Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader
CRISPR-Cas9-based genome editors can precisely target and edit genes efficiently. However, prolonged Cas9 activity poses challenges for laboratory experiments and raises safety concerns for therapeutic applications due to unintended consequences such as off-target editing, genotoxicity, immunogenici...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-09-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253125001945 |
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| author | Namita Khajanchi Vrusha Patel Ronak Dua Meha Kabra Bikash R. Pattnaik Krishanu Saha |
| author_facet | Namita Khajanchi Vrusha Patel Ronak Dua Meha Kabra Bikash R. Pattnaik Krishanu Saha |
| author_sort | Namita Khajanchi |
| collection | DOAJ |
| description | CRISPR-Cas9-based genome editors can precisely target and edit genes efficiently. However, prolonged Cas9 activity poses challenges for laboratory experiments and raises safety concerns for therapeutic applications due to unintended consequences such as off-target editing, genotoxicity, immunogenicity, and undesired on-target modifications. Here, we evaluate a novel molecular glue degradation system, called Cas9-degron (Cas9-d), designed to degrade Cas9 in the presence of the US Food and Drug Administration (FDA)-approved drug, pomalidomide (POM). This system is highly biocompatible and rapidly reduces Cas9 protein levels within 4 h of induction, resulting in a 3- to 5-fold decrease in editing at on-target sites. The reduction is reversible, as Cas9 levels are restored within 24 h after POM withdrawal. Without initiating degradation, the on-target editing efficiency and accuracy of the Cas9-d system remain intact in different human cell types, including hepatic cell lines and human induced pluripotent stem cell (hiPSC)-derived GABAergic neurons. Cells edited with the Cas9-d system were healthy and functional, exhibiting minimal toxicity from using the strategy. The Cas9-d system provides a versatile approach to adjust Cas9 levels, demonstrating its potential as an experimental tool for controlling genome editing outcomes in vitro and ex vivo. With further development, it holds promise for enhancing somatic cell genome editing in vivo. |
| format | Article |
| id | doaj-art-fd177b20c8f1428582473fdb3c93d752 |
| institution | Kabale University |
| issn | 2162-2531 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-fd177b20c8f1428582473fdb3c93d7522025-08-20T03:42:02ZengElsevierMolecular Therapy: Nucleic Acids2162-25312025-09-0136310264010.1016/j.omtn.2025.102640Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degraderNamita Khajanchi0Vrusha Patel1Ronak Dua2Meha Kabra3Bikash R. Pattnaik4Krishanu Saha5Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USADepartment of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA; Department of Integrative Biology, University of Wisconsin-Madison, Madison, WI 53706, USADepartment of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USADepartment of Pediatrics, University of Wisconsin-Madison, Madison, WI 53792, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USADepartment of Pediatrics, University of Wisconsin-Madison, Madison, WI 53792, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53705, USADepartment of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53792, USA; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA; Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; Corresponding author: Krishanu Saha, Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA.CRISPR-Cas9-based genome editors can precisely target and edit genes efficiently. However, prolonged Cas9 activity poses challenges for laboratory experiments and raises safety concerns for therapeutic applications due to unintended consequences such as off-target editing, genotoxicity, immunogenicity, and undesired on-target modifications. Here, we evaluate a novel molecular glue degradation system, called Cas9-degron (Cas9-d), designed to degrade Cas9 in the presence of the US Food and Drug Administration (FDA)-approved drug, pomalidomide (POM). This system is highly biocompatible and rapidly reduces Cas9 protein levels within 4 h of induction, resulting in a 3- to 5-fold decrease in editing at on-target sites. The reduction is reversible, as Cas9 levels are restored within 24 h after POM withdrawal. Without initiating degradation, the on-target editing efficiency and accuracy of the Cas9-d system remain intact in different human cell types, including hepatic cell lines and human induced pluripotent stem cell (hiPSC)-derived GABAergic neurons. Cells edited with the Cas9-d system were healthy and functional, exhibiting minimal toxicity from using the strategy. The Cas9-d system provides a versatile approach to adjust Cas9 levels, demonstrating its potential as an experimental tool for controlling genome editing outcomes in vitro and ex vivo. With further development, it holds promise for enhancing somatic cell genome editing in vivo.http://www.sciencedirect.com/science/article/pii/S2162253125001945MT: RNA/DNA Editinggene therapyCRISPRcell therapyCas9degron |
| spellingShingle | Namita Khajanchi Vrusha Patel Ronak Dua Meha Kabra Bikash R. Pattnaik Krishanu Saha Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader Molecular Therapy: Nucleic Acids MT: RNA/DNA Editing gene therapy CRISPR cell therapy Cas9 degron |
| title | Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader |
| title_full | Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader |
| title_fullStr | Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader |
| title_full_unstemmed | Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader |
| title_short | Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader |
| title_sort | controlling crispr cas9 genome editing in human cells using a molecular glue degrader |
| topic | MT: RNA/DNA Editing gene therapy CRISPR cell therapy Cas9 degron |
| url | http://www.sciencedirect.com/science/article/pii/S2162253125001945 |
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