A Versatile Tool for the Quantification of CRISPR/Cas9-Induced Genome Editing Events in Human Hematopoietic Cell Lines and Hematopoietic Stem/Progenitor Cells |
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| Institution: | 1. Division of Experimental Hematology and Cancer Biology, Cancer and Blood Disease Institute (CBDI), Cincinnati Children''s Hospital Medical Center (CCHMC), Cincinnati, OH, 45229, USA;2. Pathology and Molecular Medicine Program, University of Cincinnati, Cincinnati, OH, 45229, USA;3. Division of Hematology, CBDI, CCHMC, Cincinnati, OH, 45229, USA;1. Department of Emergency Medicine, Yale School of Medicine, 464 Congress Avenue, New Haven, CT 06519-1362, USA;2. Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA |
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| Abstract: | The efficient site-specific DNA double-strand breaks (DSB) created by CRISPR/Cas9 has revolutionized genome engineering and has great potential for editing hematopoietic stem/progenitor cells (HSPCs). However, detailed understanding of the variables that influence choice of DNA–DSB repair (DDR) pathways by HSPC is required for therapeutic levels of editing in these clinically relevant cells. We developed a hematopoietic-reporter system that rapidly quantifies the three major DDR pathways utilized at the individual DSB created by CRISPR/Cas9—NHEJ, MMEJ, and HDR—and show its applicability in evaluating the different DDR outcomes utilized by human hematopoietic cell lines and primary human HSPC. |
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