016-0595-3
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
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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