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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...

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Autores principales: Erbs, Valérie, Lorentz, Romain, Eisenman, Benjamin, Schaeffer, Laurence, Luppi, Laurence, Lindner, Loic, Hérault, Yann, Pavlovic, Guillaume, Wattenhofer-Donzé, Marie, Birling, Marie-Christine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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.
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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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