Dissecting oncogenic signaling networks through combinatorial and single-cell CRISPR screens

Abstract

Understanding how genes act together to control cellular behavior is central to elucidating disease mechanisms. This thesis presents two CRISPR-based platforms to study genetic interactions and transcriptional regulation in human cells. First, we evaluate the RNA-targeting CRISPR effector Cas13d for quantitative genetic interaction mapping. Cas13d enables reversible transcript-level gene silencing and supports efficient dual-gene perturbations. Using a dual-promoter gRNA expression strategy, we mapped interactions among six genes modulating response of the CML cell line K562 to imatinib. Second, we use single-cell CRISPR sequencing to reconstruct the transcriptional network downstream of RAF-MAPK signaling. Perturbation of 22 transcription factors in an inducible RAF1 HEK293 model reveals a feedback loop between EGR1 and TCF7, linking MAPK and Wnt signaling. Together, these approaches enable scalable, high-resolution analysis of genetic dependencies relevant to cancer therapy.