Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements

Mol Ther. 2019 Jan 2;27(1):137-150. doi: 10.1016/j.ymthe.2018.10.008. Epub 2018 Oct 17.


Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9+ HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.

Keywords: CRISPR/Cas9 delivery; genome editing; β-hemoglobinopathies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anemia, Sickle Cell / genetics
  • Anemia, Sickle Cell / metabolism
  • Anemia, Sickle Cell / therapy
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics
  • CRISPR-Cas Systems / physiology*
  • Gene Editing / methods
  • Genetic Therapy / methods*
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism*
  • Hemoglobinopathies / genetics
  • Hemoglobinopathies / metabolism
  • Hemoglobinopathies / therapy
  • Heterogeneous-Nuclear Ribonucleoproteins / genetics
  • Heterogeneous-Nuclear Ribonucleoproteins / metabolism
  • Humans
  • Plasmids / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • beta-Thalassemia / genetics
  • beta-Thalassemia / metabolism
  • beta-Thalassemia / therapy


  • Heterogeneous-Nuclear Ribonucleoproteins
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9