Combination treatment with synthetic gRNA/Cas12a and gRNA/Cas9 ribonucleoproteins disrupts HIV replication and expression

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. Sequential or combinatorial treatments with gRNA4/Cas9 and/or gRNA5/Cas12a RNPs elicited the most effective antiviral effect, with significant reduction of the 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.