Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Cell Rep. 2016 Oct 25;17(5):1453-1461. doi: 10.1016/j.celrep.2016.09.092.


Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (∼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.

Keywords: CRISPR/Cas9; HSC; gene therapy; genome editing; hematopoietic stem cells; homology-directed repair; human CD34; progenitor; sgRNA; transplantation.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • DNA Repair
  • Gene Deletion
  • Gene Editing / methods*
  • Gene Knockout Techniques
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Leukocyte Common Antigens / metabolism
  • Mice


  • Leukocyte Common Antigens