{"doi":"10.1016/j.xpro.2025.103831","title":"Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function","abstract":"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 • Steps for designing and cloning dual U6gRNA cassettes to delete a specific CRE • Instructions for optimized retrovirus production and transduction into CD4 + T cells • Guidance on Th17 differentiation and confirmation of CRE deletion in cultured T cells • Procedures for adoptive transfer of CRISPR-edited Th17 cells to assess in vivo function Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.","journal":"STAR Protocols","year":2025,"id":566384,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9502,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1047476,"name":"Hongmin Wu","orcid":"0000-0002-0762-8322","position":1,"is_corresponding":false},{"id":1311627,"name":"Guanpeng Wang","orcid":null,"position":2,"is_corresponding":false},{"id":378708,"name":"Zuoming Sun","orcid":"0000-0003-1896-6666","position":3,"is_corresponding":false},{"id":861624,"name":"Xiancai Zhong","orcid":"0000-0001-8312-4057","position":0,"is_corresponding":true}],"reference_count":13,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:56:36.440192Z","pmid":"40397576","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}