In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technology has become the most heated genome editing technique. Comparing to earlier genetic engineering methods, the CRISPR/Cas system is more advantageous due to its simple convenient design, high efficiency, cost-effectiveness, and the ability to perform multi-sites editing simultaneously. As the most effective gene editing tool, it utilizes a simple short RNA-guided mechanism to direct Cas-mediated DNA cleavage at the target genome locus and exploits the endogenous DNA repair pathways to achieve site-specific gene modifications. Initially discovered as a part of the bacterial adaptive immune system, the CRISPR/Cas system has now been widely used to study a broad range of biological genomes. Besides its contribution to our understanding of the basic genetic science, the application of the CRISPR/Cas system also expands rapidly into the medical fields, showing great potentials in the research of genetic diseases, viral infectious diseases, and cancers. In this review, the latest research progress of CRISPR/Cas technology is summarized based on its development, mechanism, and application in HIV/AIDS intervention. This review also examines the existing weaknesses and the future prospects of this promising technology.