The CRISPR-Cas9 system is a powerful tool for genome engineering, and its programmability and simplicity have enabled various types of gene manipulation such as gene disruption and transcriptional and epigenetic perturbation. Particularly, CRISPR-based pooled libraries facilitate high-throughput screening for functional regulatory elements in the human genome. In this review, we describe recent advances in CRISPR-Cas9 technology and its use in high-throughput genetic screening. We also discuss its potential for drug target discovery and current challenges of this technique in biomedical research.