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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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Functional sequence requirement at the mouse Bcl11a erythroid enhancer for in vivo hemoglobin switchinga, Mapping sgRNA εy enrichment scores to genomic cleavage positions. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing. b, BCL11A expression in mouse erythroid clones with deletion or inversion of individual DHSs relative to nondeleted controls. c, Transgenic human β-like globin expression in β-YAC/+62 deletion mice. For +/+, +/Δ, and Δ/Δ: at E12.5, n = 5, 11, and 3 embryos respectively; at E14.5, n = 2, 3, and 4; at E16.5, n = 2, 4, and 1; at E18.5, n = 3, 1, and 3. Error bars represent s.e.m.
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Figure 15: Functional sequence requirement at the mouse Bcl11a erythroid enhancer for in vivo hemoglobin switchinga, Mapping sgRNA εy enrichment scores to genomic cleavage positions. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing. b, BCL11A expression in mouse erythroid clones with deletion or inversion of individual DHSs relative to nondeleted controls. c, Transgenic human β-like globin expression in β-YAC/+62 deletion mice. For +/+, +/Δ, and Δ/Δ: at E12.5, n = 5, 11, and 3 embryos respectively; at E14.5, n = 2, 3, and 4; at E16.5, n = 2, 4, and 1; at E18.5, n = 3, 1, and 3. Error bars represent s.e.m.

Mentions: Upon mapping the sgRNA cleavage positions to the genome, we again observed that the majority of enhancer targeting sgRNAs demonstrated no significant εy enrichment or depletion. We observed colocalization of sets of sgRNAs with εy enrichment (Fig. 5a). There was a similar complex pattern at the m+55 ortholog as at h+55, with adjacent regions with enriching and depleting sgRNAs from the high-εy:mCherry pool at the DHS core. At the m+58 ortholog we did not observe any evidence of εy enriching or depleting sgRNAs. At the m+62 ortholog there was a marked peak, with five sgRNAs with εy enrichment scores exceeding 1.30, the median enrichment score of Bcl11a exon-2 targeting sgRNAs (Fig. 5a). This potent impact of the m+62 ortholog was in contrast to the modest impact of individual sgRNAs or DHS deletion at h+62.


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)

Functional sequence requirement at the mouse Bcl11a erythroid enhancer for in vivo hemoglobin switchinga, Mapping sgRNA εy enrichment scores to genomic cleavage positions. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing. b, BCL11A expression in mouse erythroid clones with deletion or inversion of individual DHSs relative to nondeleted controls. c, Transgenic human β-like globin expression in β-YAC/+62 deletion mice. For +/+, +/Δ, and Δ/Δ: at E12.5, n = 5, 11, and 3 embryos respectively; at E14.5, n = 2, 3, and 4; at E16.5, n = 2, 4, and 1; at E18.5, n = 3, 1, and 3. Error bars represent s.e.m.
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Figure 15: Functional sequence requirement at the mouse Bcl11a erythroid enhancer for in vivo hemoglobin switchinga, Mapping sgRNA εy enrichment scores to genomic cleavage positions. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing. b, BCL11A expression in mouse erythroid clones with deletion or inversion of individual DHSs relative to nondeleted controls. c, Transgenic human β-like globin expression in β-YAC/+62 deletion mice. For +/+, +/Δ, and Δ/Δ: at E12.5, n = 5, 11, and 3 embryos respectively; at E14.5, n = 2, 3, and 4; at E16.5, n = 2, 4, and 1; at E18.5, n = 3, 1, and 3. Error bars represent s.e.m.
Mentions: Upon mapping the sgRNA cleavage positions to the genome, we again observed that the majority of enhancer targeting sgRNAs demonstrated no significant εy enrichment or depletion. We observed colocalization of sets of sgRNAs with εy enrichment (Fig. 5a). There was a similar complex pattern at the m+55 ortholog as at h+55, with adjacent regions with enriching and depleting sgRNAs from the high-εy:mCherry pool at the DHS core. At the m+58 ortholog we did not observe any evidence of εy enriching or depleting sgRNAs. At the m+62 ortholog there was a marked peak, with five sgRNAs with εy enrichment scores exceeding 1.30, the median enrichment score of Bcl11a exon-2 targeting sgRNAs (Fig. 5a). This potent impact of the m+62 ortholog was in contrast to the modest impact of individual sgRNAs or DHS deletion at h+62.

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