{"doi":"10.7554/elife.81086","title":"Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation","abstract":"Targeted selection-based genome-editing approaches have enabled many fundamental discoveries and are used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette in budding yeast led to reduced expression and function for the adjacent gene, MRP51 , despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all mutant phenotypes detected in cells deleted for DBP1 . This behavior resembled the ‘neighboring gene effect’ (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target mutant phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that the inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). Divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette-induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests it should be considered in the design and interpretation of experiments using integrated expression cassettes in other eukaryotic systems, including human cells.","journal":"eLife","year":2022,"id":257042,"datarank":0.7029299519643109,"base_score":2.995732273553991,"endowment":2.995732273553991,"self_citation_contribution":0.4493598410330987,"citation_network_contribution":0.2535701109312122,"self_endowment_contribution":0.4493598410330987,"citer_contribution":0.2535701109312122,"corpus_percentile":null,"corpus_rank":null,"citation_count":19,"citer_count":13,"citers_with_citation_signal":9,"citers_with_endowment":9,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9507,"is_data_producer":true,"deposit_databanks":{"GEO":["GSE207267","GSE207189","GSE121189","GSE111255"]},"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":895095,"name":"Charlene Chan","orcid":"0000-0002-0479-3373","position":1,"is_corresponding":false},{"id":576850,"name":"Ella Doron‐Mandel","orcid":"0000-0002-2519-5938","position":2,"is_corresponding":false},{"id":815866,"name":"Lidia Llacsahuanga Allcca","orcid":null,"position":3,"is_corresponding":false},{"id":554829,"name":"Jenny Kim Kim","orcid":null,"position":4,"is_corresponding":false},{"id":17835,"name":"Marko Jovanovic","orcid":"0000-0001-9707-3377","position":5,"is_corresponding":false},{"id":63413,"name":"Gloria A. Brar","orcid":"0000-0002-8560-9581","position":6,"is_corresponding":false},{"id":519369,"name":"Emily Nicole Powers","orcid":"0000-0003-4497-7318","position":0,"is_corresponding":true}],"reference_count":99,"raw_metadata":null,"created_at":"2026-07-19T00:25:30.183987Z","pmid":"36503721","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":[]}