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Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells
The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for ‘à la carte’ mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to targ...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9957269/ https://www.ncbi.nlm.nih.gov/pubmed/36833328 http://dx.doi.org/10.3390/genes14020401 |
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author | Erbs, Valérie Lorentz, Romain Eisenman, Benjamin Schaeffer, Laurence Luppi, Laurence Lindner, Loic Hérault, Yann Pavlovic, Guillaume Wattenhofer-Donzé, Marie Birling, Marie-Christine |
author_facet | Erbs, Valérie Lorentz, Romain Eisenman, Benjamin Schaeffer, Laurence Luppi, Laurence Lindner, Loic Hérault, Yann Pavlovic, Guillaume Wattenhofer-Donzé, Marie Birling, Marie-Christine |
author_sort | Erbs, Valérie |
collection | PubMed |
description | The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for ‘à la carte’ mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5′ and 3′ PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes. |
format | Online Article Text |
id | pubmed-9957269 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-99572692023-02-25 Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells Erbs, Valérie Lorentz, Romain Eisenman, Benjamin Schaeffer, Laurence Luppi, Laurence Lindner, Loic Hérault, Yann Pavlovic, Guillaume Wattenhofer-Donzé, Marie Birling, Marie-Christine Genes (Basel) Article The French mouse clinic (Institut Clinique de la Souris; ICS) has produced more than 2000 targeting vectors for ‘à la carte’ mutagenesis in C57BL/6N mice. Although most of the vectors were used successfully for homologous recombination in murine embryonic stem cells (ESCs), a few have failed to target a specific locus after several attempts. We show here that co-electroporation of a CRISPR plasmid with the same targeting construct as the one that failed previously allows the systematic achievement of positive clones. A careful validation of these clones is, however, necessary as a significant number of clones (but not all) show a concatemerization of the targeting plasmid at the locus. A detailed Southern blot analysis permitted characterization of the nature of these events as standard long-range 5′ and 3′ PCRs were not able to distinguish between correct and incorrect alleles. We show that a simple and inexpensive PCR performed prior to ESC amplification allows detection and elimination of those clones with concatemers. Finally, although we only tested murine ESCs, our results highlight the risk of mis-validation of any genetically modified cell line (such as established lines, induced pluripotent stem cells or those used for ex vivo gene therapy) that combines the use of CRISPR/Cas9 and a circular double-stranded donor. We strongly advise the CRISPR community to perform a Southern blot with internal probes when using CRISPR to enhance homologous recombination in any cell type, including fertilized oocytes. MDPI 2023-02-03 /pmc/articles/PMC9957269/ /pubmed/36833328 http://dx.doi.org/10.3390/genes14020401 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Erbs, Valérie Lorentz, Romain Eisenman, Benjamin Schaeffer, Laurence Luppi, Laurence Lindner, Loic Hérault, Yann Pavlovic, Guillaume Wattenhofer-Donzé, Marie Birling, Marie-Christine Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells |
title | Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells |
title_full | Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells |
title_fullStr | Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells |
title_full_unstemmed | Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells |
title_short | Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells |
title_sort | increased on-target rate and risk of concatemerization after crispr-enhanced targeting in es cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9957269/ https://www.ncbi.nlm.nih.gov/pubmed/36833328 http://dx.doi.org/10.3390/genes14020401 |
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