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.
Highlights
Human immunodeficiency virus-1 (HIV-1) infection continues to be a major health issue with more than 35 million individuals being infected worldwide[1]
We demonstrated that tat and rev-targeting clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] lentiviral constructs successfully abolished regulatory protein expression and function, and inhibited HIV-1 replication in persistently infected CD4+ T-cell lines as well as latently infected T cells
The regulatory proteins Tat and Rev play a crucial role in HIV-1 transcription and latency maintenance, and their genomes are among the most functionally conserved across subtypes, making them a compelling site of attack to suppress HIV-1
Summary
Human immunodeficiency virus-1 (HIV-1) infection continues to be a major health issue with more than 35 million individuals being infected worldwide[1]. One strategy is to deliberately induce this reactivation, termed the “shock and kill” approach, using latency-reversing agents such as inhibitors of histone deacetylase (HDACi), and this method has shown the potent induction of T-cell activation followed by viral mRNA increases[14]; ex vivo assays suggested that the outgrowth of HIV-1 from latent reservoirs is insufficient and cytotoxic T-cell responses are not sufficiently strong to eliminate reactivated infected cells, resulting in a minimal impact on the overall reservoir size[15,16] These findings indicate that strategies for a HIV-1 cure www.nature.com/scientificreports/. We found that CRISPR transduction successfully diminished viral capsid production in persistently and latently infected CD4+ T-cell lines These results support the potential use of CRISPR to target HIV-1 regulatory genes and suppress viral replication
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