Abstract
Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes. A variety of reagents have been developed in recent years that can generate targeted double-stranded DNA cuts which can be repaired by the error-prone, non-homologous end joining repair system or via the homologous recombination-based double-strand break repair pathway provided a suitable template is available. These genome editing reagents require components for recognizing a specific DNA target site and for DNA-cleavage that generates the double-stranded break. In order to reduce potential toxic effects of genome editing reagents, it might be desirable to control the in vitro or in vivo activity of these reagents by incorporating regulatory switches that can reduce off-target activities and/or allow for these reagents to be turned on or off. This review will outline the various genome editing tools that are currently available and describe the strategies that have so far been employed for regulating these editing reagents. In addition, this review will examine potential regulatory switches/strategies that can be employed in the future in order to provide temporal control for these reagents.
Highlights
Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes
The major focus in this review is to examine the efforts that have been made in the development of programmable, endonuclease-based platforms and various molecular switches that could be employed for the temporal regulation of these DNA-cutting enzymes in order to reduce off-target activities
The first group consists of MNs, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which achieve sequence-specific DNA-binding via protein-DNA interactions [13,42]
Summary
Genome editing tools using DSB nuclease-driven reactions (Fig. 1) can be divided into two groups. The ‘MegaTev’ (Fig. 1d) architecture has been generated which involves fusion of the DNA-binding and cutting domain from a meganuclease (Mega, I-OnuI) with another nuclease domain derived from the GIY-YIG HEase (Tev, I-TevI) This protein was designed to position the two cutting domains ~30 bp apart on the DNA substrate and generate two DSBs with non-compatible single-stranded overhangs for more efficient gene disruption [108]. Similar to the MegaTev concept, Wolfs et al.have designed another dual nuclease, in which the Tev endonuclease domain is attached to the Cas nuclease domain, known as TevCas9 [109] This hybrid nuclease, when introduced within human embryonic kidney cells (HEK293) along with appropriate guide RNAs, has been shown to delete 33 to 36 bp of the target site, thereby creating two non-compatible DNA breaks at moderately higher frequencies (40%). Yes 1994 Unknown Multiple Short Electroporation, Viral transduction, Direct injection into zygotes
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