137 Novel Genome-wide CRISPR Screen for Glioblastoma Invasion

Abstract

Abstract INTRODUCTION Among high-grade pediatric brain tumors, glioblastoma multiforme (GBM) is particularly difficult to treat. Invasion of GBM cells into normal brain parenchyma renders malignant cells inaccessible to surgical intervention and poised to drive tumor recurrence. In order to better understand the molecular mechanisms and signaling pathways underlying pediatric GBM cell invasion we performed a genome-wide CRISPR/Cas9 loss of function screen for genes that inhibit invasion. METHODS CRISPR/Cas9 is an emerging technology for genome editing that can be used to knock out genes that are targeted by short RNA guide sequences (sgRNAs). First, we made an immortalized human glioma line that stably expresses the RNA-guided DNA endonuclease, Cas9, and showed that Cas9 is functional in these cells. We then used a pooled approach to knock-out individual genes on a genome-wide scale in the Cas9 GBM cell line. Pooled cells were subjected to a Boyden chamber invasion assay which separated the cell populations based on invasive phenotype. To determine which genes were enriched in the invasive cell fraction compared to the noninvasive cell population we used next generation sequencing of the sgRNAs isolated and amplified from each group. RESULTS >Analysis of sgRNA enrichment in invasive compared to noninvasive cell populations identified specific genes expected to be altered in migration, such as those involved with actin based cell projections, as well as novel candidate genes. We are currently testing the necessity and sufficiency of these genes for glioma invasion in vitro and in vivo using a xenograft mouse model. CONCLUSION In this study, we identified critical genes necessary for GBM invasion using a novel genome-wide knock-out approach. Once validated, the genes of interest may serve as prognostic surveillance markers or targets for therapeutic intervention in pediatric glioblastoma.