Lepcha, T. T.; Zhou, D.; Fiches, G. N.; Paruchuri, N.; He, J.; Shanaka, K. A.; Liu, Y.; Eleya, S.; Santoso, N. G.; Zhu, J.

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-eIF2a; 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.