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.
Highlights
There are approximately 39 million individuals worldwide that are infected with human immunodeficiency virus type-1 (HIV-1)
This review will discuss the factors that influence the molecular basis of off-target editing (Figure 1), what has been done to characterize off-target promiscuity in the context of RNA-guided targeting of integrated Human immunodeficiency virus type-1 (HIV-1) proviral sequences, and why a clearly defined bioinformatic approach with a robust experimental validation protocol to identify off-target potential should be one of the major objectives in the development of an anti-HIV-1 clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based therapy (Figure 2)
Translocation events are rare and taken together these results suggest that CRISPR-induced translocations may present minimal risk
Summary
There are approximately 39 million individuals worldwide that are infected with human immunodeficiency virus type-1 (HIV-1). This review will discuss the factors that influence the molecular basis of off-target editing (Figure 1), what has been done to characterize off-target promiscuity in the context of RNA-guided targeting of integrated HIV-1 proviral sequences, and why a clearly defined bioinformatic approach with a robust experimental validation protocol to identify off-target potential should be one of the major objectives in the development of an anti-HIV-1 CRISPR/Cas9-based therapy (Figure 2). In addition to the change of PAM stringency, previous attempts in Cas engineering have reduced Cas: target DNA interaction by neutralizing the positive charges in DNA strand binding domain in the Cas9 This conferred a higher requirement for gRNA: DNA homology for cleavage, increased the targeting specificity (Slaymaker et al, 2016). Further experimentation will be necessary to fully characterize the risks posed by CRISPR/ Cas with respect to translocation events
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
