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Review
. 2013 Nov 14;5(11):2748-66.
doi: 10.3390/v5112748.

Newer gene editing technologies toward HIV gene therapy

N Manjunath  1 , Guohua Yi, Ying Dang, Premlata Shankar
Affiliations
Review

Newer gene editing technologies toward HIV gene therapy

N Manjunath et al. Viruses. .

Abstract

Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called "Berlin patient" who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy.

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Figures

Figure 1
Figure 1
Structure and schematic representation of zinc finger nuclease (ZFN) bound to the target site. (A) Crystal structure of a six-finger zinc finger protein (ZFP) complexed with its target DNA fragment (PDB ID: 1MEY, modeled by UCSF chimera). Each single zinc finger consists of one α-helix and two antiparallel β-sheets and contacts DNA with side chains of the α-helix; (B) Schematic representation of ZFN bound to double-stranded DNA. Each zinc finger unit recognizes 3 nucleotides of DNA, each ZFN module (left and right) recognizes 9–18 nucleotides. Once both ZFN modules bind DNA, the catalytic domain of Fok I endonuclease dimerizes and cleaves the DNA at the spacer region.
Figure 2
Figure 2
Structure and schematic representation of transcription activator-like effector nucleases (TALEN) bound to the target site. (A) Crystal structure of a TALEN complexed with the target DNA fragment (PDB ID: 3UGM, modeled by UCSF chimera software). Each single TALE unit is composed of two similar α-helices with intervening two variable amino acids that specifically recognize one single nucleotide; (B) Schematic representation of TALEN bound to double stranded DNA. Each TALEN unit recognizes one single nucleotide. Once both TALEN modules bind DNA, the Fok I endonuclease dimerizes and cuts DNA at the spacer region; (C) TALEN nucleotide recognition code. Variable diresidues (RVDs) located at amino acids 12 and 13 of each TALEN unit recognizes a single nucleotide according to the code: NG for T, HD for C, NI for A and NN for G or A.
Figure 3
Figure 3
Schematic representation of clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 System. (A) In prokaryotic cells, the CRISPR locus contains an array of repeats (of the same sequence) interspersed with spacers with unique sequences. This locus also contains DNA coding for tracRNA and Cas proteins. The transcribed pre-crRNA is processed by RNAse III into mature crRNA that associates with tracRNA. This RNA duplex then binds the Cas9 protein to form a ribonucleotide complex. crRNA guides this complex to bind complementary (to spacer in crRNA) target DNA following which, the Cas9 protein cleaves the DNA to induce DSB; (B) In engineered CRISPR/Cas9 system, a plasmid encoding a chimeric RNA consisting of crRNA targeting the gene of interest fused at its 3' end to tracrRNA and a plasmid encoding the Cas9 protein are cotransfected.
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