Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2017
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
_version_ 1783270756927406080
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
work_keys_str_mv AT robertsbrock systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT hauptamanda systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT tuckerandrew systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT grancharovatanya systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT arakakijoy systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT fuquamargareta systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT nelsonangelique systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT hookwaycaroline systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT ludmannsusana systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT muelleririnaa systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT yangruian systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT horwitzrick systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT rafelskisusannem systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization
AT gunawardaneruwanthin systematicgenetaggingusingcrisprcas9inhumanstemcellstoilluminatecellorganization