{"doi":"10.1101/2023.01.08.523136","title":"Decoding CRISPR–Cas9 PAM recognition with UniDesign","abstract":"Abstract The critical first step in CRISPR–Cas mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein’s PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR–Cas engineering to relax or tighten PAM requirement for subsequence applications. Here we describe a universal computational protein design framework (UniDesign) for designing protein–nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM–PIAA interactions for eight Cas9 proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas9s. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (>70% and >80% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it as a useful tool for engineering CRISPR–Cas and other nucleic acid–interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign .","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":402287,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9493,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1053258,"name":"Jun Zhou","orcid":"0000-0002-2576-7692","position":1,"is_corresponding":false},{"id":328111,"name":"Dongshan Yang","orcid":"0000-0003-2449-8651","position":2,"is_corresponding":false},{"id":243097,"name":"Jifeng Zhang","orcid":"0000-0001-5161-4705","position":3,"is_corresponding":false},{"id":328113,"name":"Xiaofeng Xia","orcid":"0000-0001-9522-7459","position":4,"is_corresponding":false},{"id":237553,"name":"Y. Eugene Chen","orcid":"0000-0003-2357-7825","position":5,"is_corresponding":false},{"id":328114,"name":"Jie Xu","orcid":"0000-0001-9452-2878","position":6,"is_corresponding":false},{"id":477118,"name":"Xiaoqiang Huang","orcid":"0000-0002-1005-848X","position":0,"is_corresponding":true}],"reference_count":50,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T01:20:27.903246Z","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":[]}