Genome-wide CRISPR/Cas9 transcriptional activation screen reveals novel prometastatic genes in Ewing sarcoma. A, Schematic overview of the in vivo CRISPR/Cas9 genome-wide transcriptional activation screening experiment (TSS, transcription start site). B, Four Ewing sarcoma cell lines were injected into 48 hpf zebrafish embryos at three different cell densities (n = 12 for each condition) and the number of fish with metastasis to the tail was scored every day for 5 days. C, Read counts per million for each gRNA detected from the genome-wide CRISPR/Cas9 transcriptional activation screening experiment. The eight genes chosen for further study are indicated in bold. D, Eight candidate genes from the screen were stably overexpressed in TC-71 cells and tested in zebrafish xenograft assays (*, P < 0.05; **, P < 0.01; χ2 test). The red line indicates the baseline intravasation of TC-71 cells transfected with EV. The means and SEMs are shown. The dots represent individual experiments and the total number of fish in all the experiments combined is provided above each condition. E, TC-71 cells stably overexpressing eight different genes were tested via a scratch assay. The dots indicate independent experiments. The means and SEMs are shown (*, P < 0.05, Student t test). The red line indicates the average percent wound closure of TC-71 cells transfected with EV. [A, Created in BioRender. Lab, S. (2026) https://BioRender.com/pw6o2c1.]
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