Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells.

Autor:	Benitez EK; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Lomova Kaufman A; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Cervantes L; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Clark DN; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Ayoub PG; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Senadheera S; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Osborne K; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Sanchez JM; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Crisostomo RV; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Wang X; Department of General Internal Medicine and Health Services Research, University of California, Los Angeles, Los Angeles, CA, United States., Reuven N; Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel., Shaul Y; Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel., Hollis RP; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Romero Z; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States., Kohn DB; Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States.
Jazyk:	angličtina
Zdroj:	Frontiers in genome editing [Front Genome Ed] 2020 Dec 10; Vol. 2, pp. 601541. Date of Electronic Publication: 2020 Dec 10 (Print Publication: 2020).
DOI:	10.3389/fgeed.2020.601541
Abstrakt:	Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2020 Benitez, Lomova Kaufman, Cervantes, Clark, Ayoub, Senadheera, Osborne, Sanchez, Crisostomo, Wang, Reuven, Shaul, Hollis, Romero and Kohn.)
Databáze:	MEDLINE
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