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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, Distribution of NGG and NAG PAM sgRNAs mapped to genomic cleavage position. The vertical lines represent cleavage sites for sgRNAs mapped to plus and minus strands. b, Gap distance between adjacent genomic cleavage position for NAG PAM sgRNAs. c, Library composition by target sequence and PAM restriction. d. Representation of both NGG and NAG sgRNA (1,338 sgRNAs in total) within the plasmid pool by deep-sequencing. The median was 718 normalized reads and the 10th and 90th percentiles (indicated by the vertical dotted lines) ranged from 337 to 1,205 normalized reads. e, HbF distribution in HUDEP-2 cells transduced with Cas9 and individual sgRNAs, either nontargeting or targeting BCL11A exon 2. f, HbF enrichment scores of NGG sgRNAs in six biological replicates. g, Sort of library-transduced cells into HbF-high and HbF-low pools. h, Control sgRNA enrichment. Boxes demonstrate 25th, median, and 75th percentiles and whiskers minimum and maximum values. **** P < 0.0001, ns non-significant. i, NGG sgRNA representation in plasmid pool and cells at conclusion of experiment (left), and in HbF-high and HbF-low pools (right), with dotted lines at x=y and x=8y. j, Quantile-quantile plots of NGG sgRNA enrichment scores. k, Cellular dropout scores of NGG sgRNAs relative to genomic cleavage position and repetitive elements. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing.
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Figure 2: Tiled pooled in situ CRISPR-Cas9 BCL11A enhancer screena, Distribution of NGG and NAG PAM sgRNAs mapped to genomic cleavage position. The vertical lines represent cleavage sites for sgRNAs mapped to plus and minus strands. b, Gap distance between adjacent genomic cleavage position for NAG PAM sgRNAs. c, Library composition by target sequence and PAM restriction. d. Representation of both NGG and NAG sgRNA (1,338 sgRNAs in total) within the plasmid pool by deep-sequencing. The median was 718 normalized reads and the 10th and 90th percentiles (indicated by the vertical dotted lines) ranged from 337 to 1,205 normalized reads. e, HbF distribution in HUDEP-2 cells transduced with Cas9 and individual sgRNAs, either nontargeting or targeting BCL11A exon 2. f, HbF enrichment scores of NGG sgRNAs in six biological replicates. g, Sort of library-transduced cells into HbF-high and HbF-low pools. h, Control sgRNA enrichment. Boxes demonstrate 25th, median, and 75th percentiles and whiskers minimum and maximum values. **** P < 0.0001, ns non-significant. i, NGG sgRNA representation in plasmid pool and cells at conclusion of experiment (left), and in HbF-high and HbF-low pools (right), with dotted lines at x=y and x=8y. j, Quantile-quantile plots of NGG sgRNA enrichment scores. k, Cellular dropout scores of NGG sgRNAs relative to genomic cleavage position and repetitive elements. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing.

Mentions: We designed all possible sgRNAs within the human BCL11A composite enhancer DHSs (Fig. 1d, e) as restricted only by the presence of the SpCas9 NGG protospacer adjacent motif (PAM), which restricts cleavage at an average 1/8 frequency at each genomic position22,39. The NGG PAM restricted sgRNAs had a median gap between adjacent genomic cleavages of 4 bp and 90th percentile of 18 bp (Fig. 1f), which suggested that this strategy could approach saturation mutagenesis in situ. We included nontargeting sgRNAs as negative controls as well as sgRNAs tiling exon-2 of BCL11A as positive controls (Fig. 1e). The library was successfully cloned to a lentiviral vector. The basic experimental schema was to transduce HUDEP-2 cells with the lentiviral library at low multiplicity such that nearly all selected cells contained a single integrant (Fig. 1d). Following expansion, differentiation, sorting by HbF level, genomic DNA isolation, and deep sequencing of integrated sgRNAs, an HbF enrichment score was calculated for each sgRNA by comparing its representation in HbF-high and HbF-low pools (see Supplementary Information and Extended Data Fig. 2 for additional technical details).


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, Distribution of NGG and NAG PAM sgRNAs mapped to genomic cleavage position. The vertical lines represent cleavage sites for sgRNAs mapped to plus and minus strands. b, Gap distance between adjacent genomic cleavage position for NAG PAM sgRNAs. c, Library composition by target sequence and PAM restriction. d. Representation of both NGG and NAG sgRNA (1,338 sgRNAs in total) within the plasmid pool by deep-sequencing. The median was 718 normalized reads and the 10th and 90th percentiles (indicated by the vertical dotted lines) ranged from 337 to 1,205 normalized reads. e, HbF distribution in HUDEP-2 cells transduced with Cas9 and individual sgRNAs, either nontargeting or targeting BCL11A exon 2. f, HbF enrichment scores of NGG sgRNAs in six biological replicates. g, Sort of library-transduced cells into HbF-high and HbF-low pools. h, Control sgRNA enrichment. Boxes demonstrate 25th, median, and 75th percentiles and whiskers minimum and maximum values. **** P < 0.0001, ns non-significant. i, NGG sgRNA representation in plasmid pool and cells at conclusion of experiment (left), and in HbF-high and HbF-low pools (right), with dotted lines at x=y and x=8y. j, Quantile-quantile plots of NGG sgRNA enrichment scores. k, Cellular dropout scores of NGG sgRNAs relative to genomic cleavage position and repetitive elements. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Tiled pooled in situ CRISPR-Cas9 BCL11A enhancer screena, Distribution of NGG and NAG PAM sgRNAs mapped to genomic cleavage position. The vertical lines represent cleavage sites for sgRNAs mapped to plus and minus strands. b, Gap distance between adjacent genomic cleavage position for NAG PAM sgRNAs. c, Library composition by target sequence and PAM restriction. d. Representation of both NGG and NAG sgRNA (1,338 sgRNAs in total) within the plasmid pool by deep-sequencing. The median was 718 normalized reads and the 10th and 90th percentiles (indicated by the vertical dotted lines) ranged from 337 to 1,205 normalized reads. e, HbF distribution in HUDEP-2 cells transduced with Cas9 and individual sgRNAs, either nontargeting or targeting BCL11A exon 2. f, HbF enrichment scores of NGG sgRNAs in six biological replicates. g, Sort of library-transduced cells into HbF-high and HbF-low pools. h, Control sgRNA enrichment. Boxes demonstrate 25th, median, and 75th percentiles and whiskers minimum and maximum values. **** P < 0.0001, ns non-significant. i, NGG sgRNA representation in plasmid pool and cells at conclusion of experiment (left), and in HbF-high and HbF-low pools (right), with dotted lines at x=y and x=8y. j, Quantile-quantile plots of NGG sgRNA enrichment scores. k, Cellular dropout scores of NGG sgRNAs relative to genomic cleavage position and repetitive elements. Nontargeting sgRNAs pseudo-mapped with 5 bp spacing.
Mentions: We designed all possible sgRNAs within the human BCL11A composite enhancer DHSs (Fig. 1d, e) as restricted only by the presence of the SpCas9 NGG protospacer adjacent motif (PAM), which restricts cleavage at an average 1/8 frequency at each genomic position22,39. The NGG PAM restricted sgRNAs had a median gap between adjacent genomic cleavages of 4 bp and 90th percentile of 18 bp (Fig. 1f), which suggested that this strategy could approach saturation mutagenesis in situ. We included nontargeting sgRNAs as negative controls as well as sgRNAs tiling exon-2 of BCL11A as positive controls (Fig. 1e). The library was successfully cloned to a lentiviral vector. The basic experimental schema was to transduce HUDEP-2 cells with the lentiviral library at low multiplicity such that nearly all selected cells contained a single integrant (Fig. 1d). Following expansion, differentiation, sorting by HbF level, genomic DNA isolation, and deep sequencing of integrated sgRNAs, an HbF enrichment score was calculated for each sgRNA by comparing its representation in HbF-high and HbF-low pools (see Supplementary Information and Extended Data Fig. 2 for additional technical details).

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