Multiple Gene Repression in Cyanobacteria Using Crispri

Cyanobacteria are promising hosts for production of biofuels and chemicals. However, the deficiency of robust genetic tools has been a barrier to metabolic engineering of cyanobacteria. The CRISPR/Cas system has emerged and been widely used for genome editing, gene expression regulation and imaging of genomic elements. However, CRISPR-based gene editing or regulation had not yet been demonstrated in cyanobacteria. Here, we used the nuclease-deficient Cas9 (dCas9) of Streptrococcus pyogenes for the repression of genes in the model cyanobacterium Synechcocystis sp. PCC 6803. This CRISPR-interference (CRISPRi) repressed green fluorescent protein (GFP) to negligible levels. The respression effect was inducible and reversible when using a synthetic aTc-inducible promoter. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. By expressing multiple sgRNAs, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50−95% repression. Thus, CRISPRi is a robust tool and could greatly accelerate both basic research and metabolic engineering efforts in cyanobacteria. We also anticipate the development of other CRISPR-based synthetic biology tools in cyanobacteria.