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Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization
We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major ce...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638588/ https://www.ncbi.nlm.nih.gov/pubmed/28814507 http://dx.doi.org/10.1091/mbc.E17-03-0209 |
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author | Roberts, Brock Haupt, Amanda Tucker, Andrew Grancharova, Tanya Arakaki, Joy Fuqua, Margaret A. Nelson, Angelique Hookway, Caroline Ludmann, Susan A. Mueller, Irina A. Yang, Ruian Horwitz, Rick Rafelski, Susanne M. Gunawardane, Ruwanthi N. |
author_facet | Roberts, Brock Haupt, Amanda Tucker, Andrew Grancharova, Tanya Arakaki, Joy Fuqua, Margaret A. Nelson, Angelique Hookway, Caroline Ludmann, Susan A. Mueller, Irina A. Yang, Ruian Horwitz, Rick Rafelski, Susanne M. Gunawardane, Ruwanthi N. |
author_sort | Roberts, Brock |
collection | PubMed |
description | We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1–4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line–generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. |
format | Online Article Text |
id | pubmed-5638588 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-56385882017-12-30 Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization Roberts, Brock Haupt, Amanda Tucker, Andrew Grancharova, Tanya Arakaki, Joy Fuqua, Margaret A. Nelson, Angelique Hookway, Caroline Ludmann, Susan A. Mueller, Irina A. Yang, Ruian Horwitz, Rick Rafelski, Susanne M. Gunawardane, Ruwanthi N. Mol Biol Cell Articles We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1–4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line–generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The American Society for Cell Biology 2017-10-15 /pmc/articles/PMC5638588/ /pubmed/28814507 http://dx.doi.org/10.1091/mbc.E17-03-0209 Text en © 2017 Roberts, Haupt, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. |
spellingShingle | Articles Roberts, Brock Haupt, Amanda Tucker, Andrew Grancharova, Tanya Arakaki, Joy Fuqua, Margaret A. Nelson, Angelique Hookway, Caroline Ludmann, Susan A. Mueller, Irina A. Yang, Ruian Horwitz, Rick Rafelski, Susanne M. Gunawardane, Ruwanthi N. Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization |
title | Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization |
title_full | Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization |
title_fullStr | Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization |
title_full_unstemmed | Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization |
title_short | Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization |
title_sort | systematic gene tagging using crispr/cas9 in human stem cells to illuminate cell organization |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638588/ https://www.ncbi.nlm.nih.gov/pubmed/28814507 http://dx.doi.org/10.1091/mbc.E17-03-0209 |
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