Cell factories are applicable for production of a wide range of products from renewable sources and waste materials as an energy source. Optimization of cell factories operation conditions is further possible with metabolic engineering. One of the methods to achieve this is the CRISPR-Cas9 technology, which effectively introduces the desired changes to the genome of the target organism. Typically, deletions, insertions and other manipulations of target genes are performed with the CRISPR-Cas9 with the aim to increase metabolic flux through the target metabolic pathways. Using this approach, large quantities of the target product is synthesised through an environmentally benign process, via efficient cell factories. The advantages of CRISPR-Cas9 are high precision of genetic changes at targeted loci, simplicity, speed and economic efficiency of the process. In this thesis I will describe the state-of-the-art applications of CRISPR-Cas9 technology in metabolic engineering. I will also address production of efficient cell lines with potential applications in various fields such as food, cosmetic and chemical industry, production of biofuels and pharmaceuticals. Metabolic engineering is a key factor for the further development and manufacturing of desired products with cell factories.