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BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

Author

Listed:
  • Matthew C. Canver

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

  • Elenoe C. Smith

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

  • Falak Sher

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

  • Luca Pinello

    (Dana-Farber Cancer Institute and Harvard School of Public Health)

  • Neville E. Sanjana

    (Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, MIT)

  • Ophir Shalem

    (Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, MIT)

  • Diane D. Chen

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

  • Patrick G. Schupp

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

  • Divya S. Vinjamur

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

  • Sara P. Garcia

    (Dana-Farber Cancer Institute and Harvard School of Public Health)

  • Sidinh Luc

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

  • Ryo Kurita

    (RIKEN BioResource Center)

  • Yukio Nakamura

    (RIKEN BioResource Center
    Comprehensive Human Sciences, University of Tsukuba)

  • Yuko Fujiwara

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
    Howard Hughes Medical Institute)

  • Takahiro Maeda

    (Brigham and Women’s Hospital, Harvard Medical School)

  • Guo-Cheng Yuan

    (Dana-Farber Cancer Institute and Harvard School of Public Health)

  • Feng Zhang

    (Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, MIT)

  • Stuart H. Orkin

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
    Howard Hughes Medical Institute)

  • Daniel E. Bauer

    (Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School)

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.

Suggested Citation

  • Matthew C. Canver & Elenoe C. Smith & Falak Sher & Luca Pinello & Neville E. Sanjana & Ophir Shalem & Diane D. Chen & Patrick G. Schupp & Divya S. Vinjamur & Sara P. Garcia & Sidinh Luc & Ryo Kurita &, 2015. "BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis," Nature, Nature, vol. 527(7577), pages 192-197, November.
  • Handle: RePEc:nat:nature:v:527:y:2015:i:7577:d:10.1038_nature15521
    DOI: 10.1038/nature15521
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    Cited by:

    1. Tomoyuki Ohno & Taichi Akase & Shunya Kono & Hikaru Kurasawa & Takuto Takashima & Shinya Kaneko & Yasunori Aizawa, 2022. "Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    2. Inês A. M. Barbosa & Rajaraman Gopalakrishnan & Samuele Mercan & Thanos P. Mourikis & Typhaine Martin & Simon Wengert & Caibin Sheng & Fei Ji & Rui Lopes & Judith Knehr & Marc Altorfer & Alicia Lindem, 2023. "Cancer lineage-specific regulation of YAP responsive elements revealed through large-scale functional epigenomic screens," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    3. Arianna Moiani & Gil Letort & Sabrina Lizot & Anne Chalumeau & Chloe Foray & Tristan Felix & Diane Clerre & Sonal Temburni-Blake & Patrick Hong & Sophie Leduc & Noemie Pinard & Alan Marechal & Eduardo, 2024. "Non-viral DNA delivery and TALEN editing correct the sickle cell mutation in hematopoietic stem cells," Nature Communications, Nature, vol. 15(1), pages 1-21, December.
    4. Patrick C Fiaux & Hsiuyi V Chen & Poshen B Chen & Aaron R Chen & Graham McVicker, 2020. "Discovering functional sequences with RELICS, an analysis method for CRISPR screens," PLOS Computational Biology, Public Library of Science, vol. 16(9), pages 1-23, September.
    5. Jing Guo & Luyao Gong & Haiying Yu & Ming Li & Qiaohui An & Zhenquan Liu & Shuru Fan & Changjialian Yang & Dahe Zhao & Jing Han & Hua Xiang, 2024. "Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    6. Jiaoyang Liao & Shuanghong Chen & Shenlin Hsiao & Yanhong Jiang & Yang Yang & Yuanjin Zhang & Xin Wang & Yongrong Lai & Daniel E. Bauer & Yuxuan Wu, 2023. "Therapeutic adenine base editing of human hematopoietic stem cells," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    7. Colin McGaw & Anthony J. Garrity & Gabrielle Z. Munoz & Jeffrey R. Haswell & Sejuti Sengupta & Elise Keston-Smith & Pratyusha Hunnewell & Alexa Ornstein & Mishti Bose & Quinton Wessells & Noah Jakimo , 2022. "Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    8. Deborah E. Daniels & Ivan Ferrer-Vicens & Joseph Hawksworth & Tatyana N. Andrienko & Elizabeth M. Finnie & Natalie S. Bretherton & Daniel C. J. Ferguson & A. Sofia. F. Oliveira & Jenn-Yeu A. Szeto & M, 2023. "Human cellular model systems of β-thalassemia enable in-depth analysis of disease phenotype," Nature Communications, Nature, vol. 14(1), pages 1-14, December.

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