CRISPR/Cas9, including the derived CRISPRi, has evolved quickly into a popular technology to edit the genomes of prokaryotes and eukaryotes. For constructing microbial cell factories (MCF), it is especially important to regulate metabolic flux toward the target products. To achieve maximal flux channelling toward target products, the regulation of expression of multiple genes is desirable, which is labor intensive and time consuming in most cases. CRISPR/Cas9, especially CRISPRi, allows the design of multiple single strange-guided RNA (sg RNA). These bind to the target genes with different repression intensities with the help of dCas9, and thus regulate metabolic flux. The technology provides a convenient tool to enhance production of various chemicals, regulating cell morphology and changing growth pattern. It is a technology that will revolutionize the metabolic engineering of microbial cells.