Journal
CELL STEM CELL
Volume 24, Issue 5, Pages 821-+Publisher
CELL PRESS
DOI: 10.1016/j.stem.2019.04.001
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Funding
- NRSA (NIH National Research Services Award) institutional predoctoral training (T32) grant
- institutional NIH postdoctoral T32 hematology training grant
- Danish Council for Independent Research, Medical Sciences [DFF-1333-00106B, DFF-1331-00735B]
- Amon Carter Foundation
- Taube Program in Neurodegenerative Research
- Children's Health Research Institute at Stanford
- Deutsche Forschungsgemeinschaft (DFG)
- Laurie Kraus Lacob Translational Scholar Fund
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Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describemarker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.
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