Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9
Abstract CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for no...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-91153-2 |
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| author | Seph M. Palomino Katherin A. Gabriel Juliet M. Mwirigi Anna Cervantes Peter Horton Geoffrey Funk Aubin Moutal Laurent F. Martin Rajesh Khanna Theodore J. Price Amol Patwardhan |
| author_facet | Seph M. Palomino Katherin A. Gabriel Juliet M. Mwirigi Anna Cervantes Peter Horton Geoffrey Funk Aubin Moutal Laurent F. Martin Rajesh Khanna Theodore J. Price Amol Patwardhan |
| author_sort | Seph M. Palomino |
| collection | DOAJ |
| description | Abstract CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases. |
| format | Article |
| id | doaj-art-26c3298eaff343a7acae9e8d2c153a33 |
| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-26c3298eaff343a7acae9e8d2c153a332025-08-20T03:07:41ZengNature PortfolioScientific Reports2045-23222025-04-0115111310.1038/s41598-025-91153-2Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9Seph M. Palomino0Katherin A. Gabriel1Juliet M. Mwirigi2Anna Cervantes3Peter Horton4Geoffrey Funk5Aubin Moutal6Laurent F. Martin7Rajesh Khanna8Theodore J. Price9Amol Patwardhan10Department of Anesthesiology and Pain Management, University of Texas Southwestern Medical CenterDepartment of Neuroscience and Center for Advanced Pain Studies, University of Texas at DallasDepartment of Neuroscience and Center for Advanced Pain Studies, University of Texas at DallasSouthwest Transplant AllianceSouthwest Transplant AllianceSouthwest Transplant AllianceDepartment of Pharmacology and Physiology, Saint Louis UniversityDepartment of Pharmacology, University of ArizonaDepartment of Pharmacology and Therapeutics, University of FloridaDepartment of Neuroscience and Center for Advanced Pain Studies, University of Texas at DallasDepartment of Anesthesiology and Pain Management, University of Texas Southwestern Medical CenterAbstract CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.https://doi.org/10.1038/s41598-025-91153-2 |
| spellingShingle | Seph M. Palomino Katherin A. Gabriel Juliet M. Mwirigi Anna Cervantes Peter Horton Geoffrey Funk Aubin Moutal Laurent F. Martin Rajesh Khanna Theodore J. Price Amol Patwardhan Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 Scientific Reports |
| title | Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 |
| title_full | Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 |
| title_fullStr | Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 |
| title_full_unstemmed | Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 |
| title_short | Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 |
| title_sort | genetic editing of primary human dorsal root ganglion neurons using crispr cas9 |
| url | https://doi.org/10.1038/s41598-025-91153-2 |
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