{"doi":"10.1101/2024.02.14.580371","title":"Virus-free CRISPR knock-in of a chimeric antigen receptor into <i>KLRC1</i> generates potent GD2-specific natural killer cells","abstract":"Abstract Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing. KLRC1 -GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2 + human melanoma cells. Notably, KLRC1 -GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2 + melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E + solid tumors.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":494361,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9614,"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":1341179,"name":"Isabella Zingler-Hoslet","orcid":null,"position":1,"is_corresponding":false},{"id":258238,"name":"Lei Shi","orcid":"0000-0002-5312-8521","position":2,"is_corresponding":false},{"id":811242,"name":"Varun Katta","orcid":"0000-0002-4565-7335","position":3,"is_corresponding":false},{"id":1341180,"name":"Brittany E. Russell","orcid":null,"position":4,"is_corresponding":false},{"id":320685,"name":"Shengdar Q. Tsai","orcid":"0000-0001-9161-3993","position":5,"is_corresponding":false},{"id":277448,"name":"Christian M. Capitini","orcid":"0000-0002-2276-6731","position":6,"is_corresponding":false},{"id":253458,"name":"Krishanu Saha","orcid":"0000-0003-2837-0858","position":7,"is_corresponding":false},{"id":296955,"name":"Keerthana Shankar","orcid":null,"position":0,"is_corresponding":true}],"reference_count":45,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:09:11.736910Z","pmid":"38405747","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":[]}