title: CRISPR/Cas9 advances engineering of microbial cell factories.
abstract: One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
authors.forename: Tadas
authors.initials: T
authors.lastname: Jakočiūnas
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: mije@biosustain.dtu.dk.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Jensen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark; Joint BioEnergy Institute, Emeryville, CA, USA; Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA; Department of Chemical and Biomolecular Engineering & Department of Bioengineering University of California, Berkeley, CA, USA.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D064349:Genome, Microbial / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins
keywords: Bacteria; CRISPR/Cas9; Genome editing; Metabolic engineering; Recombineering; Yeast