ENCoRE: an efficient software for CRISPR screens identifies new players in extrinsic apoptosis

Dietrich Trümbach,Hagen Scherb,Susanne Pfeiffer,Wolfgang Wurst,Manuel Poppe,Joel A Schick,Sebastian Doll

doi:10.1186/s12864-017-4285-2

Abstract

BackgroundAs CRISPR/Cas9 mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight and efficient software to easily manipulate large raw next generation sequencing datasets derived from such screens into informative relational context with graphical support. The advantages of the software entitled ENCoRE (Easy NGS-to-Gene CRISPR REsults) include a simple graphical workflow, platform independence, local and fast multithreaded processing, data pre-processing and gene mapping with custom library import.ResultsWe demonstrate the capabilities of ENCoRE to interrogate results from a pooled CRISPR cellular viability screen following Tumor Necrosis Factor-alpha challenge. The results not only identified stereotypical players in extrinsic apoptotic signaling but two as yet uncharacterized members of the extrinsic apoptotic cascade, Smg7 and Ces2a. We further validated and characterized cell lines containing mutations in these genes against a panel of cell death stimuli and involvement in p53 signaling.ConclusionsIn summary, this software enables bench scientists with sensitive data or without access to informatic cores to rapidly interpret results from large scale experiments resulting from pooled CRISPR/Cas9 library screens.

Highlights

As CRISPR/Cas9 mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged
The system consists of a Single guide RNA (sgRNA) that directs the paradigm Cas9 nuclease from Streptococcus pyogenes to bind and cleave genomic DNA strands at the location corresponding to the guide sequence [10]
We demonstrate the power of Easy next generation sequencing (NGS)-to-Gene CRISPR REsults (ENCoRE) to rapidly deliver results in a cell survival screen using Tumor Necrosis Factor-alpha (TNFa) challenge, which identified known members as well as two genes not yet implicated in the extrinsic apoptosis pathway, Suppressor with morphological defects in genitalia 7 (Smg7) and Carboxylesterase 2a (Ces2a)

Summary

Introduction

As CRISPR/Cas mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight and efficient software to manipulate large raw generation sequencing datasets derived from such screens into informative relational context with graphical support. CRISPR/Cas technology has enabled rapid genetic mutation in mammalian cells and the ability to conduct genome-wide screens using tailored lentiviral libraries [1,2,3,4,5,6,7,8,9]. The resulting double strand cleavage triggers cellular repair mechanisms including nonhomologous end-joining (NHEJ), which in the imperfect sense generates insertions or deletions (indel) mutations and associated nonsense transcripts. This technology makes pooled sgRNA libraries a powerful tool to.

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