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...

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Bibliographic Details
Published in:Virus research 2018-05, Vol.250, p.21-30
Main Authors: Wang, Qiankun, Liu, Shuai, Liu, Zhepeng, Ke, Zunhui, Li, Chunmei, Yu, Xiao, Chen, Shuliang, Guo, Deyin
Format: Article
Language:English
Subjects:
Combinational gRNAs
CRISPR-Cas Systems
Gene Editing
Genetic Therapy
Genetic Vectors
Genome, Viral
HIV Infections - therapy
HIV-1 - genetics
HIV-1 provirus
Humans
Jurkat Cells
Lentivirus - genetics
Proviruses - genetics
RNA, Guide, CRISPR-Cas Systems
SaCas9/gRNA
Staphylococcus aureus - genetics
Virion - genetics
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Summary:•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.
ISSN:0168-1702
1872-7492
DOI:10.1016/j.virusres.2018.04.002