CRISPR/Cas9 inhibits multiple steps of HIV-1 infection.

CRISPR/Cas9 is an adaptive immune system that bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. In this study, we have tested this gene editing system in inhibiting HIV-1 infection by targeting the viral long terminal repeat and the gene coding sequences. We observed strong inhibition of HIV-1 infection by Cas9/gRNA, 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. We further observed that Cas9, when is solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, only that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA, and thus cannot be used to excise the latent provirus. Together, our 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.