P13.04.A REPRODUCIBLE CRISPR-SEQ PIPELINE FOR NGS FUNCTIONAL GENOMICS SCREENINGS FOR GLIOMA CELL LINES

Abstract

Abstract BACKGROUND The CRISPR/Cas9 genome editing technology has revolutionized the field of functional genomics by enabling precise editing of DNA. Over the past few years, CRISPR technology has been widely employed to identify genetic dependencies in a broad spectrum of applications paving the way for the discovery and validation of relevant molecular targets, for example potential drug targetsGlioblastoma are incurable primary tumors of the central nervous system. Functional genomics could enable the discovery of novel therapeutic targets or even combination therapies. MATERIAL AND METHODS Here, we present a bioinformatics pipeline that combines functional screens and target editing analysis to identify potential vulnerabilities and drug targets in glioblastoma or other diseases. The pipeline consists of two workflows, one performing functional screening and one performing base editing analysis. This developed CRISPR-seq pipeline is reproducible and containerized, allowing high-throughput processing. All processes are modularized in Nextflow DSL2 (Domain Specific Language) and versioned which facilitates maintainability and customization, as well as the addition of new tools. A quality control report is also automatically generated, allowing the user an overview of their ran experiments. The pipeline also helps us to keep the data FAIR (Findable, Accessible, Interoperable and Reusable) and optimized, making it easier to share with other researchers and collaborate on future studies. RESULTS This pipeline was applied to functional screens using CRISPR/Cas9 genome-wide knockout or activation libraries to detect genetic vulnerabilities that might enhance drug treatment efficacy, e.g. for ataxia telangiectasia and Rad3 related (ATR) inhibition in experimental glioma.With this pipeline, we were able to identify several genes that are involved in drug resistance in glioblastoma, by starting from raw data (fastq files) to genes presenting vulnerabilities.The results obtained through our pipeline provided important information on the disease's molecular mechanisms, which were subsequently confirmed through wet lab validation. CONCLUSION In conclusion, the crisprseq software described in this abstract is a powerful tool for identifying potential genetic dependencies. The pipeline aims at analyzing this output data in a reproducible and containerized way using Nextflow and respecting nf-core community standards. By combining functional screens and base editing analysis with bioinformatics tools, this pipeline allows for the identification of essential genes and pathways, as well as the characterization of specific mutations that contribute to the development and progression of glioblastoma.