The Conditional Knockout Analogous System: CRISPR-Mediated Knockout Together with Inducible Degron and Transcription-Controlled Expression

Abstract

The revolutionary CRISPR technology opens a new era of cell biology in mammalian cells. The InDel mutation is induced by CRISPR and results in the frameshift mutation of the gene. Owing to the nature of CRISPR induced knockout, the conditional knockout using CRISPR technology is not common. With the recent development of the small molecule-inducible degron system, an analogous system to the classical genetic conditional knockout has become feasible. By integrating CRISPR-knockout, the tetracycline-controlled transcriptional and auxin-induced degradation post-translational control of protein expression, a method imitating the conditional knockout is developed. We herein describe the detailed protocol for the generation of a conditional protein inactivation in human cancer cells. The system is especially useful to study essential gene function in aneuploidy cancer cells where gain in copy number is common.