title: BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.
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.
authors.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.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Canver
authors.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.
authors.forename: Elenoe C
authors.initials: EC
authors.lastname: Smith
authors.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.
authors.forename: Falak
authors.initials: F
authors.lastname: Sher
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02115, USA.
authors.forename: Luca
authors.initials: L
authors.lastname: Pinello
authors.affiliation: Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences and Department of Biological Engineering, MIT, Cambridge, Massachusetts 02142, USA.
authors.forename: Neville E
authors.initials: NE
authors.lastname: Sanjana
authors.affiliation: Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences and Department of Biological Engineering, MIT, Cambridge, Massachusetts 02142, USA.
authors.forename: Ophir
authors.initials: O
authors.lastname: Shalem
authors.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.
authors.forename: Diane D
authors.initials: DD
authors.lastname: Chen
authors.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.
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Schupp
authors.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.
authors.forename: Divya S
authors.initials: DS
authors.lastname: Vinjamur
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02115, USA.
authors.forename: Sara P
authors.initials: SP
authors.lastname: Garcia
authors.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.
authors.forename: Sidinh
authors.initials: S
authors.lastname: Luc
authors.affiliation: Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kurita
authors.affiliation: Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan.|Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nakamura
authors.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.|Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fujiwara
authors.affiliation: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maeda
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02115, USA.
authors.forename: Guo-Cheng
authors.initials: GC
authors.lastname: Yuan
authors.affiliation: Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences and Department of Biological Engineering, MIT, Cambridge, Massachusetts 02142, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.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.|Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Orkin
authors.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.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Bauer
mesh_terms: D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D004900:Erythroblasts / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000235:genetics; D005818:Genetic Engineering; D016678:Genome / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D009928:Organ Specificity; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012097:Repressor Proteins; D015203:Reproducibility of Results; D013045:Species Specificity
chemical_list: C486563:BCL11A protein, human; C407733:Bcl11a protein, mouse; D064130:CRISPR-Associated Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012097:Repressor Proteins; D005319:Fetal Hemoglobin