Extended Data Figure 6: Tiled pooled in situ CRISPR-Cas9 Bcl11a enhancer screen. | Nature

Extended Data Figure 6: Tiled pooled in situ CRISPR-Cas9 Bcl11a enhancer screen.

From: BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

Extended Data Figure 6

a, Schematic of the mouse Bcl11a locus (mm9, transcription from left to right) with erythroid chromatin marks (top, dark blue H3K27ac from ref. 55; middle, light blue H3K27ac from ref. 56; and bottom, black DNase I from ref. 28) and regions of primary sequence homology to the human DHSs displayed. y axes for H3K27ac tracks are both scaled to maximum 3.5 reads per million. Composite enhancer as previously defined28. b, Ranked enhancers in mouse erythroid precursors by H3K27ac signal intensity55,56, with super-enhancers shaded. Super-enhancer associated genes are indicated by Venn diagram. c, Strategy to knock-in by homology-directed repair the fluorescent protein mCherry into the mouse embryonic globin Hbb-y locus (encoding the εy embryonic globin chain). d, Distribution of NGG and NAG PAM sgRNAs mapped to genomic cleavage position with vertical lines representing cleavage sites for sgRNAs mapped to plus and minus strands. e, Distance to adjacent genomic cleavage position for NGG (left) and NAG (right) PAM sgRNAs. f, Representation of the 1,271 NGG and NAG sgRNAs within the plasmid pool by deep sequencing. The median was 735 normalized reads and the 10th and 90th percentiles (indicated by the vertical dotted lines) ranged from 393 to 1,240 normalized reads. g, Library composition by target sequence and PAM restriction. h, mCherry expression upon exposure to Cas9 and an individual NGG sgRNA targeting Bcl11a exon 2 in MEL εy:mCherry reporter cells. i, εy:mCherry sort of library transduced cells. j, Control sgRNA enrichment. Boxes demonstrate 25th, median and 75th percentiles and whiskers minimum and maximum values. ****P < 0.0001. k, Enrichment scores of NGG sgRNAs between four biological replicates.

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