CRISPRi-mediated tunable control of gene expression level with engineered single-guide RNA in Escherichia coli

Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.