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

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.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4285-2) contains supplementary material, which is available to authorized users.

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“…Requirements are different for each tool; while STARS and BAGEL require as input file the read count table, MAGeCK and ENCoRE accept raw reads and perform median normalization on read counts of the different samples to adjust for the effect of library sizes and read count distributions. Finally, ENCoRE provides a graphical interface, enabling non-bioinformaticians to perform a complete analysis starting from raw sequencing data (Trumbach et al, 2017). Of these methods, MAGeCK would appear to be the most widely used.…”

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“…To extend the functions, MAGeCK-RRA ( 147 ) was further updated to scMAGeCK ( 148 ) for single-cell CRISPR screening (a novel technique combining pooled CRISPR screening with single-cell RNA-seq, which enables the identification of gRNAs at single-cell resolution from sequencing by modifying the lentiviral vector) and MAGeCKFlute ( 137 ) with optional ranking algorithm (maximum likelihood estimation) ( 149 ), gRNA outlier removal by network essentiality scoring tool ( 150 ), and various accessory functions including upstream quality control and downstream visualization. For some novices without programming expertise, command-line programs are hard to tame and the graphical workflow, ENCoRE ( 141 ), seems more user-friendly, whereas the rough processing of gene ranking may induce unreliable results. Likewise, a universal analyzer, HiTSelect ( 138 ), is designed for both RNAi and CRISPR screens, whereas Poisson distribution used to fit the active gRNA abundance is not applicable because the mean and variance of gRNA count are always not equal.…”

“…Meanwhile, there are still positions for other tools when facing the cases they are adept at. ScreenBEAM ( 139 ) for low-quality data and ENCoRE ( 141 ) for novice users are two representative examples.…”