Multiple Gene Repression in Cyanobacteria Using CRISPRi - ACS

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Multiple gene repression in cyanobacteria using CRISPRi Lun Yao, Ivana Cengic, Josefine Anfelt, and Elton Paul Hudson ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00264 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 23, 2015

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ACS Synthetic Biology

Multiple gene repression in cyanobacteria using CRISPRi Lun Yao, Ivana Cengic, Josefine Anfelt, and Elton P. Hudson* KTH—Royal Institute of Technology. Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, Stockholm SE-171 21 Sweden. *Correspondence to [email protected] [email protected] [email protected] [email protected] [email protected] Abstract We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium Synechcocystis sp. PCC 6803. The nuclease-deficient Cas9 from the type-II CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. As an example of the potential of CRISPRi for basic and applied cyanobacteria research, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50-95% repression. This work also demonstrates that tightly repressed promoters allow for inducible and reversible CRISPRi in cyanobacteria.

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For Table of Contents Use Only

Multiple gene repression in cyanobacteria using CRISPRi Lun Yao, Ivana Cengic, Josefine Anfelt, and Elton P. Hudson


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Introduction The CRISPR/Cas system (clustered regularly interspaced short palindromic repeats/ CRISPR associated nuclease) has been adapted for targeted gene editing in mammalian, plant, fungal, and bacterial hosts.1 A simplified variant of the type-II CRISPR/Cas9 from Streptococcus pyogenes uses a chimeric single guide RNA (sgRNA) to direct the Cas9 nuclease to the host genome, where the Cas9 HNH and RuvC nuclease domains create a double-strand break.2 The sgRNA contains a structural Cas9-binding handle (42 nucleotides) and a protospacer (20 nucleotides) that hybridizes to the target sequence. Due to their small size, sgRNA fragments can be synthesized synthetically and easily incorporated into an array on a plasmid or genome. In this way, the CRISPR/Cas9 platform can be multiplexed, allowing for simultaneous cleavage, inactivation, or editing of multiple genes. Complementary to the CRISPR/Cas9 nuclease platform is CRISPRinterference (CRISPRi), which uses the same sgRNA, but with dCas9, a nucleasedeficient Cas9 in which the HNH and RuvC nuclease domains are silenced by point mutations (D10A and H840A).3 Here, the dCas9-sgRNA ribonuclueoprotein complex binds to the DNA target, but does not cleave it. CRISPRi can provide targeted gene regulation, as the dCas9-sgRNA complex can block RNA polymerase binding or elongation, resulting in gene repression. To activate a gene, a transcription factor or RNA polymerase subunit is fused or co-localized with the dCas9 and the dCas9sgRNA complex is directed to a site upstream of the target gene promoter.4 Multiplexed CRISPRi is a powerful tool for metabolic engineering, as multiple “knockdown” or “knockup” strains can be quickly realized and characterized for chemical production.5–7 Previous bioinformatics analysis identified three native CRISPR-Cas systems in Synechocystis PCC6803 (hereafter Synechocystis), one type-I (CRISPR1) and two type-III (CRISPR2 and CRISPR3).8 The crRNA array transcripts and some crRNA processing enzymes from these systems have been experimentally detected.9 The absence of a type-II CRISPR/Cas system in Synechocystis raises the possibility that the heterologous type-II CRISPRi can operate orthogonally from native RNA-



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processing enzymes. The CRISPRi platform could greatly accelerate both basic research and metabolic engineering efforts in cyanobacteria. These photoautotrophic bacteria are promising hosts for production of biofuels,10 yet exhibit productivities that are often an order of magnitude lower than E.coli and yeast.11 Recent efforts to increase biofuel and chemical productivities of cyanobacteria have turned to systems biology12 and high-throughput screening13. However, the systematic knockout of multiple genes in cyanobacteria is time consuming, due to relatively slow growth and multiple genome copies.14 For example, knockout of a gene locus in Synechocystis with an antibiotic selection marker often requires more than 2 weeks in optimal CO2 and light conditions, as recombinants must be re-plated and screened by PCR to ensure full genome segregation. Compounding the difficulty for multiple knockouts is a limited number of antibiotic resistance cassettes and a relative dearth of reliable counter-selection strategies, though some have recently been developed for model strains.15,16 A recent work developed an inducible sRNA-based repression tool in Synechococcus PCC7002 and achieved 59% repression of a target gene.17 However, multiplex repression was not reported. Despite the potential of CRISPR/Cas for accelerating metabolic engineering in cyanobacteria18, to date there has been no implementation of heterologous CRISPR/Cas-based genome editing or gene regulation in these hosts. Here we implement and characterize CRISPRi for gene repression in Synechocystis. We show that genes can be effectively repressed in multiplex format, and during nitrogen starvation conditions. Various inducible promoters were tested for both dCas9 and sgRNA and we found that a tightly repressed, low-strength promoter was most suitable for reversible repression. This work is a step toward rapid synthetic biology and metabolic engineering in cyanobacteria.

Results and Discussion Inducible repression of green fluorescent protein in Synechocystis with dCas9 The nuclease-deficient form of the Cas9 from Streptococcus pyogenes (dCas9) was used for gene knockdowns.3 Efficient gene repression using the dCas9 requires


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design of the sgRNA protospacer, as well as optimal expression levels of dCas9 and sgRNA. In initial tests, GFPmut3b (hereafter GFP) was used as a reporter and was put under the strong Ptrc promoter and integrated into the slr0168 neutral site of the Synechocystis genome. The sgRNA constructs were designed according to the protocol described by Larson et al.19 and consisted of a 5´-protospacer region, dCas9 binding handle, and Rho-independent transcription terminator described by Qi et al.3 All sgRNAs were checked for potential off-target binding using the CasOT software.20 The majority of potential off-target binding sites contained 6-7 mismatches with the sgRNA protospacer, including several in the 12 bp seed region near the PAM (see Supplemental for a list). These off-target binding sites were therefore likely not significant. Among all sgRNAs used in this study, there were 4 potential off-target binding sites with 4-5 mismatches, although each of these had at least 1 mismatch in the seed region. All sgRNAs targeted the non-template strand as this was previously reported to be more effective for repression in E. coli.3,4 Three sgRNAs were designed for GFP knockdown: sgRNA-NT1 targeted the 5´end of the gene (+86 bp from the Ptrc transcription start site), sgRNA-NT2 targeted +210 bp from the transcription start site (TSS), and sgRNA-NT0 targeted outside of the gene (Fig 1). The sgRNAs were put behind the constitutive Ptrc promoter and integrated into the slr2030-slr2031 neutral site. The dCas9 was put behind the constitutive PpsbA2 promoter21 and integrated into the psbA1 neutral site. The dCas9/sgRNA-NT1 repressed fluorescence to background levels (at least 100-fold), even though GFP was present on multiple chromosome copies in Synechocystis. The dCas9/sgRNA-NT2 also gave strong (90-fold) repression. These GFP repression levels are consistent with reports of CRISPRi blocking transcription elongation in E.coli, when using sgRNA protospacers within 300 bp of the TSS.3,4 No repression was seen with dCas9/sgRNA-NT0, which targeted outside of the GFP gene. Strains harboring only dCas9 or sgRNA-NT1 gave no repression of GFP. We next tested a variety of promoters for both dCas9 and sgRNA in order to identify optimal expression levels for targeted knockdown as well as a suitable inducible promoter. An inducible dCas9 tool would allow time-resolved studies of the effects of gene repression, reversible gene repression, and conditional



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repression of genes essential for growth. The suite of PL promoters developed by Huang et al. contain a TetR operator site and show a wide range of tightness and induction by anhydrotetracycline (aTc), as measured using a YFP reporter.22 We note that the Ptrc and PL series promoters have different transcription start sites, “GA” and “A” respectively,22 and this must be considered when designing the sgRNA protospacer. We chose three PL promoters spanning 2 orders of magnitude in transcription strength for either dCas9 or sgRNA-NT1 (Table 1). The promoter for dCas9 also included a TetR cassette. GFP was efficiently repressed when either dCas9 or sgRNA-NT1 was under the PL03 or PL31 promoter, even in the absence of the inducer aTc (Fig 2A). This indicates sufficient leakiness of these promoters so that they are unsuitable for reversible gene repression using CRISPRi. Western blotting confirmed leaky expression of dCas9 from the PL03 promoter in the absence of aTc, though expression was substantially increased when aTc was added (Fig 2C). The PL22 promoter was reported as 50-fold weaker than PL03 and basal expression of sgRNA and dCas9 from PL22 did not repress GFP. Western blotting confirmed nearly undetectable basal expression of dCas9 from PL22. In this strain, addition of aTc to 1 ug/mL resulted in 94% repression of GFP after 3 days (Fig 2B). Addition of aTc to 0.1 ug/mL also resulted in >90% repression of GFP (data not shown). The time necessary to achieve GFP protein knockdown after induction of dCas9/sgRNA was thus 4-5 cell generations, due to a high starting amount of GFP and GFP protein stability. The repression was reversible, as removal of aTc resulted in full recovery of GFP after 4 days (Fig 2B). We conclude that very tightly regulated promoters, where low basal expression is emphasized, are necessary to realize inducible and reversible gene repression by CRISPRi in Synechocystis.

Repression of carbon storage during nitrogen starvation Studies of biofuel production in Synechocystis have reported higher productivities upon eliminating biosynthesis of polyhydroxybutyrate (PHB), a carbon and reductant storage polymer that is a potential drain on acetyl-CoA precursor.10,23 PHB biosynthesis is upregulated during nutrient limitation, which


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can occur during the weeks-long culturing periods of cyanobacteria. The terminal step in PHB biosynthesis is catalyzed by polyhydroxyalkanoate (PHA) synthase, a heterodimer encoded by the phaE and phaC genes; both monomers are essential for PHB formation.24 We designed a sgRNA that targeted phaE (slr1829) at +55 bp from the predicted TSS. This sgRNA was placed under a constitutive promoter and quantification of mRNA transcripts showed a 10-fold repression of phaE in the mutant strain (Fig. 3A). When this strain was subject to nitrogen starvation, PHB biosynthesis was not detected (Fig. 3B). Thus, CRISPRi can function in the nitrogen starvation condition, which is characterized by protein degradation and cessation of ribosome synthesis.25 We next attempted to repress the carbon storage compound glycogen by introducing an sgRNA targeting the ADP-glucose pyrophosphorylase (AGPase) encoded by glgC (slr1176). Glycogen-negative mutants are useful to study the linkages between carbon and nitrogen metabolism in Synechocystis. In previous reports, glgC mutants of Synechocystis required multiple rounds of colony restreaking on selection antibiotics to achieve full genomic segregation.26 Therefore, inducible glycogen repression would be a preferred tool to achieve a glycogennegative phenotype. Transformation of the PpsbA2-dCas9 strain with a PL31-sgRNAglgC did not give resistant colonies after repeated transformation attempts, though mutants harboring only sgRNA-glgC (no dCas9) grew unimpeded. Lethality was presumably due to constitutive expression of dCas9 from the PpsbA2 promoter, resulting in permanent glgC repression. We next put dCas9 under the tightly repressed PL22 promoter, which gave transformants. We quantified both glgC mRNA and glycogen content from this strain. Without aTc inducer, glgC transcript levels were slightly reduced compared to wild type, consistent with low background expression of dCas9 (Fig 3C). Induction with 1 ug/mL aTc repressed glgC transcript levels to 10% after 24 hours. When this strain was then subject to nitrogen starvation for 2 days, it accumulated only 25% of the glycogen that wild type accumulated. The cultures where glgC was repressed did not bleach during starvation (Fig 3D). Resistance to chlorosis during nitrogen starvation is a known phenotype of glycogen-negative Synechocystis mutants.26



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Simultaneous repression of multiple putative aldehyde reductases and dehydrogenases The benefit of CRISPRi over traditional gene knockout is the ability to repress multiple genes simultaneously. As an example of how CRISPRi could be useful in both fundamental and applied studies of cyanobacteria metabolism, we sought to repress several aldehyde reductases and dehydrogenases. Cyanobacteria possess an extensive system for alleviating photooxidative stress caused by light. Part of this is a network of enzymes to directly oxidize or reduce reactive carbonyls of lipidderived peroxides formed by ROS. The ability to selectively repress a number of these enzymes would facilitate mapping of the photooxidative stress response. From a metabolic engineering perspective, cyanobacteria naturally produce alkanes27, which are potential biofuel additives. A cyanobacteria strain deficient in aldehyde reductases and dehydrogenases may have a larger aldehyde pool available for decarbonylation to alkanes.28 We selected four putative aldehyde reductases/dehydrogenases based on CyanoBase annotation, NAD(P)H-binding motif search, and alignment to known aldehyde reductases from E.coli or other cyanobacteria. Locus slr0942 encodes an aldo/keto reductase that actively reduces the aldehyde carbonyl group of several lipid peroxidation products to an alcohol using NAPDH.29 Locus sll0990 encodes a putative S-hydroxymethyl-glutathione dehydrogenase that has not been well characterized in Synechocystis, but was found to be significantly upregulated during peroxide stress.30 The gene product of sll0990 also had aldehyde reductase activity and catalyzed the conversion of acetaldehyde to ethanol when expressed in E.coli.31 Locus slr1192 encodes a well-characterized aldehyde reductase AdhA.32 Locus slr0091 encodes Alh, a dehydrogenase that oxidizes two substrate classes, longchain apocarotenals and long-change aldehydes (C9-C18). Alh is upregulated in high-light, indicating a possible protective role in detoxification of reactive aldehydes.33 We designed sgRNAs to target the 5´ end of the coding sequence of each locus and cloned them into a PpsbA2-dCas9 Synechocystis strain to create singleknockdown strains (Fig 4A). In addition to single knockdowns, we also created an sgRNA array of 3-sgRNA and 4-sgRNA for simultaneous knockdowns. In these


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arrays, each 100 bp sgRNA unit had its own promoter, dCas9 binding handle and protospacer, and terminator. Repression was greater than 10-fold for slr0942, sll0990, and slr1192 in both single- and multi-knockdown strains (Fig 4B). Notably, repression of each of these three did not affect expression of the others. The slr0091 locus is very weakly transcribed in wild-type Synechocystis relative to the other reductases, which is in agreement with previous RNA-Seq quantification.34 Repression of slr0091 was only 2-fold in the single-knockdown and the 4-sgRNA strain. The sgRNA-slr0091 region from these strains was sequenced and verified correct, allowing us to rule out mutation in the sgRNA as a reason for weak repression. A second possible cause of the weak repression is the position of the sgRNA_slr0091 protospacer, which is +167 bp from the ATG start codon. However, the near total repression of GFP using a protospacer at +210 bp (Fig. 1) suggests that sgRNA targeting at +167 bp would provide strong repression. It is likely that the TSS is not close to the slr0091 start codon. The slr0091 locus appears to be the third gene in an operon. A functional link to the upstream genes is possible. Gamma-tocopherol methyltransferase (slr0089) and 4-hydroxyphenolpyruvate dioxygenase (slr0090) are involved in the biosynthesis of tocopherols (Vitamin E), which relieve photooxidative stress, though deletion of slr0091 did not affect tocopherol levels.35 More conclusively, previous experimental determination of TSS in Synechocystis did not find one directly upstream of slr0091.36 Therefore, the most proximal TSS was upstream of slr0089, 2440 bp away from the sgRNA_slr0091 protospacer. Importantly, slr0942, sll0990, and slr1192 each have an experimentally confirmed TSS directly upstream of the reading frame, which may explain their relatively strong repression factors. Our results point to a limitation in the CRISPRi implementation in bacteria, namely that blocking of transcription elongation may not be efficient if the gene of interest is part of an operon with a distant TSS. In this case, multiple sgRNAs targeting the gene of interest may be necessary to achieve full knockdown.3

Methods Assembly of CRISPRi constructs



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All genes, promoters, terminators, and antibiotic resistance cassettes were assembled into Synechocystis targeting vectors using Biobrick assembly. Targeting vectors used the pMD19-T (simple) backbone (Takara). The dCas9 was a gift from Stanley Qi (Addgene plasmid #44249).3 An internal EcoRI site was mutated by overlap extension PCR to allow for BioBrick assembly of this part. Sequences of the Ptrc, PpsbA2, and PL promoters and RBS are listed in Supplemental. The sgRNAs were based on the sgRNA provided by Stanley Qi (Addgene plasmid #44251) and were ordered as gBlocks gene fragments from Integrated DNA Technologies or amplified by overlap extension PCR to replace the protospacer region. All sgRNA sequences are listed in Supplemental. All dCas9 constructs were inserted into the psbA1 neutral site with a spectinomycin resistance cassette. In PL-dCas9 constructs, a TetR cassette (BioBrick part BBa_J23101 and BBa_P0440)22 was added upstream of PL. All sgRNA constructs were inserted at the slr2030-slr2031 homology site with a kanamycin resistance cassette. When needed, Ptrc-eGFP was inserted into the slr0168 neutral site with a chloramphenicol resistance cassette. The flanking homology arms on targeting vectors were 900-1000 bp on each side. Using genome numbering of NCBI NC_000911, the slr0168 neutral site was at 2299815-2302815, the psbA1 site was at 3522675-3525651, and the slr2030-slr2031 homology site was at 780371-782732.

Cyanobacteria transformation and culturing methods Synechocystis sp. PCC 6803 was a gift from Dr. Martin Fulda (Göttingen, Germany). Synechcocystis strains were cultivated in BG-11, nitrate-depleted BG-11 (BG-110) or on agar plates buffered to pH 7.8 with 25 mM HEPES. Cultures were grown in a climatic chamber (Percival Climatics SE-1100) at 28 °C, 50 μE s-1 m-2 illumination, and CO2 at 1% v/v. For the growth of Synechocystis mutant strains, appropriate antibiotics (25 ug/mL kanamycin, 20 ug/mL chloramphenicol and 20 ug/mL spectinomycin) were added in BG-11, BG-110 medium or agar plates. Synechocystis natural transformation was done according to a published protocol.16 Transformant colonies appeared after approximately 1 week and were screened for the correct genotype and segregation using PCR.


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Measurement of GFP in plate and flow cytometer Colonies were scraped from agar plates and inoculated into 10 mL of BG-11. After incubation under photoautotrophic conditions for 2 days, 100 uL aliquots were taken and absorbance (Abs730 nm) and fluorescence (excitation 488 nm, emission 525 nm) were measured in a SpectraMax M5 (Molecular Devices). Alternatively, flow cytometry was used (ex 488 nm, em 525 nm; CyAN analyzer, Beckman Coultier). For the strains with PL inducible promoters, anhydrotetracycline was added to either 100 or 1000 ng/mL and the cells were incubated for 12-24 hours before measurement of cell density and fluorescence. For reversibility of repression, cells were washed to remove aTc and re-suspended in fresh BG-11 (final OD730=0.05). Fluorescence and cell density was recorded using a fluorimeter.

Measurement of PHB by Nile Red staining Cell culture grown in BG-11 to mid-log phase (10 mL at Abs730=1.0) was pelleted, washed in BG110, and resuspended in BG110. After 3 days of incubation under photoautotrophic conditions, cells were assayed for PHB using Nile-Red staining and flow cytometry (excitation 488 nm, detection 575 nm), following the protocol of Tyo et al.37

Measurement of glycogen Glycogen was quantified from biological triplicates. Cell pellets from 1 mL cultures at Abs730=1 were washed in H2O and stored in –20 °C until further processing. Glycogen was isolated from the pellets as previously described.26 Glycogen pellets were re-suspended in 200 μL H2O and mixed with 10 μL of 54% H2SO4 prior to incubation at 100 °C for 1 h. The samples were neutralized with NaOH and glycogen was quantified according to the protocol of Schlebusch et al.38 Glycogen from rabbit liver (Sigma-Aldrich) was used as standard.

Quantification of mRNA



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Total RNA was isolated from 5 mL of Synechocystis culture (Abs730= 1.0) using the GeneJet RNA purification kit (Thermo Fisher). Manufacture’s instructions were followed except that lysozyme was added to the TE buffer to 40 mg/L instead of 0.4 mg/L, as the latter gave very little cell-wall degradation and low RNA yield. DNA was removed using the Turbo DNA-free kit (Thermo Fisher). RT-qPCR was performed on 50-100 ng of total RNA with the Power SYBRGreen RNA-to-Ct kit (Thermo Fisher) and a BioRad CFX light cycler.

Western blotting of dCas9 Cell culture (15 mL at Abs730=1) was centrifuged and resuspended in CelLytic B Cell Lysis Reagent (Sigma-Aldrich) supplemented with 1 mM PMSF. Acid-washed glass beads, 425-600 μm (Sigma-Aldrich), were added and the cells were lysed by vortexing for 20 min at 4 °C, followed by centrifugation for 4 min at 13,000 x g, 4 °C. Supernatant containing 25 μg protein, determined with the bicinchoninic acid assay, was used for the Western blotting kit (Western Breeze, Life Technologies). AntiCas9 polyclonal antibody (Epigentek/BioCat) was necessary to detect dCas9, as attempts to detect the N-terminal c-myc tag using an Anti-c-myc polyclonal failed. Anti-rabbit secondary antibody (alkaline phosphatase-linked) was from Life Technologies.

Acknowledgements This work was partially supported by a SciLifeLab Fellowship to EPH (2014), the Swedish Research Council Formas (213-2011-1655) and the Swedish Foundation for Strategic Research (RBP14-0013).

Supporting Information A file containing the sequences of promoters, sgRNAs, and primers used in this study, the genotypes of all strains used in this study, and a list of potential off-target binding sites for each sgRNA.

References


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Tables Promoter Ptrc PpsbA2 PL03 PL31 PL22 Prnpb

Induced (1 ug/mL aTc) 46 10a 19.2 3.1 0.38 1

Repressed 44 10a 0.22 0.17 0.02 1

Table 1. Relative strengths of constitutive (Ptrc, PpsbA2) and TetR-repressible (PL) promoters used in this study. Values are taken from Huang et al.22 and were based on eYFP expression, except a) estimated in this study based on dCas9 expression.

Figure legends

Figure 1. CRISPRi-mediated repression of GFP in Synechocystis. Top: Placement of sgRNA protospacers on the target GFP gene. Bottom: Fluorescence from slr0168::Ptrc-GFP strains with or without slr2030::Ptrc-sgRNA and psbA1::PpsbA2dCas9. Only sgRNA targeting NT1 and NT2 efficiently repressed GFP expression. The wild type Synechocystis has weak autofluorescence on the GFP channel. Error bars are standard deviation from 2 biological replicates.

Figure 2. Inducible and reversible repression of GFP by CRISPRi. A) Strong repression of GFP by dCas9 and sgRNA-NT1 is observed when dCas9 is under PpsbA2 and TetR-PL03 promoters, even in the absence of the aTc inducer. Inducible expression was obtained with the tightly repressed promoter TetR-PL22. B) Time course for repression of GFP after aTc addition at day 0 (arrow) to TetR-PL22dCas9/PL22-sgRNA strain. After 4 days, aTc was removed (arrow) and GFP fluorescence recovered. Cell generation time is approximately 16 hours. C) Western blot with a Cas9/dCas9 antibody shows leakiness of dCas9 (150 kDa) from the TetR-


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PL03 promoter in the absence of aTc inducer and tight repression of dCas9 from the TetR-PL22 promoter.

Figure 3. Repression of carbon storage during nitrogen starvation. A) CRISPRi knockdown of PHA synthase subunit phaE (slr1829) using constitutive promoter as measured by RT-qPCR. B) Nile-Red staining of PHB in Synechcoystis wild-type and the phaE knockdown strains after 2 days of nitrogen starvation as measured by flow cytometry. C) Inducible knockdown of slr1176 (glgC) as measured by RT-qPCR 24 hours-post induction. D) Wild type and glgC knockdown strains after 2 days of nitrogen starvation. A non-chlorosis phenotype is typical of glycogen-deficient strains. Glycogen quantification showed 80% less glycogen when glgC was knocked down.

Figure 4. Multiplex repression of aldehyde reductases/dehydrogenases. dCas9 was expressed constitutively from PpsbA2-dCas9. The various PL31-sgRNA were inserted in the slr0230-slr0231 neutral site and expressed constitutively (no TetR cassette). Target mRNA was quantified using RT-qPCR. A) mRNA levels for the four genes in wildtype (WT) and from individual knockdown strains (1-sgRNA mutants). More than 10-fold repression was observed except for slr0091, which is the 3rd gene of an operon. B) mRNA levels for the four genes in a 3-sgRNA mutant, where slr0942, sll0990, and slr1192 were targeted. In the 4-sgRNA mutant, slr0942, sll0990, slr1192, and slr0091 were targeted. Repression levels were similar to those from 1-sgRNA mutants. Error bars are standard deviations from biological replicates.



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Figure 1. CRISPRi-mediated repression of GFP in Synechocystis. Top: Placement of sgRNA protospacers on the target GFP gene. Bottom: Fluorescence from slr0168::Ptrc-GFP strains with or without slr2030::PtrcsgRNA and psbA1::PpsbA2-dCas9. Only sgRNA targeting NT1 and NT2 efficiently repressed GFP expression. The wild type Synechocystis has weak autofluorescence on the GFP channel. Error bars are standard deviation from 2 biological replicates. 69x80mm (300 x 300 DPI)


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Figure 2. Inducible and reversible repression of GFP by CRISPRi. A) Strong repression of GFP by dCas9 and sgRNA-NT1 is observed when dCas9 is under PpsbA2 and TetR-PL03 promoters, even in the absence of the aTc inducer. Inducible expression was obtained with the tightly repressed promoter TetR-PL22. B) Time course for repression of GFP after aTc addition at day 0 (arrow) to TetR-PL22-dCas9/PL22-sgRNA strain. After 4 days, aTc was removed (arrow) and GFP fluorescence recovered. Cell generation time is approximately 16 hours. C) Western blot with a Cas9/dCas9 antibody shows leakiness of dCas9 (150 kDa) from the TetR-PL03 promoter in the absence of aTc inducer and tight repression of dCas9 from the TetRPL22 promoter. 155x82mm (300 x 300 DPI)



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Figure 3. Repression of carbon storage during nitrogen starvation. A) CRISPRi knockdown of PHA synthase subunit phaE (slr1829) using constitutive promoter as measured by RT-qPCR. B) Nile-Red staining of PHB in Synechcoystis wild-type and the phaE knockdown strains after 2 days of nitrogen starvation as measured by flow cytometry. C) Inducible knockdown of slr1176 (glgC) as measured by RT-qPCR 24 hours-post induction. D) Wild type and glgC knockdown strains after 2 days of nitrogen starvation. A non-chlorosis phenotype is typical of glycogen-deficient strains. Glycogen quantification showed 80% less glycogen when glgC was knocked down. 114x136mm (300 x 300 DPI)


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Figure 4. Multiplex repression of aldehyde reductases/dehydrogenases. dCas9 was expressed constitutively from PpsbA2-dCas9. The various PL31-sgRNA were inserted in the slr0230-slr0231 neutral site and expressed constitutively (no TetR cassette). Target mRNA was quantified using RT-qPCR. A) mRNA levels for the four genes in wildtype (WT) and from individual knockdown strains (1-sgRNA mutants). More than 10fold repression was observed except for slr0091, which is the 3rd gene of an operon. B) mRNA levels for the four genes in a 3-sgRNA mutant, where slr0942, sll0990, and slr1192 were targeted. In the 4-sgRNA mutant, slr0942, sll0990, slr1192, and slr0091 were targeted. Repression levels were similar to those from 1-sgRNA mutants. Error bars are standard deviations from biological replicates. 161x67mm (300 x 300 DPI)


Multiple Gene Repression in Cyanobacteria Using CRISPRi - ACS

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