Repression of HIV-1 Reactivation Mediated By KRAB Fused CRISPR/dCas9 Proteins In Lymphoid And Myeloid Cell Models

Abstract

Background: Despite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. In addition, HIV-1 latency reactivation is a major challenge towards cure efforts. Two strategies to cure HIV-1 infection, the “shock and kill” is based on the eradication of the HIV-1 from the patient, and the “block and lock". The “Block and Lock” methodology aims to control HIV-1 latency reactivation, promoting a functional cure. The KRAB fused CRISPR/dCas9 (pdCas9KRAB) system was previously produced to control cell transcription. Based on this construct we developed a CRISPR RNAs (sgRNAs), to guide the pdCas9KRAB up to five different sites in HIV-1 provirus sites to block HIV-1 latency reactivation. This process was mediated by phorbol esters and HDAC inhibitors.Results: We found five sites in the HIV-1 provirus genome (LTR1-LTR5) that minimize CRISPR off-targets and transduced them in the lymphoid and myeloid HIV-1 latency models. One of the five sgRNAs (LTR5) which binds specifically in the HIV-1 LTR NFκB binding site was able to promote a robust repression of reactivation pattern in a HIV-1 latency lymphoid model stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a reduction of 100 times in HIV-1 RNA molecules, when reactivated IngB in myeloid HIV-1 latently infected U1 cells.Conclusion: Taken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latency. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we also have shown some useful hotspots sequences in HIV-1 LTR to design sgRNAs.