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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We thank J. Hughes and D. Higgs for assistance with analysis of ChIP-seq; R. Mathieu and the Boston Children’s Hospital Hematology/Oncology-HSCI Flow Cytometry Research Facility for cell sorting; Z. Herbert and F. Abderazzaq at the Dana-Farber Cancer Institute Molecular Biology Core Facility and Center for Cancer Computational Biology, respectively, for sequencing; J. Doench for providing TALENs; C. Peng for advice with MEL reporter cell generation; F. Godinho and M. Nguyen for technical help with ESCs and transgenic mice; A. Dass, C. Lin and S. Kamran for general technical assistance; C. Brendel and D. Williams for input regarding lentiviral transduction of HSPCs; J. Desimini for graphical assistance; and J. Xu and G. Lettre for insightful discussions. M.C.C. is supported by F30DK103359-01A1. E.C.S. is supported by a Jane Coffin Childs Memorial Fund for Medical Research Fellowship. L.P. is supported by NHGRI Career Development Award K99HG008399. N.E.S. is supported by a Simons Center for the Social Brain Postdoctoral Fellowship and NIH NHGRI award K99-HG008171. O.S. is supported by a fellowship from the Klarman Family Foundation. S.L. is supported by a Leukemia & Lymphoma Society Fellow Award. T.M. is supported by NIH R01 A1084905. G.-C.Y. is supported by NIH R01HL119099 and R01HG005085. F.Z. is supported by the NIMH (5DP1-MH100706) and NIDDK (5R01-DK097768), a Waterman award from the National Science Foundation, the Keck, McKnight, Damon Runyon, Searle Scholars, Merkin, Vallee, and Simons Foundations, and Bob Metcalfe. S.H.O. is supported by P01HL032262 and P30DK049216 (Center of Excellence in Molecular Hematology). D.E.B. is supported by an NIDDK Career Development Award K08DK093705, Doris Duke Charitable Foundation Innovations in Clinical Research Award (2013137), and Charles H. Hood Foundation Child Health Research Award. Computational tools and instructions for designing CRISPR-Cas9 sgRNA libraries for conducting non-coding screening can be found at the Zhang laboratory website http://www.genome-engineering.org.

Author information

Author notes

    • Matthew C. Canver
    • , Elenoe C. Smith
    • , Falak Sher
    • , Luca Pinello
    •  & Neville E. Sanjana

    These authors contributed equally to this work.

    • Feng Zhang
    • , Stuart H. Orkin
    •  & Daniel E. Bauer

    These authors jointly supervised this work.


  1. 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

    • Matthew C. Canver
    • , Elenoe C. Smith
    • , Falak Sher
    • , Diane D. Chen
    • , Patrick G. Schupp
    • , Divya S. Vinjamur
    • , Sidinh Luc
    • , Yuko Fujiwara
    • , Stuart H. Orkin
    •  & Daniel E. Bauer
  2. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02115, USA

    • Luca Pinello
    • , Sara P. Garcia
    •  & Guo-Cheng Yuan
  3. 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

    • Neville E. Sanjana
    • , Ophir Shalem
    •  & Feng Zhang
  4. Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan

    • Ryo Kurita
    •  & Yukio Nakamura
  5. Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan

    • Yukio Nakamura
  6. Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA

    • Yuko Fujiwara
    •  & Stuart H. Orkin
  7. Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Takahiro Maeda


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D.E.B. conceived this study. N.E.S., O.S. and F.Z. conceived the pooled non-coding screening strategy using CRISPR-Cas9. M.C.C., N.E.S., O.S., F.Z., S.H.O. and D.E.B. designed and executed the pooled CRISPR screening strategy. E.C.S., F.S., Y.F., S.L., S.H.O. and D.E.B. designed, produced and analysed the transgenic mice. R.K. and Y.N. provided the HUDEP-2 cell line. M.C.C., F.S., T.M., S.H.O. and D.E.B. adapted the HUDEP-2 cell line as a model of globin gene regulation. M.C.C., F.S., D.D.C., P.G.S., D.S.V. and D.E.B. performed all experiments in cell lines. M.C.C., L.P., N.E.S., S.P.G., G.-C.Y., F.Z., S.H.O. and D.E.B. analysed the data. L.P., S.P.G. and G.-C.Y. developed the HMM. M.C.C., S.H.O., and D.E.B. wrote the manuscript with input from all authors.

Competing interests

D.E.B. and S.H.O. are inventors on a patent related to this work. N.E.S., O.S. and F.Z. are inventors on a patent application related to the screening technology. F.Z. is a founder of Editas Medicine and scientific advisor for Editas Medicine and Horizon Discovery. S.H.O. is on the Scientific Advisory Board of Editas Medicine.

Corresponding authors

Correspondence to Feng Zhang or Stuart H. Orkin or Daniel E. Bauer.

All reagents described in this manuscript have been deposited with Addgene (http://www.addgene.org).

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