{"doi":"10.1074/jbc.ra120.015386","title":"Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity","abstract":"RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both). RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both). Type 3 RNA polymerase (Pol III) promoters, such as 7SK, U6, and H1, are popular for the expression of small noncoding RNAs because of their robust level of transcription in all cell types and defined transcription initiation and termination sites, such that a precise (therapeutic) transcript is made (1Kole R. Krainer A.R. Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat. Rev. Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (852) Google Scholar, 2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (448) Google Scholar). These promoters are unique in that all critical elements are located upstream of the transcriptional initiation site, thus enabling the expression of almost any small RNA sequence, including siRNA or shRNA for RNAi purposes, and guide RNA for CRISPR-mediated genome editing applications (1Kole R. Krainer A.R. Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat. Rev. Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (852) Google Scholar, 2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (448) Google Scholar, 3Fellmann C. Lowe S.W. Stable RNA interference rules for silencing.Nat. Cell Biol. 2014; 16: 10-18Crossref PubMed Scopus (140) Google Scholar, 4Kabadi A.M. Ousterout D.G. Hilton I.B. Gersbach C.A. Multiplex CRISPR/Cas9-based genome engineering from a single lentivira","journal":"Journal of Biological Chemistry","year":2020,"id":71249,"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":18,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9483,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":377777,"name":"Yme U. van der Velden","orcid":null,"position":1,"is_corresponding":false},{"id":279772,"name":"Ming-Hui Fan","orcid":null,"position":2,"is_corresponding":false},{"id":377778,"name":"Cynthia A. van der Linden","orcid":null,"position":3,"is_corresponding":false},{"id":377779,"name":"Monique Vink","orcid":null,"position":4,"is_corresponding":false},{"id":277330,"name":"Elena Herrera-Carrillo","orcid":"0000-0001-9986-8552","position":5,"is_corresponding":false},{"id":277331,"name":"Ben Berkhout","orcid":"0000-0002-1905-8486","position":6,"is_corresponding":false},{"id":277328,"name":"Zongliang Gao","orcid":"0000-0002-8257-8986","position":0,"is_corresponding":true}],"reference_count":23,"raw_metadata":null,"created_at":"2026-07-18T21:43:57.004133Z","pmid":"33154168","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":[]}