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

Nat Commun. 2019 Oct 18;10(1):4730. doi: 10.1038/s41467-019-12726-0.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cell Differentiation / genetics*
  • Cell Proliferation / genetics*
  • Electroporation / methods
  • Female
  • GATA1 Transcription Factor / genetics
  • GATA1 Transcription Factor / metabolism
  • Gene Editing / methods*
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Mice, Inbred NOD
  • Mice, Knockout
  • Mice, SCID
  • Transplantation, Heterologous


  • GATA1 Transcription Factor
  • GATA1 protein, human