Functional screening of guide RNAs targeting the regulatory and structural HIV-1 viral genome for a cure of AIDS

Abstract

Objective There is an urgent need for the development of HIV-1 genome eradication strategies that lead to a permanent cure for HIV-1/AIDS. We previously reported that 4 guide RNAs (gRNAs) targeting HIV-1 long terminal repeats (LTR) effectively eradicated the entire HIV-1 genome. In this study, we sought to identify the best gRNAs targeting HIV-1 LTR and viral structural region and to optimize gRNA pairing that can efficiently eradicate the HIV-1 genome. Design Highly specific gRNAs were designed using bioinformatics tools and their capacity of guiding Cas9 to cleave HIV-1 proviral DNA was evaluated using high throughput HIV-1 luciferase reporter assay and rapid Direct-PCR genotyping. Methods The target seed sequences for each gRNA were cloned into lentiviral vectors. HEK293T cells were cotransfected with a pEcoHIV-NL4-3-firefly-luciferase reporter vector (1:20) over lentiviral vectors carrying Cas9 and single gRNA or various combinations of gRNAs. The EcoHIV DNA cleaving efficiency was evaluated by Direct-PCR genotyping and the EcoHIV transcription/replication activity was examined by a luciferase reporter assay. Results Most of the designed gRNAs are effective to eliminate the predicted HIV-1 genome sequence between the selected two target sites. This is evidenced by the presence of PCR genotypic deletion/insertion and the decrease of luciferase reporter activity. In particular, a combination of viral structural gRNAs with LTR gRNAs provided a higher efficiency of genome eradication and an easier approach for PCR genotyping. Conclusion Our screening strategy can specifically and effectively identify gRNAs targeting HIV-1 LTR and structural region to excise proviral HIV-1 from the host genome.

CRISPR Editing Technology in Biological and Biomedical Investigation

“…CRISPR/Cas9 can be used either to disrupt the integrated HIV-1 provirus or the cellular genes that are required for productive HIV-1 infection. For example, CRISPR/Cas9 facilitates the excision of HIV proviral DNA segments in several different cell types including CD4+ T-cells and by targeting the flanking HIV-1 LTR sequences, complete excision of proviral DNA can be effected (Ebina et al, 2013; Hu et al, 2014; Kaminski et al, 2016b; Yin et al, 2016). This approach has been demonstrated to be effective in cleaving HIV proviral DNA in latently-infected cells in HIV-infected humanized Bone marrow-Liver-Thymus (hu-BLT) mice (Yin et al, 2017).…”

“…In principle, multiplex gRNAs can be employed so that the chance of generating escape mutants can be greatly reduced, since the probability of viable mutations occurring at multiple sites is much less than for a single site. Also, if the multiplex gRNAs are designed in such a way as to produce DNA breaks resulting in a large section of DNA being deleted, this will permanently prevent escape mutations from occurring (Hu et al, 2014; Kaminski et al, 2016a,b; Yin et al, 2016, 2017). Some Cas9 variants, e.g., Cpf1, cleave at a site that is outside the target sequence so that InDel mutations introduced at the cleavage site by NHEJ will not prevent further cleavage (Zetsche et al, 2015).…”

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

“…CRISPR/Cas9 allows the excision of segments of integrated proviral DNA in different latently infected cell types by targeting sequences within the HIV-1 long terminal repeat (LTR) flanking the provirus and allowing complete provirus excision [20–22, 24–26]. These experiments were performed with T cells [20], microglia, promonocytic T cells [21], primary CD4+ T-cells cultured ex vivo [22], induced pluripotent stem cells [24], HEK293T cells [25], and Jurkat cells [26], indicating that the ability of CRISPR/Cas9 to excise HIV provirus is not restricted by cell-type–specific factors. While the fate of the eliminated DNA arising after excision of segments of integrated proviral DNA is not known for certain, we tend to think that after excision from the genome, it will be degraded.…”

“…Alternatively, if two gRNAs produce DNA breaks, allowing excision of a large section of DNA, this will permanently prevent the occurrence of escape mutations, as shown in Fig 1. Several studies have demonstrated the strong suppression of HIV-1 using a multiplex approach [21–25]. Table 1 summarizes recent studies [52–75] on the use of CRISPR/Cas9 technology for editing several human viruses using single and multiplex gRNAs, resulting in the introduction of InDels and/or excision of the segment of the viral genome.…”

Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome

“…They designed sgRNAs for LTR region, resulting in a remarkable removal and eradication of proviral genome integrated in host genome . Similarly, several studies demonstrated excision and elimination of integrated HIV genome by the CRISPR/Cas9 system in different HIV‐1 latent reservoir cells . Moreover, removing of the integrated HIV genome in in vivo has been demonstrated using transgenic mice and rats models .…”