{"doi":"10.1126/science.abf5972","title":"Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity","abstract":"Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.","journal":"Science","year":2021,"id":152003,"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":78,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9525,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":645967,"name":"Hao Liu","orcid":"0000-0001-6572-2870","position":1,"is_corresponding":false},{"id":32145,"name":"Michael S. Packer","orcid":"0000-0002-7842-997X","position":2,"is_corresponding":false},{"id":645968,"name":"Xiaozhe Xiong","orcid":"0000-0001-9493-707X","position":3,"is_corresponding":false},{"id":645969,"name":"Pyung‐Gang Lee","orcid":"0000-0001-9895-1098","position":4,"is_corresponding":false},{"id":493675,"name":"Sicai Zhang","orcid":"0000-0002-8894-5476","position":5,"is_corresponding":false},{"id":245811,"name":"Michelle F. Richter","orcid":"0000-0002-6113-6820","position":6,"is_corresponding":false},{"id":530033,"name":"G. Minasov","orcid":"0000-0001-5460-3462","position":7,"is_corresponding":false},{"id":36778,"name":"Karla J. F. Satchell","orcid":"0000-0003-3274-7611","position":8,"is_corresponding":false},{"id":298483,"name":"Min Dong","orcid":"0000-0002-1744-7293","position":9,"is_corresponding":false},{"id":32147,"name":"David R. Liu","orcid":"0000-0002-9943-7557","position":10,"is_corresponding":false},{"id":645966,"name":"Travis R. Blum","orcid":"0000-0001-7911-9204","position":0,"is_corresponding":true}],"reference_count":74,"raw_metadata":null,"created_at":"2026-07-18T23:43:22.562252Z","pmid":"33602850","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":[]}