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Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells

Author

Listed:
  • Elvin Wagenblast

    (University Health Network)

  • Maria Azkanaz

    (University Health Network)

  • Sabrina A. Smith

    (University Health Network)

  • Lorien Shakib

    (University Health Network)

  • Jessica L. McLeod

    (University Health Network)

  • Gabriela Krivdova

    (University Health Network)

  • Joana Araújo

    (University Health Network)

  • Leonard D. Shultz

    (The Jackson Laboratory)

  • Olga I. Gan

    (University Health Network)

  • John E. Dick

    (University Health Network
    University of Toronto)

  • Eric R. Lechman

    (University Health Network)

Abstract

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.

Suggested Citation

  • Elvin Wagenblast & Maria Azkanaz & Sabrina A. Smith & Lorien Shakib & Jessica L. McLeod & Gabriela Krivdova & Joana Araújo & Leonard D. Shultz & Olga I. Gan & John E. Dick & Eric R. Lechman, 2019. "Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-12726-0
    DOI: 10.1038/s41467-019-12726-0
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