CRISPR/Cas9 system targeting regulatory genes of HIV-1 inhibits viral replication in infected T-cell cultures

Abstract

The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293 T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells. Tat functional assays showed significantly reduced HIV-1 promoter-driven luciferase expression after tat-CRISPR transduction, while Rev functional assays revealed abolished gp120 expression after rev-CRISPR transduction. The target gene was mutated at the Cas9 cleavage site with high frequency and various indel mutations. Conversely, no mutations were detected at off-target sites and Cas9 expression had no effect on cell viability. CRISPR/Cas9 was further tested in persistently and latently HIV-1-infected T-cell lines, in which p24 levels were significantly suppressed even after cytokine reactivation, and multiplexing all six gRNAs further increased efficiency. Thus, the CRISPR/Cas9 system targeting HIV-1 regulatory genes may serve as a favorable means to achieve functional cures.

Measuring the Success of HIV-1 Cure Strategies

“…This approach may also be used to specifically knockout or attenuate the HIV-1 provirus, for example, by targeting the LTR to disrupt viral gene expression or excise the integrated genome (Ebina et al, 2013;Hu et al, 2014;Kaminski et al, 2016;Lebbink et al, 2017;Yin et al, 2017;Bella et al, 2018;Wang Q. et al, 2018). Alternatively, various positions of the latent provirus could be targeted by CRISPR-Cas9 to induce multiple non-homologous end joining (NHEJ) associated indels that deactivate the virus through frame shift mutation (Liao et al, 2015;Ueda et al, 2016;Wang et al, 2016;Ophinni et al, 2018). Additionally, recent work has shown that, in combination with a novel drug delivery system, CRISPR-Cas9 directed editing of proviral DNA could effectively eliminate HIV-1 infection in mouse models (Dash et al, 2019).…”

Reduce and Control: A Combinatorial Strategy for Achieving Sustained HIV Remissions in the Absence of Antiretroviral Therapy

“…CRISPR/Cas9 technology was first applied to HIV-1 in 2013 [219], with several rapidly ensuing studies (reviewed in [216]). Proviral cleavage by CRISPR was often tested in cell lines, like HEK 293T cells and HeLa [219][220][221][222][223], which unfortunately do not resemble natural targets of HIV. However, this technology was also shown to be effective in more relevant T cell lines [219][220][221][222][223] and in cell line models of HIV-1 latency [220,[222][223][224].…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…A general ideal is to make two DSBs by nucleases in proviral DNA (e.g., in the LTRs), leading to the removal of the viral genome [250]. Alternatively, targeting conserved and critical viral genes has been found to disable the provirus [251]. Nevertheless, this strategy faces profound challenges, including the diversity of viral quasi-species, the fast mutating nature of HIV-1, the transcriptionally inactive latency, the establishment of latent infection in long-lived cell subsets (such as resident macrophages in various tissues), and the lack of ideal animal models.…”