{"doi":"10.1038/s42003-024-05776-8","title":"Selection of extended CRISPR RNAs with enhanced targeting and specificity","abstract":"As CRISPR effectors like Cas9 increasingly enter clinical trials for therapeutic gene editing, a future for personalized medicine will require efficient methods to protect individuals from the potential of off-target mutations that may also occur at specific sequences in their genomes that are similar to the therapeutic target. A Cas9 enzyme's ability to recognize their targets (and off-targets) are determined by the sequence of their RNA-cofactors (their guide RNAs or gRNAs). Here, we present a method to screen hundreds of thousands of gRNA variants with short, randomized 5' nucleotide extensions near its DNA-targeting segment-a modification that can increase gene editing specificity by orders of magnitude-to identify extended gRNAs (x-gRNAs) that effectively block any activity at those off-target sites while still maintaining strong activity at their intended targets. X-gRNAs that have been selected for specific target / off-target pairs can significantly out-perform other methods that reduce Cas9 off-target activity overall, like using Cas9 variants engineered for higher specificity in general, and we demonstrate their effectiveness in clinically-relevant gRNAs. Our streamlined approach to efficiently identify highly specific and active x-gRNAs provides a way to move beyond a one-size-fits-all model of high-fidelity CRISPR for safer and more effective personalized gene therapies.","journal":"Communications Biology","year":2024,"id":463039,"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":6,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9521,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1180246,"name":"Hillary Dimig","orcid":null,"position":1,"is_corresponding":false},{"id":1180247,"name":"Miranda Roesing","orcid":null,"position":2,"is_corresponding":false},{"id":924833,"name":"Eric A. Josephs","orcid":"0000-0002-5330-6842","position":3,"is_corresponding":false},{"id":1180245,"name":"Ashley Herring‐Nicholas","orcid":null,"position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":null,"created_at":"2026-07-19T02:04:28.838123Z","pmid":"38212640","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":[]}