Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination
| Autor: | Nobuko Uchida, Martin R. Jakobsen, Anders Laustsen, Rosa Romano, Rasmus O. Bak, Volker Wiebking, Mara Pavel-Dinu, Toshinobu Nishimura, Hiromitsu Nakauchi, Camille Sindhu, Matthew H. Porteus, M. Kyle Cromer, Benjamin J. Lesch, Viktor T. Lemgart, Renata M. Martin, Daniel P. Dever, Andrew J. Tong, Vittorio Sebastiano, Sriram Vaidyanathan, Kazuya Ikeda, Joab Camarena |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Pluripotent Stem Cells
electroporation DNA Repair Genotype Genetic Vectors ESC Computational biology Biology gene targeting RNP Homology directed repair 03 medical and health sciences 0302 clinical medicine Gene Frequency Genome editing CRISPR-Associated Protein 9 Genetics Humans CRISPR genome editing Clustered Regularly Interspaced Short Palindromic Repeats Pathology Molecular Homologous Recombination Induced pluripotent stem cell Gene CRISPR/Cas9 030304 developmental biology Gene Editing 0303 health sciences iPSC Cas9 Gene targeting Cell Biology Dependovirus Tissue Donors homology-directed repair Molecular Medicine AAV6 sgRNA CRISPR-Cas Systems Stem cell Genetic Engineering 030217 neurology & neurosurgery |
| Zdroj: | Martin, R M, Ikeda, K, Cromer, M K, Uchida, N, Nishimura, T, Romano, R, Tong, A J, Lemgart, V T, Camarena, J, Pavel-Dinu, M, Sindhu, C, Wiebking, V, Vaidyanathan, S, Dever, D P, Bak, R O, Laustsen, A, Lesch, B J, Jakobsen, M R, Sebastiano, V, Nakauchi, H & Porteus, M H 2019, ' 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 . https://doi.org/10.1016/j.stem.2019.04.001 Martin, R M, Ikeda, K, Cromer, M K, Uchida, N, Nishimura, T, Romano, R, Tong, A J, Lemgart, V T, Camarena, J, Pavel-Dinu, M, Sindhu, C, Wiebking, V, Vaidyanathan, S, Dever, D P, Bak, R O, Laustsen, A, Lesch, B J, Jakobsen, M R, Sebastiano, V, Nakauchi, H & Porteus, M H 2019, ' 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, vol. 24, no. 5, pp. 821-828.e5 . https://doi.org/10.1016/j.stem.2019.04.001 |
| DOI: | 10.1016/j.stem.2019.04.001 |
| Popis: | Summary 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. |
| Databáze: | OpenAIRE |
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