Development of a CRISPR activation system for targeted gene upregulation in Synechocystis sp. PCC 6803

Abstract

The photosynthetic cyanobacterium Synechocystis sp. PCC 6803 offers a promising sustainable solution for simultaneous CO₂ fixation and compound bioproduction. While various heterologous products have now been synthesised in Synechocystis, limited genetic tools hinder further strain engineering for efficient production. Here, we present a versatile CRISPR activation (CRISPRa) system for Synechocystis, enabling robust multiplexed activation of both heterologous and endogenous targets. Following tool characterisation, we applied CRISPRa to explore targets influencing biofuel production, specifically isobutanol (IB) and 3-methyl-1-butanol (3M1B), demonstrating a proof-of-concept approach to identify key reactions constraining compound biosynthesis. Notably, individual upregulation of target genes, such as pyk1, resulted in up to 4-fold increase in IB/3M1B formation while synergetic effects from multiplexed targeting further enhanced compound production, highlighting the value of this tool for rapid metabolic mapping. Interestingly, activation efficacy did not consistently predict increases in compound formation, suggesting complex regulatory interactions influencing bioproduction. This work establishes a CRISPRa system for targeted upregulation in cyanobacteria, providing an adaptable platform for high-throughput screening, metabolic pathway optimisation and functional genomics. Our CRISPRa system provides a crucial advance in the genetic toolbox available for Synechocystis and will facilitate innovative applications in both fundamental research and metabolic engineering in cyanobacteria.

Fig. 1. The developed CRISPRa system consists of a protein fusion between Francisella novicida dCas12a and SoxS^R93A.

Through gRNA-guided dCas12a binding at a specific site upstream of the target promoter, RNA polymerase is recruited by SoxS to initiate transcription at the candidate promoter. GOI: gene of interest.

Fig. 2. GFP fluorescence at 72 hours post-induction with 3 mM rhamnose with eight gRNAs targeting P_trc with dCas12a-SoxS.

a Seven gRNAs were designed to target a 328-bp region upstream P_trc driving GFP expression, with an additional gRNA targeting the coding sequence. b GFP fluorescence was measured in the absence or presence of the rhamnose inducer across 8 candidate gRNAs. c Fold-activation of GFP was determined relative to the negative control (EV) for each candidate gRNA. GFP green fluorescent protein, NTS non-template strand, EV negative control—sBB_CA1 expressing plasmid pBB_CA, which contains dCas12a-SoxS and a CRISPR array lacking a targeting gRNA, CDS coding sequence. Error bars indicate standard deviation (− rhamnose samples: EV: n = 6; −48, −108, −156, CDS: n = 10; −97 NTS, −144 NTS, −251, −328, −108/−156: n = 3; + rhamnose samples: EV: n = 8; −97 NTS: n = 5; other gRNAs: n = 10). p value representation: ns > 0.05; * < 0.05; ** < 0.01; *** < 0.001. p value was calculated by comparing each sample to the negative control unless otherwise stated.

Fig. 3. Correlation between promoter strength and activation levels with the dCas12a-SoxS CRISPRa system.

a Four synthetic promoters of the Biobrick J23 series were selected and tested for activation with dCas12a-SoxS using a common gRNA. b Fluorescence (grey) and activation (red) levels were measured following rhamnose induction of the dCas12a-SoxS system for the four J23 promoters. EV negative control—respective background strains expressing plasmid pBB_CA. Error bars indicate standard deviation (n = 5). p value representation: ns > 0.05; * < 0.05; ** < 0.01; *** < 0.001.

Fig. 4. Relative isobutanol (IB) and 3-methyl-1-butanol (3M1B) titres on days 4 and 8 in response to CRISPRa-mediated kivD^S286T upregulation.

Single, dual, and triple CRISPRa targeting using different gRNA combinations were assessed in three background strains: a ddh_kivD (harbouring one copy of kivD^S286T), b HX11 (two copies of kivD^S286T), and c HX51 (three copies of kivD^S286T), as shown in the corresponding construct schematics. The gRNA(s) expressed in each resulting strain are represented on the×axis. For dual targeting, both gRNAs are named. Triple targeting of ddh, NS1, and sll1564 loci in HX51 is referred to as “Triple”. Relative titres were calculated in comparison to the respective negative control (EV) for each background strain. EV negative control—respective background strain expressing plasmid pBB_CA, UP upstream homology arm, DN downstream homology arm. Error bars indicate standard deviation (n = 3). p value representation: * < 0.05; ** < 0.01.

Fig. 5. Relative kivD^S286T transcript levels at day 4 in CRISPR-activated strains.

Gene expression was quantified with different primer sets designed to bind within the coding sequence (Total), the Flag tag (Flag) or the His tag (His) to differentiate which kivD^S286T copy was activated when applicable. EV negative control—respective background strain expressing plasmid pBB_CA. Error bars indicate standard deviation (n = 3). p value representation: * < 0.05; ** < 0.01; *** < 0.001.

Fig. 6. Simplified biosynthetic pathway for isobutanol and 3-methyl-1-butanol formation in Synechocystis.

The key heterologous enzyme KivD (red) catalyses the conversion of 2-ketoisovalerate and 2-ketoisocaproate into isobutylaldehyde and 3-methylbutyraldehyde, respectively, which are subsequently converted into isobutanol and 3-methyl-1-butanol by Synechocystis alcohol dehydrogenase (Adh). Genes (blue) targeted by the CRISPRa system were selected to enhance pyruvate formation and NADPH regeneration, hypothesised as key bottlenecks for IB/3M1B biosynthesis. Adh alcohol dehydrogenase, CBB cycle Calvin-Benson-Bassham cycle, DHAP dihydroxyacetone phosphate, FNR ferredoxin-NADP⁺ oxidoreductase, G3P glyceraldehyde-3-phosphate, KivD α-ketoisovalerate decarboxylase, ME malic enzyme, PEP phosphoenolpyruvate, PK pyruvate kinase, PntAB pyridine nucleotide transhydrogenase, PSI photosystem I, TCA cycle tricarboxylic acid cycle.

Fig. 7. Isobutanol and 3-methyl-1-butanol production in response to CRISPRa targeting of select genes involved in pyruvate formation and NADPH regeneration.

a Isobutanol titres on days 2, 3, and 4. b 3-methyl-1-butanol titres on days 2, 3, and 4. c Fold-increase of isobutanol and 3-methyl-1-butanol relative to the negative control (EV) on day 2. d IB/3M1B ratio for pyk1 activation. gene.1 and gene.2 refers to gRNAs #1 and #2, respectively, when applicable. EV negative control—HX11 expressing plasmid pBB_CA, IB isobutanol, 3M1B 3-methyl-1-butanol. Error bars indicate standard deviation (n = 3). p value representation: * < 0.05; ** < 0.01; *** < 0.001.

Fig. 8. IB/3M1B biosynthesis in response to CRISPRa multiplexing for simultaneous down- and upregulation of several target genes.

a Targeting of pyk1 or pyk2 with two gRNAs. pyk2 strains are represented with bars with a dashed outline. b Simultaneous activation of pyk1 and pyk2 or pyk2 and me. Fold-improvement are shown as the mean fold-change in IB and 3M1B production. c Downregulation of slr6040 and acnSP using single or multiplexed gRNAs. d Fold-change of IB and 3M1B biosynthesis in multiplexed strains compared to single gRNA-targeted strains. EV negative control—HX11 expressing plasmid pBB_CA, IB isobutanol, 3M1B 3-methyl-1-butanol. Error bars represent standard deviation (single activation: n = 2; single repression and multiplexed targeting: n = 3). p value representation: n.s. > 0.05; * < 0.05; ** < 0.01; *** < 0.001.