CRISPR-Based Screening in Three-Dimensional Organoid Cultures to Identify TGF-β Pathway Regulators.

Abstract

The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-β signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-β pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease.