Determining on- and Off-Target Excision Events of Anti-HIV-1 Grnas Used Infor CRISPR/Cas9-Mediated Excision Therapy

While antiretroviral therapy (ART) has reduced viral loads to undetectable levels, in most patients, it does not remove the integrated HIV proviral DNA from the latent reservoirs, which is athe major barrier to a cure. The CRISPR/Cas9 gene-editing system has been shown to excise the entire HIV-1 genome by targeting the long terminal repeats (LTRs). However, published anti-HIV-1 gRNAs have not been shown to effectively target the LTRs of the viral quasispecies within or between infected individuals. Our in silico analysis has demonstrated only two previously published anti-HIV-1 gRNAs targeting the LTR can cut >90% of the patient-derived HIV-1 subtype B LTRs from the Los Alamos National Laboratory (LANL) HIV database. To improve on these results next-generation sequencing was performed on 269 patients enrolled in the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort to understand and design gRNAs to account for thevariation in the HIV-1 LTR . A novel gRNA design pipeline was designed to select for optimal gRNAs and filter out those with predicted off-target binding. These results informed the design of a 4 and 10 selected molecular gRNA targets (SMRT) package. Utilizing an in silico experimental approach, the SMRT-4 and -10 packages are able to excise all subtype B LTRs from LANL and the CARES Cohort, without any predicted off-target excision. Functional studies have demonstrated that the SMRT 4 gRNAs performedperforming better at reducing expression and mutating the LTR than other published gRNAs in a number of different assays. However, few studies have fully examined the potential off-target excision by anti-HIV-1 gRNAs. Therefore, to determine if there is off-target excision, genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-Seq) will be used to determine on- and off-target excision of SMRT gRNAs and others on a whole-genome level with NGS sequence coverage. Overall, these studies will advance a second-generation of CRISPR/Cas9 therapeutics and inform the design and testingdesing of novel anti-HIV-1 gRNAs.