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      Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.

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          Abstract

          Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.

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          Affiliations
          [1 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA.
          [2 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
          [3 ] Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
          [4 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
          [5 ] Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA.
          [6 ] Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
          [7 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
          [8 ] Institution for Experimental Medical Research, Immunology section, Lund University, 221 84, Lund, Sweden.
          [9 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Broad Institute, Cambridge, MA 02142, USA.
          [10 ] Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA; Broad Institute, Cambridge, MA 02142, USA; Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
          [11 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address: derrick.rossi@childrens.harvard.edu.
          [12 ] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Broad Institute, Cambridge, MA 02142, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address: chad_cowan@harvard.edu.
          Journal
          Cell Stem Cell
          Cell stem cell
          1875-9777
          Nov 6 2014
          : 15
          : 5
          S1934-5909(14)00455-X 10.1016/j.stem.2014.10.004 25517468 4269831 NIHMS635971
          Copyright © 2014 Elsevier Inc. All rights reserved.

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