{"doi":"10.1089/aid.2020.0055","title":"Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates","abstract":"Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 ( Sp Cas9) gRNAs directed to the 5′ and 3′ long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells ( in vivo ). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed Sp Cas9-127 HBX2 ) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.","journal":"AIDS Research and Human Retroviruses","year":2020,"id":84602,"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":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9482,"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":434925,"name":"Yun Yue Chen","orcid":null,"position":1,"is_corresponding":false},{"id":434926,"name":"Christine A. Hodge","orcid":null,"position":2,"is_corresponding":false},{"id":434927,"name":"Taylor R. Hudson","orcid":null,"position":3,"is_corresponding":false},{"id":434928,"name":"Susan K. Eszterhas","orcid":null,"position":4,"is_corresponding":false},{"id":433885,"name":"Matthew S. Hayden","orcid":"0000-0003-1670-6430","position":5,"is_corresponding":false},{"id":433886,"name":"Alexandra L. Howell","orcid":"0000-0002-2362-2596","position":6,"is_corresponding":false},{"id":434924,"name":"Katherine J. Sessions","orcid":null,"position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":null,"created_at":"2026-07-18T21:55:38.506333Z","pmid":"32640832","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":[]}