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Cell Rep. 2016 Oct 25;17(5):1453-1461. doi: 10.1016/j.celrep.2016.09.092.

Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9.

Author information

1
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.
2
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.
3
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.
4
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
5
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.
6
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.
7
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.
8
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.
9
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.

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.

KEYWORDS:

CRISPR/Cas9; HSC; gene therapy; genome editing; hematopoietic stem cells; homology-directed repair; human CD34; progenitor; sgRNA; transplantation

PMID:
27783956
PMCID:
PMC5087995
DOI:
10.1016/j.celrep.2016.09.092
[Indexed for MEDLINE]
Free PMC Article

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