Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader

CRISPR-Cas9-based genome editors can precisely target and edit genes efficiently. However, prolonged Cas9 activity poses challenges for laboratory experiments and raises safety concerns for therapeutic applications due to unintended consequences such as off-target editing, genotoxicity, immunogenicity, and undesired on-target modifications. Here, we evaluate a novel molecular glue degradation system, called Cas9-degron (Cas9-d), designed to degrade Cas9 in the presence of the US Food and Drug Administration (FDA)-approved drug, pomalidomide (POM). This system is highly biocompatible and rapidly reduces Cas9 protein levels within 4 h of induction, resulting in a 3- to 5-fold decrease in editing at on-target sites. The reduction is reversible, as Cas9 levels are restored within 24 h after POM withdrawal. Without initiating degradation, the on-target editing efficiency and accuracy of the Cas9-d system remain intact in different human cell types, including hepatic cell lines and human induced pluripotent stem cell (hiPSC)-derived GABAergic neurons. Cells edited with the Cas9-d system were healthy and functional, exhibiting minimal toxicity from using the strategy. The Cas9-d system provides a versatile approach to adjust Cas9 levels, demonstrating its potential as an experimental tool for controlling genome editing outcomes in vitro and ex vivo. With further development, it holds promise for enhancing somatic cell genome editing in vivo.