Blog
About

  • Record: found
  • Abstract: found
  • Article: not found

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

Read this article at

ScienceOpenPublisherPubMed
Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      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.

      Related collections

      Author and article information

      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.

      Comments

      Comment on this article