Cyclic GMP-AMP synthase (cGAS) is a major sensor of cytosolic DNA from invading pathogens and damaged cellular organelles. Activation of cGAS promotes liquid-like phase separation and formation of membraneless cytoplasmic structures. Here, we found that cGAS bound G3BP1, a double-stranded nucleic acid helicase involved in the formation of stress granules. Loss of G3BP1 blocked subcellular cGAS condensation and suppressed the interferon response to intracellular DNA and DNA virus particles in cells. Furthermore, an RNA-dependent association with PKR promoted G3BP1 foci formation and cGAS-dependent interferon responses. Together, these results indicate that PKR promotes the formation of G3BP1-dependent, membraneless cytoplasmic structures necessary for the DNA-sensing function of cGAS in human cells. These data suggest that there is a previously unappreciated link between nucleic acid sensing pathways, which requires the formation of specialized subcellular structures.
CRISPR-Cas provides bacteria and archaea with immunity against invading phages and foreign plasmid DNA and has been successfully adapted for gene editing in a variety of species. The class 2 type VI CRISPR-Cas effector Cas13a targets and cleaves RNA, providing protection against RNA phages. Here we report the repurposing of CRISPR-Cas13a to inhibit human immunodeficiency virus type 1 (HIV-1) infection through targeting HIV-1 RNA and diminishing viral gene expression. We observed strong inhibition of HIV-1 infection by CRISPR-Cas13a in human cells. We showed that CRISPR-Cas13a not only diminishes the level of newly synthesized viral RNA, either from the transfected plasmid DNA or from the viral DNA, which is integrated into cellular DNA, but it also targets and destroys the viral RNA that enters cells within viral capsid, leading to strong inhibition of HIV-1 infection. Together, our results suggest that CRISPR-Cas13a provides a potential novel tool to treat viral diseases in humans.
CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.
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