{"doi":"10.3389/fimmu.2022.821190","title":"Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication","abstract":"Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo . Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.","journal":"Frontiers in Immunology","year":2022,"id":260731,"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":15,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9511,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":914478,"name":"Yasmine Rashad","orcid":"0000-0001-5757-8098","position":1,"is_corresponding":false},{"id":442089,"name":"Fayez M. Saleh","orcid":"0000-0001-8417-8874","position":2,"is_corresponding":false},{"id":914911,"name":"Kurtis A. Willingham","orcid":null,"position":3,"is_corresponding":false},{"id":914912,"name":"Antonia Reilich","orcid":null,"position":4,"is_corresponding":false},{"id":442091,"name":"Dong Lin","orcid":"0000-0003-4433-6342","position":5,"is_corresponding":false},{"id":442093,"name":"Reza Izadpanah","orcid":"0000-0002-4520-8150","position":6,"is_corresponding":false},{"id":442096,"name":"Eckhard Alt","orcid":"0000-0002-6687-139X","position":7,"is_corresponding":false},{"id":442097,"name":"Stephen E. Braun","orcid":"0000-0002-4521-2678","position":8,"is_corresponding":false},{"id":740256,"name":"Stefan H. Scheller","orcid":null,"position":0,"is_corresponding":true}],"reference_count":81,"raw_metadata":null,"created_at":"2026-07-19T00:26:07.666421Z","pmid":"35386712","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":[]}