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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MDPI AG
2023-02-01
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| Online Access: | https://www.mdpi.com/2073-4425/14/2/401 |
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| author | Valérie Erbs Romain Lorentz Benjamin Eisenman Laurence Schaeffer Laurence Luppi Loic Lindner Yann Hérault Guillaume Pavlovic Marie Wattenhofer-Donzé Marie-Christine Birling |
| author_facet | Valérie Erbs Romain Lorentz Benjamin Eisenman Laurence Schaeffer Laurence Luppi Loic Lindner Yann Hérault Guillaume Pavlovic Marie Wattenhofer-Donzé Marie-Christine Birling |
| author_sort | Valérie Erbs |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-11T08:47:17Z |
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| id | doaj.art-c7f412b873e24e81a40e073edcd96636 |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-11T08:47:17Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-c7f412b873e24e81a40e073edcd966362023-11-16T20:42:28ZengMDPI AGGenes2073-44252023-02-0114240110.3390/genes14020401Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES CellsValérie Erbs0Romain Lorentz1Benjamin Eisenman2Laurence Schaeffer3Laurence Luppi4Loic Lindner5Yann Hérault6Guillaume Pavlovic7Marie Wattenhofer-Donzé8Marie-Christine Birling9CNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceCNRS, INSERM, Université de Strasbourg, CELPHEDIA, PHENOMIN-Institut Clinique de la Souris (ICS), 1 rue Laurent Fries, 67404 Illkirch Graffenstaden, FranceThe 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.https://www.mdpi.com/2073-4425/14/2/401gene targetingdouble-strand breakembryonic stem cellsgenome editingCRISPR/Cas9homologous recombination |
| spellingShingle | Valérie Erbs Romain Lorentz Benjamin Eisenman Laurence Schaeffer Laurence Luppi Loic Lindner Yann Hérault Guillaume Pavlovic Marie Wattenhofer-Donzé Marie-Christine Birling Increased On-Target Rate and Risk of Concatemerization after CRISPR-Enhanced Targeting in ES Cells Genes gene targeting double-strand break embryonic stem cells genome editing CRISPR/Cas9 homologous recombination |
| 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 | gene targeting double-strand break embryonic stem cells genome editing CRISPR/Cas9 homologous recombination |
| url | https://www.mdpi.com/2073-4425/14/2/401 |
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