Computational Selection of CRISPR/Cas9 Guide-RNAs to Target Highly Variable Integrated HIV-1 Provirus Using Next Generation Sequencing

HIV-1 persistence and/or latency even after long-term antiretroviral therapy is a major hurdle to a cure. Genomic editing techniques, like the CRISPR/Cas9 system, hold promise to permanently excise the integrated provirus from a host cell. The CRISPR/Cas9 system could be directed to target the HIV-1 sequence by a guide-RNA designed to be homologous to the virus.integrated provirus. However, targeting is stymied by the rapid accumulation of mutations introduced during HIV-1 replication creating a set of distinct genomic variants that varies within an individual through time and between individuals. Presented here is a computational methodology for designing gRNA sequences to effectively cleave a patient’s HIV-1 quasispecies. PBMC genomic DNA was isolated from patients in the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort and the long terminal repeat (LTR) of the HIV-1 quasispecies was sampled using Next Generation Sequencing (NGS) to a depth of at least 10,000X across 269 patients. gRNAs were computationally selected by examining their predicted binding potential across a random training set of 100 CARES Cohort patient samples. This screening selected a package of 4 or 10 gRNAs, which in silico cleaved the entire detectable quasispecies of the remaining, unseen, 169 CARES samples an average of 3.4 +/- 1.7 or 5.4 +/- 3 times, respectively. The package was further tested against an independent national sampling of North American subtype B LTRs from the Los Alamos National Database and in silico was shown to cleave all LANL LTR sequences. Functional expression of the gRNAs in a P4R5 cell line model have shown knockdown of LTR activity. This work presents a step towards understanding the complex task of using excision therapy to target HIV-1 quasispecies in the infected patient population.