Publication Date:
2015-09-17
Description:
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.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4644101/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4644101/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Canver, Matthew C -- Smith, Elenoe C -- Sher, Falak -- Pinello, Luca -- Sanjana, Neville E -- Shalem, Ophir -- Chen, Diane D -- Schupp, Patrick G -- Vinjamur, Divya S -- Garcia, Sara P -- Luc, Sidinh -- Kurita, Ryo -- Nakamura, Yukio -- Fujiwara, Yuko -- Maeda, Takahiro -- Yuan, Guo-Cheng -- Zhang, Feng -- Orkin, Stuart H -- Bauer, Daniel E -- 5DP1-MH100706/DP/NCCDPHP CDC HHS/ -- 5R01-DK097768/DK/NIDDK NIH HHS/ -- F30DK103359-01A1/DK/NIDDK NIH HHS/ -- K08DK093705/DK/NIDDK NIH HHS/ -- K99 HG008171/HG/NHGRI NIH HHS/ -- K99-HG008171/HG/NHGRI NIH HHS/ -- K99HG008399/HG/NHGRI NIH HHS/ -- P01 HL032262/HL/NHLBI NIH HHS/ -- P01HL032262/HL/NHLBI NIH HHS/ -- P30DK049216/DK/NIDDK NIH HHS/ -- R01 A1084905/PHS HHS/ -- R01 HL032259/HL/NHLBI NIH HHS/ -- R01HG005085/HG/NHGRI NIH HHS/ -- R01HL119099/HL/NHLBI NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2015 Nov 12;527(7577):192-7. doi: 10.1038/nature15521. Epub 2015 Sep 16.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉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. ; Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02115, USA. ; 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. ; Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan. ; Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. ; Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA. ; Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26375006" target="_blank"〉PubMed〈/a〉
Keywords:
Animals
;
Base Sequence
;
CRISPR-Associated Proteins/*metabolism
;
CRISPR-Cas Systems/genetics
;
Carrier Proteins/*genetics
;
Cells, Cultured
;
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
;
Enhancer Elements, Genetic/*genetics
;
Erythroblasts/metabolism
;
Fetal Hemoglobin/genetics
;
*Genetic Engineering
;
Genome/genetics
;
Humans
;
Mice
;
Molecular Sequence Data
;
Mutagenesis/*genetics
;
Nuclear Proteins/*genetics
;
Organ Specificity
;
RNA, Guide/genetics
;
Reproducibility of Results
;
Species Specificity
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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