Abstract: The CRISPR/Cas9 technology has developed rapidly in recent years with fast, simple and accurate editing functions to allow gene knockout, knock in, activation and interference. It has become a powerful genetic screening tool and been widely used in various models including cell lines, mice and zebrafish. The application of CRISPR system in constructing genome library for high-throughput screening is the main strategy for target gene research of diseases, especially neoplasms. Here we summarize the rationales and recent development of CRISPR/Cas9 library screening technology, the strategies for improving the off-target effects, the basic workflow of library screening and the application of this technology in tumor research.