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

  • Cell Stem Cell. 2019 May 2;24(5):821-828.e5. doi: 10.1016/j.stem.2019.04.001.
Renata M Martin  1 ,  Kazuya Ikeda  1 ,  M Kyle Cromer  1 ,  Nobuko Uchida  2 ,  Toshinobu Nishimura  3 ,  Rosa Romano  1 ,  Andrew J Tong  1 ,  Viktor T Lemgart  1 ,  Joab Camarena  1 ,  Mara Pavel-Dinu  1 ,  Camille Sindhu  1 ,  Volker Wiebking  1 ,  Sriram Vaidyanathan  1 ,  Daniel P Dever  1 ,  Rasmus O Bak  1 ,  Anders Laustsen  4 ,  Benjamin J Lesch  1 ,  Martin R Jakobsen  4 ,  Vittorio Sebastiano  5 ,  Hiromitsu Nakauchi  3 ,  Matthew H Porteus  6
Affiliations
  • 1. Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • 2. ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA.
  • 3. Department of Genetics, Stanford University, Stanford, CA 94305, USA.
  • 4. Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark; Aarhus Research Centre of Innate Immunology, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark.
  • 5. Department of Obstetrics & Gynecology, Stanford University, Stanford, CA 94305, USA.
  • 6. Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: [email protected].
PMID: 31051134 DOI: 10.1016/j.stem.2019.04.001
Abstract

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.

Keywords
AAV6; CRISPR/Cas9; ESC; RNP; electroporation; gene targeting; genome editing; homology-directed repair; iPSC; sgRNA.