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Wildtype heterogeneity contributes to clonal variability in genome edited cells

Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among differen...

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Autores principales: Westermann, Lukas, Li, Yong, Göcmen, Burulca, Niedermoser, Matthias, Rhein, Kilian, Jahn, Johannes, Cascante, Isabel, Schöler, Felix, Moser, Niklas, Neubauer, Björn, Hofherr, Alexis, Behrens, Yvonne Lisa, Göhring, Gudrun, Köttgen, Anna, Köttgen, Michael, Busch, Tilman
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9616811/
https://www.ncbi.nlm.nih.gov/pubmed/36307508
http://dx.doi.org/10.1038/s41598-022-22885-8
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author Westermann, Lukas
Li, Yong
Göcmen, Burulca
Niedermoser, Matthias
Rhein, Kilian
Jahn, Johannes
Cascante, Isabel
Schöler, Felix
Moser, Niklas
Neubauer, Björn
Hofherr, Alexis
Behrens, Yvonne Lisa
Göhring, Gudrun
Köttgen, Anna
Köttgen, Michael
Busch, Tilman
author_facet Westermann, Lukas
Li, Yong
Göcmen, Burulca
Niedermoser, Matthias
Rhein, Kilian
Jahn, Johannes
Cascante, Isabel
Schöler, Felix
Moser, Niklas
Neubauer, Björn
Hofherr, Alexis
Behrens, Yvonne Lisa
Göhring, Gudrun
Köttgen, Anna
Köttgen, Michael
Busch, Tilman
author_sort Westermann, Lukas
collection PubMed
description Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype–phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.
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spelling pubmed-96168112022-10-30 Wildtype heterogeneity contributes to clonal variability in genome edited cells Westermann, Lukas Li, Yong Göcmen, Burulca Niedermoser, Matthias Rhein, Kilian Jahn, Johannes Cascante, Isabel Schöler, Felix Moser, Niklas Neubauer, Björn Hofherr, Alexis Behrens, Yvonne Lisa Göhring, Gudrun Köttgen, Anna Köttgen, Michael Busch, Tilman Sci Rep Article Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype–phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility. Nature Publishing Group UK 2022-10-28 /pmc/articles/PMC9616811/ /pubmed/36307508 http://dx.doi.org/10.1038/s41598-022-22885-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Westermann, Lukas
Li, Yong
Göcmen, Burulca
Niedermoser, Matthias
Rhein, Kilian
Jahn, Johannes
Cascante, Isabel
Schöler, Felix
Moser, Niklas
Neubauer, Björn
Hofherr, Alexis
Behrens, Yvonne Lisa
Göhring, Gudrun
Köttgen, Anna
Köttgen, Michael
Busch, Tilman
Wildtype heterogeneity contributes to clonal variability in genome edited cells
title Wildtype heterogeneity contributes to clonal variability in genome edited cells
title_full Wildtype heterogeneity contributes to clonal variability in genome edited cells
title_fullStr Wildtype heterogeneity contributes to clonal variability in genome edited cells
title_full_unstemmed Wildtype heterogeneity contributes to clonal variability in genome edited cells
title_short Wildtype heterogeneity contributes to clonal variability in genome edited cells
title_sort wildtype heterogeneity contributes to clonal variability in genome edited cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9616811/
https://www.ncbi.nlm.nih.gov/pubmed/36307508
http://dx.doi.org/10.1038/s41598-022-22885-8
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