{"doi":"10.1016/j.omtm.2022.02.010","title":"A combinatorial CRISPR-Cas12a attack on HIV DNA","abstract":"CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack. Here, we tested the antiviral activity of seven dual crRNA combinations and analyzed the HIV proviral genomes for mutations at the target sites. We demonstrated that dual crRNA combinations exhibit more robust antiviral activity than a single crRNA attack and, more important, that the dual-crRNA therapy can prevent virus escape in long-term cultures. We confirmed the absence of any replication-competent virus in these apparently cured cultures. Surprisingly, we did not detect excision of the HIV sequences located between two Cas12a cleavage sites. Instead, we observed almost exclusively HIV inactivation by “hypermutation,” that is, the introduction of indel mutations at both target sites due to the error-prone cellular DNA repair machinery. CRISPR-Cas12a is an alternative class 2 gene editing tool that may cause less off-target effects than the original Cas9 system. We have previously demonstrated that Cas12a attack with a single CRISPR RNA (crRNA) can neutralize all infectious HIV in an infected T cell line in cell culture. However, we demonstrated that HIV escapes from most crRNAs by acquisition of a mutation in the crRNA target sequence, thus providing resistance against Cas12a attack. Here, we tested the antiviral activity of seven dual crRNA combinations and analyzed the HIV proviral genomes for mutations at the target sites. We demonstrated that dual crRNA combinations exhibit more robust antiviral activity than a single crRNA attack and, more important, that the dual-crRNA therapy can prevent virus escape in long-term cultures. We confirmed the absence of any replication-competent virus in these apparently cured cultures. Surprisingly, we did not detect excision of the HIV sequences located between two Cas12a cleavage sites. Instead, we observed almost exclusively HIV inactivation by “hypermutation,” that is, the introduction of indel mutations at both target sites due to the error-prone cellular DNA repair machinery. IntroductionIn the past decades, scientists have made great strides in developing HIV treatment and prevention methods, but so far, there is still no cure for AIDS. Combination antiretroviral therapy can effectively suppress HIV replication, but cannot achieve a cure because it is not capable of inactivating HIV genomes that are stably integrated into the host cell genome. We and others developed clustered regularly interspersed short palindromic repeat (CRISPR) strategies to target the HIV proviral genome, aiming to achieve permanent HIV inactivation. We previously reported complete virus control in a T cell line upon stable transduction with a lentiviral vector that encodes an antiviral CRISPR system. The designed antiviral guide RNAs (gRNAs) guide the endonuclease in a sequence-specific manner to the integrated HIV proviral genome and trigger an endonuclease attack. For the original Cas9 system, we needed to combine two gRNAs to achieve a cure.1Wang G. Zhao N. Berkhout B. Das A.T. A combinatorial CRISPR-Cas9 attack on HIV-1 DNA extinguishes all infectious provirus in infected T cell cultures.Cell Rep. 2016; 17: 2819-2826Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar,2Binda C.S. Klaver B. Berkhout B. Das A.T. CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA.Viruses. 2020; 12: 330Crossref Scopus (12) Google Scholar This was originally supposed to occur via excision of the HIV sequences between the two cleavage sites, but we documented that this route toward HIV inactivation is in fact only th","journal":"Molecular Therapy — Methods & Clinical Development","year":2022,"id":250362,"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":26,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9528,"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":277331,"name":"Ben Berkhout","orcid":"0000-0002-1905-8486","position":1,"is_corresponding":false},{"id":277330,"name":"Elena Herrera-Carrillo","orcid":"0000-0001-9986-8552","position":2,"is_corresponding":false},{"id":279772,"name":"Ming-Hui Fan","orcid":null,"position":0,"is_corresponding":true}],"reference_count":32,"raw_metadata":null,"created_at":"2026-07-19T00:24:28.242323Z","pmid":"35356755","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":[]}