Metabolically engineered bacterial strains are rapidly emerging as a pivotal focus in the biosynthesis of an array of bio-based ingredients. Presently, CRISPR (clustered regularly interspaced short palindromic repeats) and its associated RNA-guided endonuclease (Cas9) are regarded as the most promising tool, having ushered in a transformative advancement in genome editing. Because of CRISPR-Cas9's accuracy and adaptability, metabolic engineers are now able to create novel regulatory systems, optimize pathways more effectively, and make extensive genome-scale alterations. Nevertheless, there are still obstacles to overcome in the application of CRISPR-Cas9 in novel microorganisms, particularly those industrial microorganism hosts that are resistant to traditional genetic manipulation techniques. How to further extend CRISPR-Cas9 to these microorganisms is an urgent problem to be solved. This article first introduces the mechanism and application of CRISPR-Cas9, and then discusses how to optimize CRISPR-Cas9 as a genome editing tool, including how to reduce off-target effects and how to improve targeting efficiency by optimizing design. Through offering a comprehensive perspective on the revolutionary effects of CRISPR-Cas9 in microbial cell factories, we hope to stimulate additional research and development in this exciting area.