UMMS Affiliation

Department of Molecular, Cell and Cancer Biology

Publication Date


Document Type



Amino Acids, Peptides, and Proteins | Cancer Biology | Cell Biology | Computational Biology | Congenital, Hereditary, and Neonatal Diseases and Abnormalities | Genetics and Genomics | Hematology | Hemic and Lymphatic Diseases | Nucleic Acids, Nucleotides, and Nucleosides


Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress gamma-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and beta-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rgamma-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.


Gene Therapy, Hematopoiesis and Stem Cells, Red Cells, Iron, and Erythropoiesis, cd34 antigens, fetal hemoglobin, transplantation, heterologous, genome editing, donors, child, globins, dna, transplantation, genes

Rights and Permissions

Copyright The American Society of Hematology. Publisher PDF posted as allowed by the publisher's copyright and author rights information at

DOI of Published Version



Blood Adv. 2019 Nov 12;3(21):3379-3392. doi: 10.1182/bloodadvances.2019000820. Link to article on publisher's site

Journal/Book/Conference Title

Blood advances


Full author list omitted for brevity. For the full list of authors, see article.

Related Resources

Link to Article in PubMed

PubMed ID