{"doi":"10.1016/j.xpro.2025.104201","title":"Protocol to generate stable knockout lines in the human-parasitic nematode Strongyloides stercoralis","abstract":"A major limitation to the study of gene function in parasitic nematodes was the inability to make stable mutant lines. Here, we present a protocol for generating stable knockout lines in the human-parasitic nematode Strongyloides stercoralis . We describe steps for generating CRISPR components and microinjecting them into worms. We also detail procedures for identifying potential gene disruptions and propagating mutants by host passage in gerbils to generate stable homozygous knockout lines. This protocol enables studies of gene function in S. stercoralis . For complete details on the use and execution of this protocol, please refer to Banerjee et al. 1 • Protocol for generating stable mutant lines of S. stercoralis using CRISPR • Steps for generating CRISPR constructs and microinjecting into free-living adults • Instructions for infecting hosts with activated F 1 iL3 progeny • Guidance on isolation of F 3 homozygous knockout iL3s and stable line maintenance Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. A major limitation to the study of gene function in parasitic nematodes was the inability to make stable mutant lines. Here, we present a protocol for generating stable knockout lines in the human-parasitic nematode Strongyloides stercoralis . We describe steps for generating CRISPR components and microinjecting them into worms. We also detail procedures for identifying potential gene disruptions and propagating mutants by host passage in gerbils to generate stable homozygous knockout lines. This protocol enables studies of gene function in S. stercoralis .","journal":"STAR Protocols","year":2025,"id":529334,"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":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9497,"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":892756,"name":"Breanna Walsh","orcid":"0000-0002-2114-9934","position":1,"is_corresponding":false},{"id":901760,"name":"Ruhi Patel","orcid":"0000-0002-8612-0016","position":2,"is_corresponding":false},{"id":322224,"name":"Michelle L. Castelletto","orcid":null,"position":3,"is_corresponding":false},{"id":321376,"name":"Elissa A. Hallem","orcid":"0000-0003-0260-3174","position":4,"is_corresponding":false},{"id":564490,"name":"Navonil Banerjee","orcid":"0000-0001-9613-5652","position":0,"is_corresponding":true}],"reference_count":31,"raw_metadata":null,"created_at":"2026-07-19T02:50:56.971987Z","pmid":"41240344","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":[]}