Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function
Summary: Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in m...
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| Language: | English |
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Elsevier
2025-06-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725002370 |
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| author | Xiancai Zhong Hongmin Wu Guanpeng Wang Zuoming Sun |
| author_facet | Xiancai Zhong Hongmin Wu Guanpeng Wang Zuoming Sun |
| author_sort | Xiancai Zhong |
| collection | DOAJ |
| description | Summary: Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction, preparation of retroviruses, viral delivery, and Th17 differentiation. We then detail procedures for in vivo functionality analysis.For complete details on the use and execution of this protocol, please refer to Zhong et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-0769e323575145dd8d918b9e2e0e3d3d |
| institution | DOAJ |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-0769e323575145dd8d918b9e2e0e3d3d2025-08-20T03:08:20ZengElsevierSTAR Protocols2666-16672025-06-016210383110.1016/j.xpro.2025.103831Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector functionXiancai Zhong0Hongmin Wu1Guanpeng Wang2Zuoming Sun3Department of Immunology & Theranostics, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USADepartment of Immunology & Theranostics, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USADepartment of Immunology & Theranostics, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USADepartment of Immunology & Theranostics, Arthur Riggs Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA; Corresponding authorSummary: Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction, preparation of retroviruses, viral delivery, and Th17 differentiation. We then detail procedures for in vivo functionality analysis.For complete details on the use and execution of this protocol, please refer to Zhong et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166725002370ImmunologyMolecular BiologyCRISPR |
| spellingShingle | Xiancai Zhong Hongmin Wu Guanpeng Wang Zuoming Sun Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function STAR Protocols Immunology Molecular Biology CRISPR |
| title | Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function |
| title_full | Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function |
| title_fullStr | Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function |
| title_full_unstemmed | Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function |
| title_short | Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function |
| title_sort | protocol for crispr mediated deletion of cis regulatory element in murine th17 cells for in vivo assessment of effector function |
| topic | Immunology Molecular Biology CRISPR |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725002370 |
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