title: Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9.
abstract: Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (∼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.
authors: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA.
authors: Michael C
authors: MC
authors: Gundry
authors: Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA; Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, 06156 Perugia, Italy.
authors: Lorenzo
authors: L
authors: Brunetti
authors: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Integrative Molecular and Biomedical Sciences Program, Baylor College of Medicine, Houston, TX 77030, USA.
authors: Angelique
authors: A
authors: Lin
authors: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA.
authors: Allison E
authors: AE
authors: Mayle
authors: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
authors: Ayumi
authors: A
authors: Kitano
authors: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA; Texas Children's Hospital and Houston Methodist Hospital, Houston, TX 77030, USA; Institute for Medical Immunology, Charité University Medicine Berlin, 13353 Berlin, Germany.
authors: Dimitrios
authors: D
authors: Wagner
authors: Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA; Translational Biology and Molecular Medicine Program, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA.
authors: Joanne I
authors: JI
authors: Hsu
authors: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
authors: Kevin A
authors: KA
authors: Hoegenauer
authors: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA; Texas Children's Hospital and Houston Methodist Hospital, Houston, TX 77030, USA.
authors: Cliona M
authors: CM
authors: Rooney
authors: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA; Texas Children's Hospital and Houston Methodist Hospital, Houston, TX 77030, USA. Electronic address: goodell@bcm.edu.
authors: Margaret A
authors: MA
authors: Goodell
authors: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address: nakada@bcm.edu.
authors: Daisuke
authors: D
authors: Nakada
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004260:DNA Repair; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D055786:Gene Knockout Techniques; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D051379:Mice
chemical_list: D017493:Leukocyte Common Antigens
keywords: CRISPR/Cas9; HSC; gene therapy; genome editing; hematopoietic stem cells; homology-directed repair; human CD34; progenitor; sgRNA; transplantation