Advances in CRISPR (clustered regularly interspaced short palindromic repeats) technology have transformed the field of genome engineering, offering new opportunities for targeted gene editing and regulation. Simplicity of use, rapid design, high efficiency, and great variety of applications have led to widespread implementation of CRISPR for cell biology studies, human disease research, and diverse biotechnological applications. In this book chapter, we describe the development of novel CRISPR-based technologies for mammalian cell engineering to facilitate efficient knockout, knock-in, activation, and repression of genes as well as epigenetic modifications. We also discuss CRISPR screens and novel RNA-guided endonucleases, which have expanded the CRISPR toolbox. A specific attention will be devoted to recent applications of CRISPR technologies in the genome engineering of mammalian cell lines, particularly Chinese hamster ovary (CHO) cells, toward improved production of biopharmaceuticals.