Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6

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DOI

  • Rasmus O Bak
  • Daniel P Dever, Department of Pediatrics, Stanford University, Stanford, United States.
  • ,
  • Andreas Reinisch, Department of Medicine, Division of Hematology, Stanford University, Stanford, United States.
  • ,
  • David Cruz Hernandez, Department of Medicine, Division of Hematology, Stanford University, Stanford, United States.
  • ,
  • Ravindra Majeti, Department of Medicine, Division of Hematology, Stanford University, Stanford, United States.
  • ,
  • Matthew H Porteus, Department of Pediatrics, Stanford University, Stanford, United States.

Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.

Original languageEnglish
JournaleLife
Volume6
ISSN2050-084X
DOIs
Publication statusPublished - 28 Sep 2017
Externally publishedYes

    Research areas

  • Journal Article

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