Development of a CRISPR/Cas9-degron system that enables in vivo specific gene depletion in leukemia models.

Abstract

The CRISPR/Cas9 system has transformed genome editing, yet precise temporal control of Cas9 activity remains challenging. We developed a Cas9-degron platform that couples degron-tagged Cas9 with a dTAG-based chemical degradation strategy. In the presence of dTAG, Cas9 is rapidly and near-completely degraded, preventing editing; upon dTAG withdrawal, Cas9 activity is restored, enabling precise temporal control. Using this system, we achieved selective in vivo gene depletion in acute myeloid leukemia (AML) models and confirmed that SETDB1, a histone H3K9 methyltransferase, is essential for the in vivo growth of both human (MOLM13) and murine (cSAM) AML cells. By maintaining SETDB1 intact prior to transplantation and depleting it afterward, we avoided culture-induced pre-selection bias inherent to sgRNA transduction and validated its critical role in AML progression within the in vivo context. The Cas9-degron retains activity and delivery efficiency comparable to conventional Cas9 in the absence of dTAG. Thus, this versatile system provides a superior alternative to conventional Cas9 and a powerful platform for in vivo CRISPR screening, gene function studies, and potentially temporally controlled gene therapy.