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Stem Cells. 2018 Oct 29. doi: 10.1002/stem.2935. [Epub ahead of print]

Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair.

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Department of Molecular and Medical Pharmacology, University of California Los Angeles (UCLA), Los Angeles, California, USA.
Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.
Institute of Genomics and Proteomics, UCLA, Los Angeles, California, USA.
Department of General Internal Medicine and Health Services Research, UCLA, Los Angeles, California, USA.
Innovative Genomics Institute, University of California Berkeley, Berkeley, California, USA.
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, USA.
Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, UCLA, Los Angeles, California, USA.


Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the field of gene therapy, and can be applied to diseases where increased HDR/NHEJ ratio is critical for therapeutic success. Stem Cells 2018.


Adult hematopoietic stem cells; CD34+; CRISPR; Cell cycle; Clinical translation; Gene therapy; Hematopoietic stem cells (HSCs); Stem/progenitor cell


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