title: Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi).
abstract: Corynebacterium glutamicum 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 C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk 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 C. glutamicum 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.
authors.affiliation: Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.|MIT Synthetic Biology Center , 500 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Sara
authors.initials: S
authors.lastname: Cleto
authors.affiliation: Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.|MIT Synthetic Biology Center , 500 Technology Square, Cambridge, Massachusetts 02139, United States.|Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University , 33615 Bielefeld, Germany.
authors.forename: Jaide Vk
authors.initials: JV
authors.lastname: Jensen
authors.affiliation: Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University , 33615 Bielefeld, Germany.
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
authors.affiliation: Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.|MIT Synthetic Biology Center , 500 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Lu
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D048230:Corynebacterium glutamicum / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D008239:Lysine / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D012313:RNA / Q000235:genetics
chemical_list: D018698:Glutamic Acid; D012313:RNA; D008239:Lysine
keywords: C. glutamicum; CRISPRi; amino acid; metabolic engineering; sgRNA/dCas9
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