Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening
Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluor...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-04-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/27/8/2434 |
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| author | Kinga Vojnits Mio Nakanishi Deanna Porras Yeonjoon Kim Zhuohang Feng Diana Golubeva Mick Bhatia |
| author_facet | Kinga Vojnits Mio Nakanishi Deanna Porras Yeonjoon Kim Zhuohang Feng Diana Golubeva Mick Bhatia |
| author_sort | Kinga Vojnits |
| collection | DOAJ |
| description | Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes. |
| first_indexed | 2024-03-09T10:30:47Z |
| format | Article |
| id | doaj.art-113796d73917474782fdeb01cc581c96 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T10:30:47Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-113796d73917474782fdeb01cc581c962023-12-01T21:15:55ZengMDPI AGMolecules1420-30492022-04-01278243410.3390/molecules27082434Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content ScreeningKinga Vojnits0Mio Nakanishi1Deanna Porras2Yeonjoon Kim3Zhuohang Feng4Diana Golubeva5Mick Bhatia6Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, CanadaDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, CanadaDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, CanadaDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, CanadaDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, CanadaDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, CanadaDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON L8N 3Z5, CanadaApplication of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.https://www.mdpi.com/1420-3049/27/8/2434genome editingCRISPR/Cas9OCT4 locusAAVS1 locushuman pluripotent stem cellsEGFP reporter lines |
| spellingShingle | Kinga Vojnits Mio Nakanishi Deanna Porras Yeonjoon Kim Zhuohang Feng Diana Golubeva Mick Bhatia Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening Molecules genome editing CRISPR/Cas9 OCT4 locus AAVS1 locus human pluripotent stem cells EGFP reporter lines |
| title | Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening |
| title_full | Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening |
| title_fullStr | Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening |
| title_full_unstemmed | Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening |
| title_short | Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening |
| title_sort | developing crispr cas9 mediated fluorescent reporter human pluripotent stem cell lines for high content screening |
| topic | genome editing CRISPR/Cas9 OCT4 locus AAVS1 locus human pluripotent stem cells EGFP reporter lines |
| url | https://www.mdpi.com/1420-3049/27/8/2434 |
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