Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

Abstract

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

CRISPR Editing Technology in Biological and Biomedical Investigation

“…In a more specific strategy, Kaminski et al (2016c) developed a new approach that turns on Cas expression by the target gene. In this case, Cas9 was under the control of a promoter that is activated by the HIV-1 transactivator protein Tat allowing targeting of HIV-1 only during the course of viral infection when Tat is expressed (Kaminski et al, 2016c). …”

“…Now that CRISPR effector endonucleases are available to target RNA, e.g., FnCas9 directed by an engineered RNA-targeting gRNA, inhibition of human RNA viruses such as hepatitis C virus within eukaryotic cells should be possible (Price et al, 2015). Our research has focused on several pathogenic human viruses, including JCV, which is a circular double-stranded DNA virus (Wollebo et al, 2015b), HIV-1, which is a retrovirus (Hu et al, 2014; Kaminski et al, 2016a,b,c; Khalili et al, 2015, 2017; White and Khalili, 2016; White et al, 2015, 2016; Yin et al, 2016, 2017; Zhang et al, 2015a) and HSV-1 a herpesvirus comprised of a double-stranded DNA of greater than 100 Kbp in size (McGeoch et al, 2006). …”

Gene Editing Approaches against Viral Infections and Strategy to Prevent Occurrence of Viral Escape

“…Genetic editing applications, including CRISPR/Cas9, can disrupt both episomal and integrated DNA viruses and can be applied to several human viruses [2], including papillomaviruses HPV16 and HPV18 [11–13], hepatitis B virus (HBV) [14–17], Epstein-Barr virus (EBV) [18, 19], HIV-1 [7, 20–27], polyomavirus JC (JCV) [28], Herpes simplex virus-1 [29, 30], and other herpesviruses [30]. …”

“…CRISPR/Cas9 was used in two murine models in which tail-vein or intraperitoneal injection of transgenic animals with virus vector expressing Cas9 and a multiplex of gRNAs resulted in the cleavage of integrated HIV-1 DNA provirus in many tissues, indicating proof-of-concept for in vivo eradication of integrated HIV-1 DNA by CRISPR/Cas9 [23]. Kaminski et al [27] personalized CRISPR/Cas9 activity by placing the Cas9 gene under the control of an HIV-1 promoter, which is activated by the HIV-1 Tat. Functional activation of CRISPR/Cas9 by Tat occurred during HIV-1 infection, resulting in the excision of a designated segment of integrated HIV-1 proviral DNA and consequently suppressing viral expression [27].…”

“…Kaminski et al [27] personalized CRISPR/Cas9 activity by placing the Cas9 gene under the control of an HIV-1 promoter, which is activated by the HIV-1 Tat. Functional activation of CRISPR/Cas9 by Tat occurred during HIV-1 infection, resulting in the excision of a designated segment of integrated HIV-1 proviral DNA and consequently suppressing viral expression [27]. Thus, regulated expression of Cas9 by Tat provides a novel and safe strategy for ablation of HIV-1 at an early stage of acute infection and silent proviral reactivation in latently infected cells.…”