Authors Keyi Tang (1), Kenneth Baillie (1), Jeffrey Schoenebeck (1) (2), Maciej Parys (1) (2), Nicholas Parkinson (1) (2)
Affiliations 1. Roslin Institute, University of Edinburgh, United Kingdom 2. Royal (Dick) School of Veterinary Studies, University of Edinburgh, United Kingdom
Presentation Type Talk
Abstract
Oral melanoma is one of the most common oral tumours in dogs. It is a highly aggressive tumor with high proliferation rate and frequent metasis to regional lymph nodes and the lungs. Currently there is a limited number of effective treatments for mucosal melanomas in canines, novel treatment approaches are urgently needed. In this study we are using a genome-wide approach to identify new genetic dependencies that could be used as therapeutic targets using CRISPR-based whole genome screening. We have created, first of its kind, canine CRISPR-Cas9 whole genome knockout library which was cloned into a LentiCrispr v2 vector. The library contains 119916 distinct single guide RNAs (sgRNA) targeting 20702 genes, with majority of genes targeted by six guides. Five canine oral melanoma cell lines (CMGD2, CMGD5, CML10, Ivy and Harvey) and Canine Fibroblast as a negative control were used for screening. Melanoma cell lines were grown in both 2-dimention (2D) and 3-dimention (3D) for another comparison as CRISPR phenotypes in 3D may more accurately mimic in-vivo tumor environment. Cells were transfected, selected and expanded for 2 weeks to allow for phenotypic effect to get pronounced. DNA was extracted from the cells after transfection and after 2 weeks expansion. The DNA was subsequently used for sequencing library creation. Sequencing was performed using Illumina sequencing. Data obtained was analysed using MAGECK pipeline. We have identified five genes, RACK1, CDK4, CDK6, CRK, and MEK1, as potential molecular targets for further validation. We selected 2-3 targeted therapy drugs, to target pathways associated with the genes identified.