chromosomes as homogeneously staining regions (HSRs), respectively. The ecDNA status of both cell lines was confirmed by DNA fluorescence in situ hybridization (DNA FISH), followed by CRISPR ribonucleoprotein (RNP) electroporation targeting GeneX for permanent gene disruption. Cell growth curve analysis showed that GeneX disruption significantly reduced the long-term proliferation of ecDNA-positive MSTO211H-DM cells but had no significant effect on MSTO211H-HSR cells, supporting a role for GeneX in ecDNA-dependent cancer cell proliferation. To extend these findings through reversible transcriptional repression, we constructed an all-in-one doxycycline-inducible CRISPR interference (CRISPRi) system designed to reduce GeneX expression at the RNA level, in which the sgRNA and dCas9-KRAB expression cassettes were incorporated into a single lentiviral vector. To allow fluorescence-based identification of transduced cells, the blasticidin resistance cassette was replaced with the fluorescent reporter mNeonGreen, enabling subsequent selection by fluorescence-activated cell sorting (FACS). This doxycycline-inducible CRISPRi system provides a platform for future studies of reversible GeneX repression. Together, these studies establish both a functional assessment and a genetic tool for dissecting the role of GeneX in ecDNA biology, evaluating its potential as a therapeutic target in ecDNA-driven cancers.
- Subject
- MSTO
- Type
- course
- Edition
- 2026
- Source
- undergraduateresearch.duke.edu