Yeast Saccharomyces cerevisiae (S. cerevisiae) is a crucial industrial platform for producing a wide range of chemicals, fuels, pharmaceuticals, and nutraceutical ingredients. It is also commonly used as a model organism for fundamental research. In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) system has become the preferred technology for genetic manipulation in S. cerevisiae owing to its high efficiency, precision, and user-friendliness. This system, along with its extensive toolbox, has significantly accelerated the construction of pathways, enzyme optimization, and metabolic engineering in S. cerevisiae. Furthermore, it has allowed researchers to accelerate phenotypic evolution and gain deeper insights into fundamental biological questions, such as genotype-phenotype relationships. In this review, we summarize the latest advancements in the CRISPR-Cas toolbox for S. cerevisiae and highlight its applications in yeast cell factory construction and optimization, enzyme and phenotypic evolution, genome-scale functional interrogation, gene drives, and the advancement of biotechnologies. Finally, we discuss the challenges and potential for further optimization and applications of the CRISPR-Cas system in S. cerevisiae.