title: CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942.
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.
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan.
authors.forename: Chun-Hung
authors.initials: CH
authors.lastname: Huang
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan.
authors.forename: Claire R
authors.initials: CR
authors.lastname: Shen
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan.
authors.forename: Hung
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan.
authors.forename: Li-Yu
authors.initials: LY
authors.lastname: Sung
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan.
authors.forename: Meng-Ying
authors.initials: MY
authors.lastname: Wu
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 30013, Taiwan. yuchen@che.nthu.edu.tw.
authors.forename: Yu-Chen
authors.identifier: http://orcid.org/0000-0002-9997-4467
authors.initials: YC
authors.lastname: Hu
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D060847:Metabolic Engineering; D019802:Succinic Acid / Q000378:metabolism*; D046940:Synechococcus / Q000235:genetics* / Q000378:metabolism*
chemical_list: D019802:Succinic Acid
keywords: CRISPRi; Cyanobacteria; Gene regulation; Metabolic engineering; PCC 7942; sgRNA