<i>Corynebacterium glutamicum</i> Metabolic Engineering with CRISPR Interference (CRISPRi)

<i>Corynebacterium glutamicum</i> is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in <i>C. glutamicum</i>. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of <i>pgi</i> and <i>pck</i> up to 98%, and of <i>pyk</i> up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for <i>C. glutamicum</i> metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection