Application of gene-editing technologies to HIV-1

Abstract

Purpose of review: This review will highlight some of the recent advances in genome engineering with applications for both clinical and basic science investigations of HIV-1.

Recent findings: Over the last year, the field of HIV cure research has seen major breakthroughs with the success of the first phase I clinical trial involving gene editing of CCR5 in patient-derived CD4(+) T cells. This first human use of gene-editing technology was accomplished using zinc finger nucleases (ZFNs). Zinc finger nucleases and the advent of additional tools for genome engineering, including transcription activator-like effector nucleases (TALENS) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, have made gene editing remarkably simple and affordable. Here we will discuss the different gene-editing technologies, the use of gene editing in HIV research over the past year, and potential applications of gene editing for both in-vitro and in-vivo studies.

Summary: Genome-engineering technologies have rapidly progressed over the past few years such that these systems can be easily applied in any laboratory for a variety of purposes. For HIV-1, upcoming clinical trials will determine if gene editing can provide the long-awaited functional cure. In addition, manipulation of host genomes, whether in vivo or in vitro, can facilitate development of better animal models and culture methods for studying HIV-1 transmission, pathogenesis, and virus-host interactions.

Figure 1

Designer nuclease systems for genome engineering. (A) Zinc-finger nucleases (ZFNs) are composed of tandem zinc finger domains fused to one-half of a FokI domain. Each zinc-finger domain binds three nucleotides. The ZFN arms must be positioned 5–6 bases apart for efficient FokI heterodimerization, activation, and double-stranded break formation. (B) TALENs are formed by the modular assembly of TALE domains, with each domain recognizing a specific nucleotide via two variable amino acids (Repeat Variable Diresidue). Like ZFNs, the two arms must be in close proximity for cleavage to occur. (C) The CRISPR/Cas9 system utilizes an ~85bp RNA-guide molecule (gRNA) to target the Cas9 endonuclease to the site of cleavage. The 5′ 20-bp of the gRNA recognizes the target sequence, while the remaining structure serves as a scaffold for Cas9 binding. DSB formation occurs 5–10 nucleotides upstream of the PAM.