Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones
Abstract Background CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such “disease in a dish” models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the hu...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-04-01
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| Series: | Stem Cell Research & Therapy |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13287-023-03327-2 |
| _version_ | 1797836631506419712 |
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| author | Elie Frank Michel Cailleret Constantin Nelep Pascal Fragner Jérome Polentes Elise Herardot Lina El Kassar Karine Giraud-Triboult Christelle Monville Karim Ben M’Barek |
| author_facet | Elie Frank Michel Cailleret Constantin Nelep Pascal Fragner Jérome Polentes Elise Herardot Lina El Kassar Karine Giraud-Triboult Christelle Monville Karim Ben M’Barek |
| author_sort | Elie Frank |
| collection | DOAJ |
| description | Abstract Background CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such “disease in a dish” models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious. Methods Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones. Results We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches. Conclusions This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening. Graphical Abstract |
| first_indexed | 2024-04-09T15:12:02Z |
| format | Article |
| id | doaj.art-a0978364e13741ee98c9f4e6a67499fa |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-04-09T15:12:02Z |
| publishDate | 2023-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-a0978364e13741ee98c9f4e6a67499fa2023-04-30T11:09:05ZengBMCStem Cell Research & Therapy1757-65122023-04-0114111410.1186/s13287-023-03327-2Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clonesElie Frank0Michel Cailleret1Constantin Nelep2Pascal Fragner3Jérome Polentes4Elise Herardot5Lina El Kassar6Karine Giraud-Triboult7Christelle Monville8Karim Ben M’Barek9INSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesALS Automated Lab Solutions GmbHINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesINSERM U861, I-Stem, AFM, Institute for Stem cell Therapy and Exploration of Monogenic DiseasesAbstract Background CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such “disease in a dish” models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious. Methods Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones. Results We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches. Conclusions This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening. Graphical Abstracthttps://doi.org/10.1186/s13287-023-03327-2CRISPR/Cas9Gene editionHuman pluripotent stem cellClonal isolation |
| spellingShingle | Elie Frank Michel Cailleret Constantin Nelep Pascal Fragner Jérome Polentes Elise Herardot Lina El Kassar Karine Giraud-Triboult Christelle Monville Karim Ben M’Barek Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones Stem Cell Research & Therapy CRISPR/Cas9 Gene edition Human pluripotent stem cell Clonal isolation |
| title | Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones |
| title_full | Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones |
| title_fullStr | Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones |
| title_full_unstemmed | Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones |
| title_short | Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones |
| title_sort | semi automated optimized method to isolate crispr cas9 edited human pluripotent stem cell clones |
| topic | CRISPR/Cas9 Gene edition Human pluripotent stem cell Clonal isolation |
| url | https://doi.org/10.1186/s13287-023-03327-2 |
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