Abstract:
: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.
journal_name
Elifejournal_title
eLifeauthors
Bak RO,Dever DP,Reinisch A,Cruz Hernandez D,Majeti R,Porteus MHdoi
10.7554/eLife.27873subject
Has Abstractpub_date
2017-09-28 00:00:00issn
2050-084Xpii
27873journal_volume
6pub_type
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