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Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Renata M Martin, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Kazuya Ikeda, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • M Kyle Cromer, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Nobuko Uchida, ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA.
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  • Toshinobu Nishimura, Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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  • Rosa Romano, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Andrew J Tong, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Viktor T Lemgart, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Joab Camarena, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Mara Pavel-Dinu, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Camille Sindhu, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Volker Wiebking, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Sriram Vaidyanathan, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Daniel P Dever, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Rasmus O Bak
  • Anders Laustsen
  • Benjamin J Lesch, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
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  • Martin R Jakobsen
  • Vittorio Sebastiano, Department of Obstetrics & Gynecology, Stanford University, Stanford, CA 94305, USA.
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  • Hiromitsu Nakauchi, Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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  • Matthew H Porteus, Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: mporteus@stanford.edu.

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 describe marker-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.

Original languageEnglish
JournalCell Stem Cell
Volume24
Issue5
Pages (from-to)821-828.e5
Number of pages13
ISSN1066-5099
DOIs
Publication statusPublished - 2 May 2019

    Research areas

  • AAV6, CRISPR/Cas9, ESC, RNP, electroporation, gene targeting, genome editing, homology-directed repair, iPSC, sgRNA

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