{"doi":"10.1093/nar/gkaf649","title":"Directed evolution expands CRISPR–Cas12a genome-editing capacity","abstract":"CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.","journal":"Nucleic Acids Research","year":2025,"id":511595,"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":15,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9519,"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":764145,"name":"Kai Chen","orcid":"0000-0002-3325-3536","position":1,"is_corresponding":false},{"id":259596,"name":"Honglue Shi","orcid":"0000-0003-3847-1652","position":2,"is_corresponding":false},{"id":869669,"name":"Kevin Wasko","orcid":"0000-0001-6629-6850","position":3,"is_corresponding":false},{"id":1369621,"name":"Isabel Esain-Garcia","orcid":"0000-0003-1931-4563","position":4,"is_corresponding":false},{"id":849169,"name":"Marena Trinidad","orcid":"0000-0001-7839-4642","position":5,"is_corresponding":false},{"id":849171,"name":"Kaihong Zhou","orcid":"0000-0001-9267-9206","position":6,"is_corresponding":false},{"id":850121,"name":"Jinjuan Ye","orcid":null,"position":7,"is_corresponding":false},{"id":2407,"name":"Jennifer A. Doudna","orcid":"0000-0001-9161-999X","position":8,"is_corresponding":false},{"id":302175,"name":"Enbo Ma","orcid":"0000-0002-4374-796X","position":0,"is_corresponding":true}],"reference_count":65,"raw_metadata":null,"created_at":"2026-07-19T02:47:55.918021Z","pmid":"40671523","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":[]}