{"doi":"10.1016/j.xpro.2025.104164","title":"Protocol to rapidly verify silent gene reporter in human pluripotent stem cells using CRISPR activation","abstract":"Validating human pluripotent stem cell (hPSC) reporters targeting silent genes typically requires inducing gene expression through cell state transitions, which can be time consuming and complex. Here, we present a rapid workflow to verify reporter knockins at unexpressed loci in hPSCs using CRISPR-mediated transcriptional activation (CRISPRa). We detail steps for designing and cloning single-guide RNA (sgRNA), delivery of CRISPRa into reporter cells, and detection of reporter gene. In this protocol, we illustrate this process using KLF17-GFP reporter hPSCs. For complete details on the use and execution of this protocol, please refer to Wu et al. 1 • Steps for design and cloning of guide RNAs targeting promoters of genes of interest • Procedures for activating silent reporters in hPSCs using the SAM-TET1 CRISPRa system • Guidance for detecting reporter gene expression within 48 h Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Validating human pluripotent stem cell (hPSC) reporters targeting silent genes typically requires inducing gene expression through cell state transitions, which can be time consuming and complex. Here, we present a rapid workflow to verify reporter knockins at unexpressed loci in hPSCs using CRISPR-mediated transcriptional activation (CRISPRa). We detail steps for designing and cloning single-guide RNA (sgRNA), delivery of CRISPRa into reporter cells, and detection of reporter gene. In this protocol, we illustrate this process using KLF17-GFP reporter hPSCs.","journal":"STAR Protocols","year":2025,"id":547980,"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":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9587,"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":510340,"name":"Aaron Zhong","orcid":"0000-0002-2468-0923","position":1,"is_corresponding":false},{"id":1241829,"name":"Bernny Ramirez","orcid":null,"position":2,"is_corresponding":false},{"id":1441213,"name":"Su Su Wai","orcid":null,"position":3,"is_corresponding":false},{"id":291183,"name":"Ting Zhou","orcid":"0000-0003-1423-1507","position":4,"is_corresponding":false},{"id":1241337,"name":"Youjun Wu","orcid":"0000-0002-3557-1159","position":0,"is_corresponding":true}],"reference_count":13,"raw_metadata":null,"created_at":"2026-07-19T02:53:53.962932Z","pmid":"41201092","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":[]}