{"doi":"10.1101/2022.11.14.516516","title":"An RNA interference (RNAi) toolkit and its utility for functional genetic analysis of <i>Leishmania (Viannia)</i>","abstract":"ABSTRACT RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia , and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis . We developed vectors facilitating generation of long-hairpin or “stem-loop” (StL) RNAi knockdown constructs, using Gateway ™ site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as QDPR , a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation ( A600 ), and essential genes of the intraflagellar transport ( IFT ) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyltransferase ( HGPRT ) and reporter firefly luciferase ( LUC ) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful ‘hypomorphic’ approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a useful tool that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":299609,"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":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9532,"is_data_producer":false,"deposit_databanks":null,"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":689510,"name":"Katherine Owens","orcid":"0000-0002-5695-0203","position":1,"is_corresponding":false},{"id":617777,"name":"Soojin Jang","orcid":"0000-0002-2921-7196","position":2,"is_corresponding":false},{"id":943419,"name":"Joseph E Marcus","orcid":"0000-0002-1789-603X","position":3,"is_corresponding":false},{"id":789654,"name":"Erin A. Brettmann","orcid":"0000-0001-8603-0670","position":4,"is_corresponding":false},{"id":689512,"name":"Stephen M. Beverley","orcid":"0000-0001-5319-0811","position":5,"is_corresponding":false},{"id":689509,"name":"Lon‐Fye Lye","orcid":"0000-0003-0704-3532","position":0,"is_corresponding":true}],"reference_count":52,"raw_metadata":null,"created_at":"2026-07-19T00:31:44.904250Z","pmid":null,"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":[]}