Targeted RNA degradation of HIV-1 TAR element at 5’UTR by CRISPR-Cas13X blocks HIV-1 lytic reactivation

Human immunodeficiency virus type 1 (HIV-1) latency remains a major barrier to a cure. Here, we developed a hypercompact CRISPR-Cas13X.1 system to target the HIV-1 5′ untranslated region (5′UTR), focusing on the transactivation response (TAR) RNA element required for Tat-dependent transcription. Following validation of a single-vector system, we screened CRISPR RNAs (crRNAs) targeting distinct 5′UTR elements and identified crRNA-TAR-2 as the most effective inhibitor of Tat-dependent reporter activity. Mechanistic analyses demonstrated that TAR targeting disrupted Tat–TAR interactions. In HIV-1 infection models, Cas13X.1-mediated TAR targeting reduced HIV-1 Gag precursor (p55) and capsid (p24) protein expression, as well as gag and tat transcript levels, accompanied by activation of the PKR–eIF2α antiviral pathway. TAR targeting also suppressed viral gene expression and reactivation in multiple latency models and reduced viral load in HIV-1-infected primary peripheral blood mononuclear cells (PBMCs). These findings establish HIV-1 TAR RNA as a target for Cas13X.1-mediated antiviral activity and support RNA-targeting CRISPR systems as a potential strategy to suppress HIV-1 infection and reactivation.