Abstract
To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.
Highlights
To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas)
Target guided RNA (gRNA) were designed against the enhanced green fluorescent protein (EGFP) coding region as well as the non-coding long terminal repeat (LTR) of an EGFP reporter lentivirus (Fig. 1b)
Using more than five independent T-cell donors, our results showed that CRISPR/Cas[9] targeting human immunodeficiency virus (HIV)-1 LTR R regions efficiently reduced virus production by more than threefold compared with the controls (Fig. 4c)
Summary
Bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas[9] system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas[9] can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge These results unveil the potential of the CRISPR/Cas[9] system as a new therapeutic strategy against viral infections. The nature of lentiviral infections, whereby the virus, such as human immunodeficiency virus (HIV), invades immune cells and integrates into the host genome to establish its latent infection, has created a huge obstacle with respect to developing efficient vaccines[4,5]. By using the same backbone of EGFP reporter in previous lentivirus assays (Supplementary Fig. 1d,f,h), the CRISPR/Cas[9] system showed its ability to inactivate the expression of invading plasmids at a comparable level, to disrupt the infectious lentivirus (Supplementary Fig. 1e,g,i)
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