Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute

Abstract

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR/Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by being a comprehensive pipeline that contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control, normalization, batch effect removal, copy number bias correction, gene hit identification, and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires approximately two hours on a desktop computer running Linux or Mac OS and with R support. The MAGeCKFlute package is available at http://www.bioconductor.org/packages/release/bioc/html/MAGeCKFlute.html.

GO-CRISPR: a highly controlled workflow to discover gene essentiality in loss-of-function screens

“…To fully illustrate the advantages of GO-CRISPR and TRACS, we have analyzed the triplicate replicates of T0 and Tf solely in Cas9-expressing cells using MAGeCK-RRA, MAGeCK-MLE (Wang et al, 2019) and BAGEL (Hart et al, 2015) as this represents a commonly used CRISPR screen workflow that lacks guide only controls (Figure S3). A basic premise for genome-wide CRISPR screens using only Cas9-expressing cells is that poorly represented sgRNAs, or stochastic changes unrelated to the experiment in question, will be removed through statistical cutoffs.…”

Integrative proteomics reveals principles of dynamic phospho-signaling networks in human erythropoiesis

“…Read counts for each sgRNA were normalized against total read counts across all samples. Mageck method were used for differential analysis for sgRNA and Gene ranking (Li et al, 2014c;Wang et al, 2019). A P < 0.05 was considered to be statistically significant.…”

Experimental Approaches to Identify Host Factors Important for Influenza Virus

“…Ideally, multiple replicates of the screen are performed. Several bioinformatic approaches have been developed to robustly identify sgRNAs that are enriched over successive rounds of selection, notably MAGeCK and STARS (Doench et al 2016;Wang et al 2019). The identification of multiple sgRNAs targeting the same gene provides additional support for the specificity of the knockout and importance of the gene during infection.…”