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CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells

Author

Listed:
  • Daniel P. Dever

    (Stanford University)

  • Rasmus O. Bak

    (Stanford University)

  • Andreas Reinisch

    (Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University)

  • Joab Camarena

    (Stanford University)

  • Gabriel Washington

    (Stanford University)

  • Carmencita E. Nicolas

    (Stanford University)

  • Mara Pavel-Dinu

    (Stanford University)

  • Nivi Saxena

    (Stanford University)

  • Alec B. Wilkens

    (Stanford University)

  • Sruthi Mantri

    (Stanford University)

  • Nobuko Uchida

    (Stem Cells, Inc. 7707 Gateway Blvd., Suite 140
    †Present address: Institute of Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.)

  • Ayal Hendel

    (Stanford University)

  • Anupama Narla

    (Stanford University School of Medicine)

  • Ravindra Majeti

    (Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University)

  • Kenneth I. Weinberg

    (Stanford University)

  • Matthew H. Porteus

    (Stanford University)

Abstract

The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

Suggested Citation

  • Daniel P. Dever & Rasmus O. Bak & Andreas Reinisch & Joab Camarena & Gabriel Washington & Carmencita E. Nicolas & Mara Pavel-Dinu & Nivi Saxena & Alec B. Wilkens & Sruthi Mantri & Nobuko Uchida & Ayal, 2016. "CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells," Nature, Nature, vol. 539(7629), pages 384-389, November.
  • Handle: RePEc:nat:nature:v:539:y:2016:i:7629:d:10.1038_nature20134
    DOI: 10.1038/nature20134
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    Citations

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    Cited by:

    1. Sandra Wimberger & Nina Akrap & Mike Firth & Johan Brengdahl & Susanna Engberg & Marie K. Schwinn & Michael R. Slater & Anders Lundin & Pei-Pei Hsieh & Songyuan Li & Silvia Cerboni & Jonathan Sumner &, 2023. "Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    2. 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.
    3. Juan A. Perez-Bermejo & Oghene Efagene & William M. Matern & Jeffrey K. Holden & Shaheen Kabir & Glen M. Chew & Gaia Andreoletti & Eniola Catton & Craig L. Ennis & Angelica Garcia & Trevor L. Gerstenb, 2024. "Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    4. M. Kyle Cromer & Valentin V. Barsan & Erich Jaeger & Mengchi Wang & Jessica P. Hampton & Feng Chen & Drew Kennedy & Jenny Xiao & Irina Khrebtukova & Ana Granat & Tiffany Truong & Matthew H. Porteus, 2022. "Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    5. Ron Baik & M. Kyle Cromer & Steve E. Glenn & Christopher A. Vakulskas & Kay O. Chmielewski & Amanda M. Dudek & William N. Feist & Julia Klermund & Suzette Shipp & Toni Cathomen & Daniel P. Dever & Mat, 2024. "Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    6. Daniel Allen & Orli Knop & Bryan Itkowitz & Nechama Kalter & Michael Rosenberg & Ortal Iancu & Katia Beider & Yu Nee Lee & Arnon Nagler & Raz Somech & Ayal Hendel, 2023. "CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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