Designing Safer CRISPR/Cas9 Therapeutics for HIV: Defining Factors That Regulate and Technologies Used to Detect Off-Target Editing

Abstract

Human immunodeficiency virus type-1 (HIV-1) infection has resulted in the death of upward of 39 million people since being discovered in the early 1980s. A cure strategy for HIV-1 has eluded scientists, but gene editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offer a new approach to developing a cure for HIV infection. While the CRISPR/Cas9 system has been used successfully in a number of different types of studies, there remains a concern for off-target effects. This review details the different aspects of the Cas9 system and how they play a role in off-target events. In addition, this review describes the current technologies available for detecting off-target cleavage events and their advantages and disadvantages. While some studies have utilized whole genome sequencing (WGS), this method sacrifices depth of coverage for interrogating the whole genome. A number of different approaches have now been developed to take advantage of next generation sequencing (NGS) without sacrificing depth of coverage. This review highlights four widely used methods for detecting off-target events: (1) genome-wide unbiased identification of double-stranded break events enabled by sequencing (GUIDE-Seq), (2) discovery of in situ Cas off-targets and verification by sequencing (DISCOVER-Seq), (3) circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-Seq), and (4) breaks labeling in situ and sequencing (BLISS). Each of these technologies has advantages and disadvantages, but all center around capturing double-stranded break (DSB) events catalyzed by the Cas9 endonuclease. Being able to define off-target events is crucial for a gene therapy cure strategy for HIV-1.

The HIV-1 provirus excised by a single CRISPR/Cas9 RNA guide persists in the host cell and may be reactivated

“…CRISPR technology is becoming the leading gene editing tool, with increasingly expanding fields of application 11, 41 . These include HIV therapy, where CRISPR might allow excision of the integrated HIV genome, which can be excised from cellular DNA by taking advantage of CRISPR/Cas9 site-specific cleavage 42, 43 .…”

“…This approach has proven to be effective and potent in vitro , whereas a number of limitations may be relevant when it is transposed in vivo . CRISPR/Cas9 may exhibit possible off-target activity and defiant gene rearrangement following DNA repair 41, 44 ; moreover, diversity and mutations of HIV genome may constrain target selection 45–47 . Lastly, site of integration and transcriptional activity of the provirus may impact on susceptibility to CRISPR cleavage 47–50 .…”

“…Thus, to effectively eliminate HIV infection in vivo , it would be advisable to convey multiple RNA guides into a single cell and ensure chopping of the provirus. Although rapid progress in CRISPR/Cas9 technology 41, 51 and improvement of delivery strategies 52, 53 , will provide a way to circumvent these drawbacks, current technologies of in vivo delivery do not allow to control the number and amounts of RNA guides introduced into single cells. We found that, if cut with a single RNA guide, the excised HIV-1 provirus persists in cells for a protracted period.…”

Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics

“…57 Yin et al reported that the CRISPR-Cas9 tool could inhibit multiple steps of HIV-1 infection, 58 and several research labs have made excellent efforts to improve the use of CRISPR-Cas9 for treating HIV infection. 59–61 Recently, CRISPR-Cas12 and CRISPR-Cas13 systems have been used to inhibit HIV infection. 62 , 63 …”

Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

“…The lentivirus-expressed Staphylococcus aureus Cas9 (saCas9)/gRNAs composed of multiple gRNAs targeting the conserved region in LTR and viral domain of HIV-1 effectively removed the latent HIV-1 virus, inhibited virus reactivation, and significantly improved the efficiency of destroying the HIV-1 genome [ 35 ]. More recent work and findings on gene therapy and editing of HIV-1 have been thoroughly reviewed and reported elsewhere [ 36 , 37 , 38 , 39 , 40 ]. In conclusion, these studies have demonstrated that the CRISPR/Cas9 system can be successfully applied to target and edit the HIV-1 genome, to inhibit HIV-1 infection, eliminate the virus, and even to induce the transcriptional activation of the latent virus to eliminate the virus, showing its potential use for HIV-1 therapy.…”