Comparative CRISPR-based systems for HIV gene editing

HIV-1 persists as integrated proviral DNA within long-lived cellular reservoirs, preventing eradication despite effective antiretroviral therapy. CRISPR-based genome editing offers a promising strategy to disrupt the proviral genome. This dissertation systematically evaluates several programmable CRISPR systems, including Cas12b, CjCas9, SaCas9, and ultracompact TnpB/Fanzor nucleases. It also investigates lipid nanoparticle (LNP) delivery strategies to advance curative HIV-1 editing approaches.
Cas12b, CjCas9, and SaCas9 each demonstrated potent antiviral activity, with single guide RNA (gRNA) strategies sufficient to fully inactivate HIV-1 in T cells. Among these systems, SaCas9 displayed the strongest inhibition, eliminating all detectable wild-type proviruses using a single gRNA. Dual-gRNA approaches further enhanced editing efficiency. Cas12b and CjCas9 primarily induced INDELs (insertions and deletions), with limited proviral DNA excision. Notably, excision efficiencies varied among SaCas9 gRNA pairs. Analysis of the binding and cleavage kinetics of the Cas nuclease-gRNA complex revealed that efficient excision requires rapid, synchronized cleavage at both targets. The Gag3 + Pol5 pair achieved near-complete excision (97%), whereas kinetically mismatched pairs produced individual mutations at both target sites.
TnpB and Fanzor were evaluated for their ability to inactivate HIV. However, their low antiviral activity indicates that substantial RNA and protein engineering will be necessary to enhance their HIV-inactivating potential. LNP-based delivery platforms were developed, including a selective organ targeting (SORT) formulation that achieved >90% mRNA expression across multiple cell types. Additionally, anti-CD4 DARPin-conjugated LNPs were explored. Overall, these findings represent a step forward in HIV-1 gene-editing strategies, while highlighting that further work is required to achieve clinical application.