Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus

Abstract

Even though highly active anti-retroviral therapy is able to keep HIV-1 replication under control, the virus can lie in a dormant state within the host genome, known as a latent reservoir, and poses a threat to re-emerge at any time. However, novel technologies aimed at disrupting HIV-1 provirus may be capable of eradicating viral genomes from infected individuals. In this study, we showed the potential of the CRISPR/Cas9 system to edit the HIV-1 genome and block its expression. When LTR-targeting CRISPR/Cas9 components were transfected into HIV-1 LTR expression-dormant and -inducible T cells, a significant loss of LTR-driven expression was observed after stimulation. Sequence analysis confirmed that this CRISPR/Cas9 system efficiently cleaved and mutated LTR target sites. More importantly, this system was also able to remove internal viral genes from the host cell chromosome. Our results suggest that the CRISPR/Cas9 system may be a useful tool for curing HIV-1 infection.

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…3). The LTR-R region contains the TAR sequence that is relatively conserved among the HIV-1 subtypes, which could serve as a common target site for anti-viral disruptions 17,34,35 . This consequence matches the fact that LTR serves as a critical and universal element for lentivirus expressional regulations by the transcriptional machinery from both virus-encoded proteins and the host cells during the HIV infections 36,37 .…”

“…Recently, several research groups have successfully applied the type II CRISPR systemSpCas9 protein from Streptococcus pyogenes with guided RNA (gRNA)-for targeted genome editing in diverse cell types and organisms, including human cells [12][13][14] . Most recently, a couple of studies have demonstrated the excision of the HIV-1 provirus from the host cell genome using gene-editing tools [15][16][17][18] . Here we apply the CRISPR/Cas9 system to directly target and disrupt the reverse-transcribed products of the lentiviral RNA genome during their life cycle within host cells.…”

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

“…Using a pair of sgRNAs targeting the LTR of HIV-1, it was shown that HIV-1 provirus can be removed from the genome of infected cell lines (Ebina et al, 2013;Hu et al, 2014). By combining TALEN or CRISPR/Cas9 with PiggyBac technology, researches have generated inducedpluripotent stem cells (iPSC) homozygous for the naturally occurring CCR5 D32 variant resistant to HIV-1 infection .…”

“…In eukaryotes, the DSBs are more commonly repaired by the mechanism of error-prone non-homologous end joining (NHEJ), therefore generating sequence changes, for instance insertions and deletions (indels), around the DSBs . Owing to the simplicity of manipulation and versatility, the CRISPR/Cas9 system has been utilized as an attractive tool for various applications, such as genome-wide screening (Shalem et al, 2014;Zhou et al, 2014), gene repression and activation (Cheng et al, 2013;Doench et al, 2014;Gilbert et al, 2014), targeted fluorescence imaging (Tanenbaum et al, 2014) and novel approaches against pathogens including hepatitis B virus (Lin et al, 2014a;Seeger & Sohn, 2014), human papillomavirus (Kennedy et al, 2014), Epstein-Barr virus (Wang & Quake, 2014;Yuen et al, 2015), malaria (Wagner et al, 2014) and HIV-1 (Ebina et al, 2013;Hu et al, 2014;Ye et al, 2014).…”

Crispr-Cas System: A Feasible Solution for Getting Rid of Persistent Viral Infection?

“…CRISPR-Cas system may be used to cleave viral DNAs in either episomal form or integrated form, as long as the sgRNA has been carefully designed to target a specific sequence in the viral genomic DNA. Indeed, studies showed that Cas9/sgRNAcan be adapted for disrupting the LTR elements of Human Immunodeficiency Virus (HIV) provirus and causing a significant loss of LTR-driven expression (Ebina et al, 2013). A similar study showed that using Cas9 and sgRNA targeting the LTR U3 region, a 9.7 kb fragment of the integrated HIV proviral DNA can be excised (Hu et al, 2014a).…”