Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
2016; Cell Press; Volume: 17; Issue: 5 Linguagem: Inglês
10.1016/j.celrep.2016.09.092
ISSN2639-1856
AutoresMichael C. Gundry, Lorenzo Brunetti, Angelique Lin, Allison Mayle, Ayumi Kitano, Dimitrios L. Wagner, Joanne I. Hsu, Kevin A. Hoegenauer, Cliona M. Rooney, Margaret A. Goodell, Daisuke Nakada,
Tópico(s)Virus-based gene therapy research
ResumoHighlights•Gene knockout of primary murine HSPCs with efficiencies routinely higher than 60%•Gene knockout of primary human HSPCs and T cells from 75% to 90%•CRISPR/Cas9 homology-directed repair in >20% of primary human HSPCsSummaryOur 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.Graphical abstract
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