{"doi":"10.1016/j.molcel.2025.03.024","title":"Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing","abstract":"RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.","journal":"Molecular Cell","year":2025,"id":510324,"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":21,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9547,"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":1367362,"name":"Noor Al-Sayyad","orcid":null,"position":1,"is_corresponding":false},{"id":869669,"name":"Kevin Wasko","orcid":"0000-0001-6629-6850","position":2,"is_corresponding":false},{"id":849169,"name":"Marena Trinidad","orcid":"0000-0001-7839-4642","position":3,"is_corresponding":false},{"id":504306,"name":"Erin Doherty","orcid":"0000-0002-1555-4124","position":4,"is_corresponding":false},{"id":1366534,"name":"Kamakshi Vohra","orcid":"0009-0004-4368-2694","position":5,"is_corresponding":false},{"id":1037834,"name":"Ron Boger","orcid":"0000-0002-4467-271X","position":6,"is_corresponding":false},{"id":344852,"name":"David Colognori","orcid":"0000-0001-6995-7079","position":7,"is_corresponding":false},{"id":267755,"name":"Joshua C. Cofsky","orcid":"0000-0001-5403-8555","position":8,"is_corresponding":false},{"id":987426,"name":"Petr Skopintsev","orcid":"0000-0002-6043-157X","position":9,"is_corresponding":false},{"id":267756,"name":"Zev Bryant","orcid":"0000-0001-6751-3693","position":10,"is_corresponding":false},{"id":2407,"name":"Jennifer A. Doudna","orcid":"0000-0001-9161-999X","position":11,"is_corresponding":false},{"id":259596,"name":"Honglue Shi","orcid":"0000-0003-3847-1652","position":0,"is_corresponding":true}],"reference_count":60,"raw_metadata":null,"created_at":"2026-07-19T02:47:36.538276Z","pmid":"40273916","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":[]}