The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA

Abstract

BackgroundHighly active antiretroviral therapy (HAART) has transformed HIV-1 infection from a deadly disease to a manageable chronic illness, albeit does not provide a cure. The recently developed genome editing system called CRISPR/Cas9 offers a new tool to inactivate the integrated latent HIV-1 DNA and may serve as a new avenue toward cure.FindingsWe tested 10 sites in HIV-1 DNA that can be targeted by CRISPR/Cas9. The engineered CRISPR/Cas9 system was introduced into the JLat10.6 cells that are latently infected by HIV-1. The sequencing results showed that each target site in HIV-1 DNA was efficiently mutated by CRISPR/Cas9 with the target site in the second exon of Rev (called T10) exhibiting the highest degree of mutation. As a result, HIV-1 gene expression and virus production were significantly diminished with T10 causing a 20-fold reduction.ConclusionsThe CRISPR/Cas9 complex efficiently mutates and deactivates HIV-1 proviral DNA in latently infected Jurkat cells. Our results also revealed a highly efficient Cas9 target site within the second exon of Rev that represents a promising target to be further explored in the CRISPR/Cas9-based cure strategy.

The clinical applications of genome editing in HIV

“…82 More conventional anti-HIV nucleases have also been described based on homing endonuclease, ZFNs, TALENs, and CRISPR/Cas9, targeting different regions of the HIV-1 genome, and with a demonstrated ability to reduce HIV-1 content in various cell lines. [83][84][85][86][87][88] Although these studies provide the initial proof of principle that gene disruption could be used to disrupt or excise an integrated HIV-1 genome, significant challenges will exist in the clinical translation of these approaches. No efficient method yet exists to enable in vivo delivery of nucleases to HIV target cells, and delivery to the subset of cells that comprise the latent reservoir will be especially problematic, due both to the location of these cells in inaccessible organ niches and the lack of identifying surface markers of viral infection.…”

Clinical Applications of Genome Editing to HIV Cure

“…Anti-HIV nucleases have been developed based on evolved Cre recombinase, ZFNs, TALENs, and CRISPR/Cas9, targeting different regions of the HIV-1 genome, and have been shown to reduce integrated HIV-1 content in various cell lines. [62][63][64][65][66][67][68][69] Significant challenges exist in the clinical translation of this approach. Targeted nucleases against HIV can promote viral escape and accelerate evolution, observed in culture over an extended period of time.…”

Disruption or Excision of Provirus as an Approach to HIV Cure

“…For HIV, introduction of a single targeted endonuclease has been reported to result in disruption of *10-50% of provirus. [13][14][15] Incomplete disruption may occur because repair of the induced DSBs is generally precise, and as such, the original target gene sequence may be retained even after multiple rounds of cleavage and repair over the duration of endonuclease expression. If so, increasing endonuclease expression levels, persistence, or specific activity may result in higher mutagenesis, as does the inclusion of DNA end-processing enzymes such as Trex2.…”