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Fig. 1 | Genome Biology

Fig. 1

From: Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

Fig. 1

Identification of X-chromosomal MAPK regulators through a pooled CRISPR knockout screen. a Schematic depiction of the screen workflow: A female mESC line carrying a stably integrated fluorescent MAPK reporter, where expression of GFP is controlled by an SRE-Elk responsive promoter, was transduced with a construct expressing the Cas9 endonuclease. Cells were further transduced with a custom sgRNA library targeting the majority of X-chromosomal genes. GFP-high cells were sorted by flow cytometry, cultured for an additional 2 days and sorted again (double-sorted). The sgRNA cassette was amplified from genomic DNA and sgRNA abundance in the unsorted and double-sorted populations was determined by deep sequencing. The screen was performed in three independent replicates. b Composition of the GeCKOx sgRNA library, targeting X-linked genes and positive control genes known to regulate the MAPK pathway, with 6 sgRNAs per gene. As negative controls, non-targeting sgRNAs were included in the library. c Volcano plot of the screen results, where screen hits (FDR < 0.05, MAGeCK) are labeled in red (positive controls) or blue (X-linked genes)

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