{"doi":"10.1016/j.omtn.2020.01.004","title":"Development of a Facile Approach for Generating Chemically Modified CRISPR/Cas9 RNA","abstract":"The RNA-guided, modified type II prokaryotic CRISPR with CRISPR-associated proteins (CRISPR/Cas9) system represents a simple gene-editing platform with applications in biotechnology and also potentially as a therapeutic modality. The system requires a small guide RNA (sgRNA) and a catalytic Cas9 protein to induce non-homologous end joining (NHEJ) at break sites, resulting in the formation of inactivating mutations, or through homology-directed repair (HDR) can engineer in specific sequence changes. Although CRISPR/Cas9 is a powerful technology, the effects can be limited as a result of nuclease-mediated degradation of the RNA components. Significant research has focused on the solid-phase synthesis of CRISPR RNA components with chemically modified bases, but this approach is technically challenging and expensive. Development of a simple, generic approach to generate chemically modified CRISPR RNAs may broaden applications that require nuclease-resistant CRISPR components. We report here the development of a novel, functional U-replaced trans-activating RNA (tracrRNA) that can be in vitro transcribed with chemically stabilizing 2′-fluoro (2′F)-pyrimidines. These data represent a unique and facile approach to generating chemically stabilized CRISPR RNA. The RNA-guided, modified type II prokaryotic CRISPR with CRISPR-associated proteins (CRISPR/Cas9) system represents a simple gene-editing platform with applications in biotechnology and also potentially as a therapeutic modality. The system requires a small guide RNA (sgRNA) and a catalytic Cas9 protein to induce non-homologous end joining (NHEJ) at break sites, resulting in the formation of inactivating mutations, or through homology-directed repair (HDR) can engineer in specific sequence changes. Although CRISPR/Cas9 is a powerful technology, the effects can be limited as a result of nuclease-mediated degradation of the RNA components. Significant research has focused on the solid-phase synthesis of CRISPR RNA components with chemically modified bases, but this approach is technically challenging and expensive. Development of a simple, generic approach to generate chemically modified CRISPR RNAs may broaden applications that require nuclease-resistant CRISPR components. We report here the development of a novel, functional U-replaced trans-activating RNA (tracrRNA) that can be in vitro transcribed with chemically stabilizing 2′-fluoro (2′F)-pyrimidines. These data represent a unique and facile approach to generating chemically stabilized CRISPR RNA.","journal":"Molecular Therapy — Nucleic Acids","year":2020,"id":109877,"datarank":0.42233990555198797,"base_score":2.0794415416798357,"endowment":2.0794415416798357,"self_citation_contribution":0.31191623125197543,"citation_network_contribution":0.11042367430001257,"self_endowment_contribution":0.31191623125197543,"citer_contribution":0.11042367430001257,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":7,"citers_with_citation_signal":7,"citers_with_endowment":7,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9602,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":429216,"name":"Citradewi Soemardy","orcid":null,"position":1,"is_corresponding":false},{"id":382492,"name":"Kevin V. Morris","orcid":"0000-0002-0157-0553","position":2,"is_corresponding":false},{"id":428073,"name":"Tristan Scott","orcid":"0000-0002-7843-9609","position":0,"is_corresponding":true}],"reference_count":35,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:12:54.309019Z","pmid":"32069700","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":[]}