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Cell Rep ; 17(5): 1453-1461, 2016 10 25.
Article in English | MEDLINE | ID: mdl-27783956

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.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Hematopoietic Stem Cells/metabolism , Animals , DNA Repair , Gene Deletion , Gene Knockout Techniques , Humans , Leukocyte Common Antigens/metabolism , Mice
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