Fan Li1,2, Vikrant Singh1, Vivienne Lu1, Jinying Chen1,3, Sandy S.C. Hung4, Joanne L. Dickinson1, Phillippa Taberlay5, Anthony L. Cook6, Alex W. Hewitt1,4,7, Guei-Sheung Liu1,3,4,7
Purpose: Uveal melanoma is a potentially fatal eye cancer. A leading genetic technique “genome-wide CRISPR/Cas9 screen” was applied to systematically interrogate each gene in the genome, to identify those essential for growth and proliferation of uveal melanoma.
Method: To identify essential uveal melanoma gene pathways, clonal human uveal melanoma cell line (OCM-1) and a GeCKO (genome-wide CRISPR knockout) screening strategy were employed. OCM-
1 cells stably expressing Cas9 and genome-wide sgRNA library were generated by lentiviral-based gene transduction and cultured for at least 12 pas- sages. Stable transgene integration in pooled formats facilitates screen readout using Next-Generation Sequencing following the negative selection and Model-based Analysis of Genome-wide CRISPR- Cas9 Knockout (MAGeCK) computational tool was used to identify genes that cause cells to be depleted during selection. Moreover, bioinformatic STRING analysis inputted with the identiﬁed genes was per- formed to further explore the potential pathways that involve in the survival of the uveal melanoma. Results: A total of 81 genes were found by MAGeCK with at least 3 or more targeted sgRNA deletions during selection. Bioinformatics STRING analysis revealed that the genes targeted by these identiﬁed sgRNAs are involved in many biological pathways, including mRNA processing and splicing. Conclusion: Here, we have identiﬁed genes essen- tial for the proliferation and survival of uveal mela- noma via a forward genetic “genome-wide CRISPR/ Cas9 screen”. Our work may provide new insights into the molecular mechanisms of uveal melanoma and reveal new therapeutic target to treat this poten- tially devastating disease.