CRISPR/Cas9 advances engineering of microbial cell factories

• Published in: Metabolic engineering 2016-03, Vol.34, p.44-59
• Main Authors: Jakočiūnas, Tadas, Jensen, Michael K., Keasling, Jay D.
• Format: Article
• Language: English
• Subjects: Bacteria; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR-Associated Proteins - genetics; CRISPR-Cas Systems - genetics; CRISPR/Cas9; Genetic Enhancement - methods; Genome editing; Genome, Microbial - genetics; Metabolic engineering; Metabolic Engineering - methods; Metabolic Networks and Pathways - genetics; Recombineering; Yeast
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2015.12.003

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. •Cas9 and gRNA expression was adopted for genome engineering of yeast and bacteria.CRISPR/Cas9 offers efficient marker-free integration of donor DNA in yeast.CRISPR/Cas9 and recombineering improves genome engineering efficiency in E. coliNuclease-deficient Cas9 was used for redirecting flux through biosynthetic pathways.•Cas9 and gRNA expression strategies have been adopted for genome engineering of yeast and bacteria.•CRISPR/Cas9 offers efficient integration of donor DNA without the use of markers in yeast.•CRISPR/Cas9 combined with recombineering improves genome engineering efficiency in E. coli.•Nuclease-deficient Cas9 can be used for redirecting flux through biosynthetic pathways.