Background: HIV presents one of the highest mutation rates, which in combination with a fast replication rate and a large population size accelerates viral evolution. Combination antiretroviral therapy can overcome the plasticity of the virus population and profoundly control viral replication. However, conventional treatment lacks the ability to stop viral production and clear the latent reservoir, which remains the major obstacle towards cure. Novel strategies, such as CRISPR/Cas9 genome editing, are required to permanently disrupt the HIV genome in the latently infected cells. In this study we investigated the ability of HIV to escape the CRISPR/Cas9 endonucleases targeting different steps in the viral life cycle.
Methods: The CRISPR/Cas9 system is comprised of a Cas9 protein, which in combination with a guideRNA (gRNA), is able to cleave a complementary dsDNA sequence. gRNAs were designed to target HIV-LTR, protease, reverse transcriptase, integrase and matrix. The CRISPR/Cas9 system was cloned in a lentivirus vector and transduced in SupT1 cells and the J.Lat Full-Length Clone 15-4. In the latter cells, impact of CRISPR/Cas9 on HIV reactivation was investigated. Transduced SupT1 cells were infected with HIV and viral replication and escape was monitored. On- and off-targeting efficiency (three genes per gRNA) and viral escape was assessed by deep sequencing.
Results: The CRISPR/Cas9 endonuclease induced efficient HIV genome editing (75%-99%), while off-target efficiency was < 0.9%. Subsequent TNFα-induced HIV reactivation was significantly reduced (single gRNA (40%-95%); two gRNAs (>98%)). We also demonstrated a significant reduction of HIV replication after six days of culture in SupT1 cells (single gRNA (82%-97%); two gRNAs (93%-99%)). Reduction in viral replication could be directly correlated to the number of CRISPR/Cas9 induced changes in the target sites (mostly indels). Independent of the potency of the gRNAs, selection of resistance was observed for all single gRNAs. However, all combinations of two potent gRNAs resulted in the generation of such a large number of CRISPR/Cas9 induced mutations (mostly indels) that viral replication could not be rescued after months of in vitro selection.
Conclusions: This is the first study to demonstrate that combining potent gRNAs targeting different steps in the viral lifecycle can prevent the selection of viral resistance.