PhD Seminar: Matthew Guo
PhD Seminar: Matthew Guo
Date: Thursday, September 5th, 2024 at 2:00 PM
Location: Mount Sinai Hospital 60 Murray Street, 3rd floor conference room, L3-201/202/203.
Abstract:
CRISPR Screen of Somatic Cell Reprogramming Reveals Novel Biphasic Regulation of Fate by Cell Cycle Genes.
Understanding the intricate mechanistic details that underlie cellular reprogramming is crucial for the safe and efficient exploitation of iPSCs. In this study, I employed an unbiased whole-genome CRISPR-Cas9 knockout screen to analyze the reprogramming MEFs, revealing previously unknown regulators. Using sgRNAs as distinctive genetic barcodes, I scrutinized temporal dynamics in clonal populations while monitoring guides that selectively drop-in or drop-out from the population. This methodology facilitated the robust identification of sgRNA enrichments across distinct phases of the reprogramming process. Interestingly, a subset of genes associated with cell cycle control, previously implicated as negative regulators of reprogramming, demonstrated bi-phasic effects during the screening process. Through a combination of pharmacokinetic and rescue experiments focusing on Cdkn2a, I began unravelling the mechanistic details of this gene. Evidently, Cdkn2a plays a role in two pluripotency-associated pathways. Its suppression activates CDK4/6-cyclin D complexes and disrupts the equilibrium between TGF-β and BMP signalling pathways. This disturbance subsequently results in gene expression patterns indicative of XEN cells. Furthermore, disruptions in Cdkn2a function impede the oscillation between a pluripotent state and a transient 2-cell-like state, thereby compromising the overall efficiency of the reprogramming process. This newly identified regulation of reprogramming, orchestrated by cell cycle genes, as elucidated through the CRISPR screening, not only provides fresh insights into the intricacies of reprogramming but also significantly contributes to our broader understanding of cellular plasticity and the fundamental processes governing normal embryogenesis.