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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.
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.
Afiliación
  • Martin RM; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Ikeda K; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Cromer MK; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Uchida N; ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA.
  • Nishimura T; Department of Genetics, Stanford University, Stanford, CA 94305, USA.
  • Romano R; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Tong AJ; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Lemgart VT; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Camarena J; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Pavel-Dinu M; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Sindhu C; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Wiebking V; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Vaidyanathan S; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Dever DP; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Bak RO; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Laustsen A; 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.
  • Lesch BJ; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
  • Jakobsen MR; 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.
  • Sebastiano V; Department of Obstetrics & Gynecology, Stanford University, Stanford, CA 94305, USA.
  • Nakauchi H; Department of Genetics, Stanford University, Stanford, CA 94305, USA.
  • Porteus MH; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: mporteus@stanford.edu.
Cell Stem Cell ; 24(5): 821-828.e5, 2019 05 02.
Article en En | MEDLINE | ID: mdl-31051134
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Dependovirus / Células Madre Pluripotentes / Sistemas CRISPR-Cas / Proteína 9 Asociada a CRISPR / Genotipo Tipo de estudio: Diagnostic_studies Límite: Humans Idioma: En Revista: Cell Stem Cell Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos
Texto completo
Añadir a Mi BVS
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Dependovirus / Células Madre Pluripotentes / Sistemas CRISPR-Cas / Proteína 9 Asociada a CRISPR / Genotipo Tipo de estudio: Diagnostic_studies Límite: Humans Idioma: En Revista: Cell Stem Cell Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos