{"doi":"10.1101/2025.06.18.660386","title":"Rapid Cell-Free Combinatorial Mutagenesis Workflow Using Small Oligos Suitable for High-Iteration, Active Learning-Guided Protein Engineering","abstract":"Active learning-guided protein engineering e2iciently navigates the challenging fitness landscape by screening designs iteratively in a model-guided design-build-test-learn cycle. However, while high iterations boost performance, current workflows reliance on tedious and costly cell-based cloning and expression steps limits the iterations they can practically implement. To address this problem, we present a novel combinatorial mutagenesis workflow that uses small (∼20-40 bp) mutagenic annealed-oligo fragments and cell-free expression to rapidly and conveniently screen protein variants in <9 hours. Using bulk-prepared mutagenic oligos eliminates the need for cloning, PCR-based mutagenesis, or ordering costly genes each screening round. Their >80% size reduction from current fragment-based shu2ling strategies also helps avoid including multiple mutations on the same fragment, reducing the number one must order to cover the design space. By screening 3-10 fragment assemblies for two di2erent proteins, we show our approach is a general, scalable, and cost-e2ective platform for high-iteration protein engineering.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":555230,"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":0.9486,"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":1133987,"name":"Sepehr Hejazi","orcid":"0000-0001-8792-0587","position":1,"is_corresponding":false},{"id":1454005,"name":"Bret Lange","orcid":null,"position":2,"is_corresponding":false},{"id":1454006,"name":"Basmala Aldamak","orcid":null,"position":3,"is_corresponding":false},{"id":815314,"name":"Nigel F. Reuel","orcid":"0000-0003-3438-2919","position":4,"is_corresponding":false},{"id":1133986,"name":"Ryan Godin","orcid":"0000-0002-9093-6384","position":0,"is_corresponding":true}],"reference_count":55,"raw_metadata":null,"created_at":"2026-07-19T02:54:59.329539Z","pmid":"40667080","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":[]}