Abstract
Eradication of HIV is challenging because of the integration of proviral DNA in reservoir cells. In this study, we evaluated the antiviral effects of synthetic gRNA/Cas12a and gRNA/Cas9 ribonucleoproteins (RNPs) in HIV-infected T cells and demonstrated their specificity and efficacy in disrupting HIV gene replication and expression. Notably, sequential or combinatorial treatments with gRNA4/Cas9 and/or gRNA5/Cas12a RNPs elicited the most effective antiviral effect, with significant reduction in integrated proviral DNA, HIV mRNA, early and late reverse transcripts, and p24 protein levels. DNA sequencing revealed a high rate of insertion and deletion or knockout frequencies at the HIV target genes. Gene alignment analysis showed a high level of conservation with both gRNA4/Cas9 and gRNA5/Cas12a target sequences across diverse regional HIV strains, indicating their potential to target different HIV strains across the world. This study indicates that synthetic gRNA4/Cas9 and gRNA5/Cas12a RNPs can be used for HIV gene disruption and viral eradication.