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CRISPR Interference (Crispri) for Gene Regulation and Succinate Production in Cyanobacterium S

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CRISPR Interference (Crispri) for Gene Regulation and Succinate Production in Cyanobacterium S Huang et al. Microb Cell Fact (2016) 15:196 DOI 10.1186/s12934-016-0595-3 Microbial Cell Factories RESEARCH Open Access CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942 Chun‑Hung Huang, Claire R. Shen, Hung Li, Li‑Yu Sung, Meng‑Ying Wu and Yu‑Chen Hu* 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 pro‑ caryotic cells. Results: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes express‑ ing 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 produc‑ tion 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. Keywords: CRISPRi, PCC 7942, Cyanobacteria, sgRNA, Metabolic engineering, Gene regulation Background Synechococcus elongatus PCC 7942 has been genetically Cyanobacteria are photoautotrophic procaryotes that modified as a “cell factory” to divert native metabolic can exploit sunlight and CO2 as the sole energy and car- pathways to produce 2,3-butanediol, 2-methyl-1-butanol, bon sources to convert CO2 into organic compounds isopropanol, free fatty acid, 1,2-propanediol, isopropanol, via photosynthesis [1]. Many cyanobacterial strains are isobutyraldehyde and isobutanol, etc. [3]. amenable to natural transformation and homologous Typically, PCC 7942 is engineered by knocking in genes recombination for gene manipulation. Thanks to these encoding synthetic pathways and/or knocking out genes attributes, genetically engineered cyanobacteria have in competing pathways [4, 5]. However, generation of a drawn increasing attention as a chassis for the production single gene knockout mutant may take >3 weeks using of biofuels and bio-derived chemicals [2]. For instance, conventional methods [6] due to its long doubling time and oligoploidy nature [1]. Sometimes deletion of cer- tain genes essential for metabolic balances is not feasible *Correspondence: [email protected] or easily achieved as the deletion might be lethal to the Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan cells. Furthermore, in many cases intermediate levels of © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. Microb Cell Fact (2016) 15:196 Page 2 of 11 enzyme expression may result in better product titer [7]. Methods Therefore, tunable and balanced gene expression is desir- Microorganisms able for high productivity, product titer, and conversion All molecular cloning experiments were performed using yield, and controllable gene repression/knockdown may E. coli DH5α strain (Sigma). Unless otherwise noted, for be preferable than gene deletion for certain biotechno- suspension culture S. elongatus PCC 7942 (Invitrogen) logical applications and synthetic biological manipula- was cultivated in a 250 ml shake flask containing 40 ml tions [8]. BG-11 medium [4, 5] with or without antibiotics (gyra- CRISPR-Cas9 is a newly developed RNA-guided tory shaking at 100 rpm, with sterile air containing 0.04% genome editing system [9, 10]. CRISPR-Cas9 system CO2) in a 30 °C incubator (600SR, Hipoint) with illu- comprises the Cas9 nuclease, transacting RNA (tracr- mination from continuous cool white fluorescent light 2 RNA) and CRISPR RNA (crRNA). crRNA/tracrRNA (intensity ≈ 70 μmol//m s). For solid culture, PCC 7942 complexes with Cas9 and, guided by the spacer sequence cells were streaked onto 90 mm plates containing 40 cm3 on crRNA, orchestrate to recognize protospacer-adja- BG-11/agar medium supplemented with 1 mM sodium cent motif (PAM) and bind to proximal complemen- thiosulfate and appropriate antibiotic, and incubated tary sequence. After the recognition and binding, Cas9 with continuous illumination (intensity 70 µmol//m2 s) nuclease triggers double strand break (DSB) at the chro- for 7–9 days until colonies developed. mosomal DNA [11]. Coupled with an editing template DNA, such CRISPR-Cas9-mediated DSB is exploited for Plasmids construction programmable genome engineering of diverse cell types pdCas9-bacteria plasmid (Addgene #44249) harbored and model organisms [12–16], as well as for gene and cell chloramphenicol resistance gene (CmR) and dCas9 gene therapy [17–19]. We have also employed CRISPR to engi- (derived from S. pyogenes) driven by the PLtetO1 pro- neer the PCC 7942 genome [20] and effectively inserted moter [24]. The sequences homologous to the 5′ (5-NSI) DNA fragments as large as 7 kb into Escherichia coli and 3′ (3-NSI) end of PCC 7942 NSI site (neutral site I), genome [21]. together with the intervening origin of replication (ori), Furthermore, the catalytic domains of Cas9 are were PCR-amplified from pSYN_1 plasmid (Invitro- mutated to generate the inactive Cas9 (dCas9) lacking gen) with flankingAvr II and SpeI sites. pdCas9-bacte- the endonuclease activity. dCas9 is used in conjunction ria and the PCR product were separately digested with with the chimeric single guide RNA (sgRNA) wherein the AvrII/SpeI and ligated together (Additional file 1: Figure mature crRNA is fused to a partial tracrRNA to mimic S1). The resultant pLtetO1-dCas9 contained the CmR and the natural crRNA:tracrRNA duplex. By co-expression, PLtetO1-dCas9 expression cassettes flanked by the 5-NSI the dCas9-sgRNA complex specifically binds to the tar- and 3-NSI homology arms (Additional file 1: Figure S1). get gene at the promoter or coding sequence and acts as We next PCR-amplified the smtP promoter (including a roadblock to the elongating RNA polymerase, hence the promoter smtA and the repressor smtB) from the aborting transcription initiation or elongation [22]. This PCC 7942 chromosome, which was cloned into pLtetO1- new technology, termed CRISPR interference (CRISPRi), dCas9 by AflII/BglII digestion to replace the PLtetO1 pro- was recently repurposed to repress sequence-specific moter, yielding pSdCas9 (Additional file 1: Figure S2). genes in diverse eucaryotic and procaryotic cells, for The eyfp gene under the control of PconII promoter was rewiring metabolic networks [23, 24] and high-through- PCR-amplified from pconII-EYFP’ (see Additional file 1: put interrogation of genome-wide gene functions [25, Supplementary Methods) and subcloned into pSdCas9 26]. Very recently, CRISPRi has also exploited for gene by AvrII/SmaI (Additional file 1: Figure S3). The resultant regulation in cyanobacteria Synechcocystis sp. PCC 6803 pSdCas9-CY’ harbored the expression cassettes compris- R [27] and Synechococcus sp. PCC 7002 [7]. However, ing Cm , dCas9 under Psmt and eyfp under PconII, which whether CRISPRi functions in PCC 7942 has yet to be were flanked by homology arms targeting the NSI site explored. (5-NSI and 3-NSI). In this study, we harnessed the CRISPRi system to pgRNA-bacteria plasmid (Addgene, #44251) contained effectively knockdown exogenous and endogenous genes the ampicillin-resistance gene (ApR) and an sgRNA back- in PCC 7942 via appropriate sgRNA design. Selective bone driven by PJ23119 promoter. The sgRNA backbone repression of glgc, sdhA and sdhB genes increased the comprised the base-pairing (spacer) region (20 bp), succinate production by PCC 7942, hence demonstrating dCas9 handle (42 bp) and the S. pyogenes terminator the feasibility of employing CRISPRi for the metabolic (40 bp) as described [22]. To replace the spacer sequence engineering of PCC 7942 and production of bio-derived on the sgRNA backbone with new spacer sequences tar- chemicals. geting different regions on the PCC 7942 chromosome, Huang et al. Microb Cell Fact (2016) 15:196 Page 3 of 11 we designed a reverse primer Ec_R and forward primers quantified using Nanodrop 2000 (Thermo), and 2000 ng Ec_F with different new
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