119. HIV-1 Proviral DNA Excision and Deactivation Using a CRISPR/Cas9 “Nickase” Targeted to the 5’ and 3’ LTR

Permanent integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected HIV cells capable of reactivation. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. Therapies aimed at targeting the latent virus offer a promising approach to a “functional cure” of HIV/AIDS. Several gene editing technologies, including ZFNs, TALENs, Tre-recombinases and RNA-guided CRISPR/Cas9 tools have been used to disrupt the HIV-1 genome and suppress viral gene expression and replication in latently-infected cells. While CRISPR/Cas9 represents a powerful and facile DNA editing technology, toxicities associated with the Cas9 nuclease activity remains a concern. Here we explore the use of a “nickase” Cas9 (nCas9), which has a mutated RuvC I catalytic domain, and requires the combination of two adjacent small guide RNAs (sgRNAs) for on-target editing and specificity. We initially identified 9 sgRNA sequences targeted to conserved sites within the HIV TAR element, found on both the 5’ and 3’ LTRs. Using integrated and episomal LTR-driven luciferase reporters, we identified functional sgRNA “nickase pairs” capable of inducing an 80% reduction in gene expression, as well as excision of the proviral DNA. Using HIV-1 latency models, plasmids expressing nCas9 and the sgRNA pairs were nucleofected into HIV-1 integrated cell lines J1.1, CHME5 and U1 and treated with the histone deacetylase inhibitor trichostatin A (TSA) to activate infection. All treated cells showed significant reduction (up to 80% for J1.1) in infectious viral output. Cleavage was further verified using ChIP assays to detect DNA damage response proteins recruited to the targeted locus in all cell lines. Lastly, using a custom algorithm for detecting off-target cleavage sites (https://cm.jefferson.edu/Off-Spotter/), we identified 8 sites for each sgRNA within the “nickase pair”. No off-target editing was observed at these loci following high throughput NGS analysis. In conclusion, we showed that CRISPR/Cas technology represents a powerful tool at eradicating and/or deactivating the integrated HIV-1 provirus and that using the Cas9 nickase is just as effective as wild-type Cas9 but with the added advantage of providing a significant additional layer of safety for future in vivo applications.