Genome scale screening identification of SaCas9/gRNAs for targeting HIV-1 provirus and suppression of HIV-1 infection

•The SaCas9/gRNAs can disrupt latent HIV-1 provirus and suppress HIV-1 proviral reactivation.•Dual or triple gRNAs in an all-in-one lentiviral vector can inhibit HIV-1 infection.•Combinational SaCas9/gRNAs have more efficacy in targeting of HIV-1 genome The CRISPR/Cas9 gene-editing approach has been widely used in anti-HIV-1 gene therapy research. However, the major challenges facing the therapeutic application of CRISPR/Cas9 are the precise genome cleavage efficacy and efficient delivery of Cas9/gRNA specifically to the HIV-infected cells. Recently, a small size Cas9 from Staphylococcus aureus (SaCas9) has shown promise in genome editing in eukaryotic cells, suggesting a potential usage in blocking HIV-1 infection by targeting the HIV-1 genome. Here, we designed 43 guide RNAs (gRNAs) against the HIV-1 genome, thereby identifying 8 gRNAs that efficiently and specifically disrupt the target DNA by SaCas9. In addition, we found the selected gRNAs induce SaCas9 to disrupt the latent HIV-1 provirus and suppress HIV-1 proviral reactivation in latently infected Jurkat C11 cells. We further confirmed that the dual or triple gRNAs in an all-in-one lentiviral vector could reduce viral production in TZM-bl cells as well as in Jurkat T cells. Moreover, we did not detect any off-target cleavages in the predicted sites, suggesting that through all-in-one lentiviral vector-mediated HIV-1 genome editing, the selected SaCas9/gRNAs can provide an alternative and flexible strategy for anti-HIV gene therapy.