- Martin, Renata M;
- Ikeda, Kazuya;
- Cromer, M Kyle;
- Uchida, Nobuko;
- Nishimura, Toshinobu;
- Romano, Rosa;
- Tong, Andrew J;
- Lemgart, Viktor T;
- Camarena, Joab;
- Pavel-Dinu, Mara;
- Sindhu, Camille;
- Wiebking, Volker;
- Vaidyanathan, Sriram;
- Dever, Daniel P;
- Bak, Rasmus O;
- Laustsen, Anders;
- Lesch, Benjamin J;
- Jakobsen, Martin R;
- Sebastiano, Vittorio;
- Nakauchi, Hiromitsu;
- Porteus, Matthew H
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.