Multiple Gene Repression in Cyanobacteria Using CRISPRi

We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium <i>Synechcocystis</i> sp. PCC 6803. The nuclease-deficient Cas9 from the type-II CRISPR/Cas of <i>Streptrococcus pyogenes</i> 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

Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria

Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing <i>Synechocystis</i> cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO₂ is diverted to ethanol or <i>n</i>-butanol. Among the most successful strategies was partial repression of citrate synthase <i>gltA.</i> Strong repression (>90%) of <i>gltA</i> at low culture densities increased carbon partitioning to <i>n</i>-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO₂ fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the <i>gltA</i> gene, we could modulate GltA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes

ICAM-1 regulates macrophage polarization by suppressing MCP-1 expression via miR-124 upregulation

Intercellular adhesion molecule-1 is the adhesion molecule mediating leukocyte firm adhesion to endothelial cells, plays a critical role in subsequent leukocyte transmigration. ICAM-1 is also expressed in other cells including macrophages; however, the role of this adhesion molecule in mediating macrophage functions remains enigmatic. We report that ICAM-1 regulates macrophage polarization by positively modulating miR-124 expression. We found higher expression levels of monocyte chemotactic protein-1 in lungs of mice lacking ICAM-1. Consistent with this result, siRNA mediated depletion of ICAM-1 in macrophage resulted in increased expression levels of MCP-1. Moreover, ICAM-1 controlled miR-124 expression and downregulated MCP-1 mRNA and protein expression by binding of miR-124 to MCP-1 3’ untranslated region. ICAM-1 also induced the transcription factor Sp1 expression, which is important for miR-124 expressing in macrophages. Furthermore, ICAM-1 depletion led to M1 macrophage polarization, in contrast, miR-124 mimics promoted M2 macrophage polarization. Exogenous administration of miR-124 mimics into the lungs prevented lipopolysaccharide-induced myeloperoxidase activity in vivo, suggesting that miR-124 is important for dampening acute lung injury. These results collectively show that adhesion molecule ICAM-1 downregulates MCP-1 expression by controlling Sp1 mediated miR-124 levels, which in turn regulate M2 macrophage polarization. Targeting ICAM-1 and downstream miR-124 may present a new therapeutic strategy for acute lung injury