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BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan GC, Zhang F, Orkin SH, Bauer DE - Nature (2015)

Bottom Line: Despite conserved function of the composite enhancers, their architecture diverges.The crucial human sequences appear to be primate-specific.The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

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Tiled pooled in situ CRISPR-Cas9 Bcl11a enhancer screena, Schematic of the mouse Bcl11a locus (mm9, transcription from left to right) with erythroid chromatin marks (top, dark blue H3K27ac from Kowalczyk et al55, middle, light blue H3K27ac from Dogan et al56, and bottom, black DNase I from Bauer et al28) 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 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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Figure 6: Tiled pooled in situ CRISPR-Cas9 Bcl11a enhancer screena, Schematic of the mouse Bcl11a locus (mm9, transcription from left to right) with erythroid chromatin marks (top, dark blue H3K27ac from Kowalczyk et al55, middle, light blue H3K27ac from Dogan et al56, and bottom, black DNase I from Bauer et al28) 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 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.

Mentions: To test functional conservation of the BCL11A enhancer, we examined the orthologous mouse Bcl11a enhancer in greater detail. Erythroid DNase I sensitivity is only observed at those sequences homologous to h+55 and h+62 and not h+58 (Extended Data Fig. 6a), consistent with the reduced sequence homology within the h+58 Active region (Fig. 3a-c). We performed a pooled CRISPR enhancer saturating mutagenesis screen in MEL εy:mCherry reporter cells, similar to the human screen described above (Extended Data Fig. 6, 7; Supplementary Information).


BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan GC, Zhang F, Orkin SH, Bauer DE - Nature (2015)

Tiled pooled in situ CRISPR-Cas9 Bcl11a enhancer screena, Schematic of the mouse Bcl11a locus (mm9, transcription from left to right) with erythroid chromatin marks (top, dark blue H3K27ac from Kowalczyk et al55, middle, light blue H3K27ac from Dogan et al56, and bottom, black DNase I from Bauer et al28) 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 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4644101&req=5

Figure 6: Tiled pooled in situ CRISPR-Cas9 Bcl11a enhancer screena, Schematic of the mouse Bcl11a locus (mm9, transcription from left to right) with erythroid chromatin marks (top, dark blue H3K27ac from Kowalczyk et al55, middle, light blue H3K27ac from Dogan et al56, and bottom, black DNase I from Bauer et al28) 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 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.
Mentions: To test functional conservation of the BCL11A enhancer, we examined the orthologous mouse Bcl11a enhancer in greater detail. Erythroid DNase I sensitivity is only observed at those sequences homologous to h+55 and h+62 and not h+58 (Extended Data Fig. 6a), consistent with the reduced sequence homology within the h+58 Active region (Fig. 3a-c). We performed a pooled CRISPR enhancer saturating mutagenesis screen in MEL εy:mCherry reporter cells, similar to the human screen described above (Extended Data Fig. 6, 7; Supplementary Information).

Bottom Line: Despite conserved function of the composite enhancers, their architecture diverges.The crucial human sequences appear to be primate-specific.The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

Show MeSH
Related in: MedlinePlus