As a new interdisciplinary subject, microbial synthetic biology aims to transform existing microbial cells or create new microbial cells to provide them with specific physiological functions or the ability to produce target products. To achieve this process, efficient, rapid and accurate gene manipulation tools are important. Since its creation, CRISPR/Cas9 system has been widely used in synthetic biology in recent years due to its ability to accurately identify and cut specific DNA sequences, as well as its characteristics of simple operation, low cost, high efficiency and diverse functions. In this paper, various gene editing techniques derived from CRISPR/Cas9 and their specific applications in microbial synthetic biology are reviewed. At the same time, the commonly used industrial model microorganisms and the corresponding transformation methods of chassis cells are introduced. A variety of industrial microbial chassis cells exist in nature and have their own metabolic characteristics to produce suitable products. However, natural chassis cells also have problems such as intermediate metabolite toxicity, end-product feedback inhibition, and branch pathway, which lead to low target yield, or the host itself does not have the ability to produce the product. Therefore, it is necessary to modify chassis cells by gene editing method, so as to obtain more excellent strains. The advantages of the edited cells compared to the control in the synthesis of target products were listed, and the characteristics of each gene editing technology were summarized in order to select the appropriate gene editing methods for the researchers in the transformation of microbial chassis cells. Finally, the existing problems of CRISPR gene editing technology, such as PAM dependence, off-target effect and universality of application, are put forward.