Institut for Biomedicin

Rasmus Bak

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

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

DOI

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

OriginalsprogEngelsk
TidsskriftCell Stem Cell
Vol/bind24
Nummer5
Sider (fra-til)821-828.e5
Antal sider13
ISSN1066-5099
DOI
StatusUdgivet - 2 maj 2019

Bibliografisk note

Copyright © 2019. Published by Elsevier Inc.

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