The past and presence of gene targeting: from chemicals and DNA via proteins to RNA

Abstract

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Keywords: CRISPR/dCas; TALEs (transcription activator-like effectors); ZFPs (zinc finger proteins); genome editing; polyamides.

Figure 1.

Co-transfection of reporter plasmids with mTFO-MunI allowed MunI digestion of the murine GAA (mGAA) gene and induced HR. A2780 cells were co-transfected with reporter plasmids and 0.25 µM TFO-conjugate in the absence or presence of a HR fragment. Total amount of DNA was 300 ng in each well. After 48 h, luciferase activity was determined. The luciferase expression of pGL2-CMV-mGAA was set at 100% for each independent experiment. Transfections were performed in triplicate for two independent experiments. One experiment is shown as representative (mean + s.d.). *, p < 0.05, **, p < 0.01. (Online version in colour.)

Figure 2.

Surface plasmon resonance (SPR analysis) of TFO binding to immobilized DNA targets at 37°C in MES buffer, pH 6.5. The sensorgrams show complex formation following injection of 35 ml 4 mM TFO (indicated by a black horizontal bar; (a) TFO-GU (targeting VEGF); (b) TFO-TM (targeting VEGF); (c) mTFO (targeting mGAA); (d) hTFO (targeting human GAA (hGAA)) over surfaces containing immobilized biotinylated hGAA (green line), mGAA (blue line) and VEGF (red line) target hairpin duplexes, after which dissociation of the complexes is monitored [41].

Figure 3.

Topoisomerase I-mediated DNA cleavage to study the specificity of TFO-camptothecin (CPT) conjugates. (a) Structure of the TFO-CPT conjugate used in this study. (b) Cleavage products mediated by topoisomerase I (Topo I). Adenine/guanine-specific Maxam-Gilbert chemical cleavage reactions were used as markers (lane G+A). Target duplexes were incubated with 10 units of Topo I and in the presence of 50 µM CPT, or 5 µM TFO-CPT conjugates. The orientation of the triplex is indicated. Arrows indicate Topo I-mediated DNA cleavage at the target site (TFO-CPT 3′ cleaves at the 3′ end of the triple helix and TFO-CPT 5′ cleaves at the 5′ end of the triple helix). (Online version in colour.)

Figure 4.

Induction of DNA damage by TFO-CPT or scPvuII after transfection. A2780 cells were treated with 2 µM of the hTFO-CPT conjugate (and hTFO for control) or 1 µg of scPvuII. After 3 h, cells were collected and stained for phosphorylated H2AX (γH2AX) and analyzed by FACS. Transfections were performed in duplicate for three independent experiments. Data are represented as means of the means + s.e.m. Statistical comparison was performed between cells treated with Saint Mix only and cells treated with TFO only, hTFO-CPT or scPvuII. *, p < 0.05, **, p < 0.01. The chemical conjugation of the TFO to CPT has been described in detail elsewhere (see Vekhoff et al. [43]. In short, 3′ thiophosphorylated TFOs were conjugated to 10-(6-bromohexyloxy)-CPT and purified by reversed-phase high performance liquid chromatography (HPLC): hTFO-CPT: retention time 18.6 min; mTFO-CPT: retention time 18.4 min. The conjugates were characterized by UV spectroscopy, denaturing polyacrylamide gel electrophoresis and mass spectroscopy (ES-MS). ES-MS: hTFO-CPT: found [M-H] 4621.74; calculated: 4621.1. mTFO-CPT: found [M-H] 6988.24; calculated: 6988.8. SM, the delivery agent Saint Mix; scPvuII, single chain PvuII.