{"doi":"10.1016/j.jbiotec.2025.09.012","title":"A next-generation platform for highly optimized CRISPR-mediated transcriptional repression","abstract":"CRISPR interference (CRISPRi), the fusion of transcriptional repressor domains to nuclease-inactive Cas9, is a powerful genetic tool enabling site-specific suppression of gene expression. However, its performance remains inconsistent across cell lines, gene targets, or single guide RNAs (sgRNAs) employed. This study described the development process of a novel, highly optimized CRISPRi repressor for mammalian gene regulation through a multi-pronged protein engineering approach: (1) truncating established domains, (2) characterizing candidate domains, (3) creating combinatorial domain fusion libraries, and (4) optimizing NLS configuration. First, by evaluating several truncations of MeCP2, a well-established repressor, we see that the ultra-compact NCoR/SMRT interaction domain (NID) significantly enhances CRISPRi gene knockdown performance, exceeding levels observed with canonical MeCP2 subdomains by an average of ∼40 %. Incorporating this optimized MeCP2 NID truncation with a diverse panel of authenticated repressor domains, we next assemble and screen combinatorial multi-domain libraries, discovering four new repressor fusions. Upon follow-up nuclear localization signal (NLS) configuration analysis, we see that affixing one carboxy-terminal NLS enhances gene knockdown efficiency of the repressors by an average of ∼50 %. Through rigorous validation of NLS-tagged repressor fusions across several cell lines, multiple sgRNA targets, and genome-wide dropout screens, we establish that our strongest system, dCas9-ZIM3-NID-MXD1-NLS, achieves superior gene silencing capabilities over alternative CRISPRi platforms. In addition to developing dCas9-ZIM3-NID-MXD1-NLS, a uniquely potent transcriptional repressor, we envision that the multi-domain engineering approach utilized in this study will be valuable framework enabling future strides in CRISPR platform development.","journal":"Journal of Biotechnology","year":2025,"id":575888,"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":0.9528,"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":1369961,"name":"Krithika Karunakaran","orcid":null,"position":1,"is_corresponding":false},{"id":1372745,"name":"Yann Ferry","orcid":null,"position":2,"is_corresponding":false},{"id":1369962,"name":"Paula Mizote","orcid":null,"position":3,"is_corresponding":false},{"id":1080929,"name":"C. Allen","orcid":"0000-0002-6285-8841","position":4,"is_corresponding":false},{"id":1369963,"name":"Sophie Briggs","orcid":null,"position":5,"is_corresponding":false},{"id":590503,"name":"John Blazeck","orcid":"0000-0002-6110-2938","position":6,"is_corresponding":false},{"id":1369960,"name":"Andrew Kristof","orcid":null,"position":0,"is_corresponding":true}],"reference_count":43,"raw_metadata":null,"created_at":"2026-07-19T02:57:52.712371Z","pmid":"41005541","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":[]}