Molecular scissors, such as meganucleases, zinc-finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN), are valuable tools for generating double-strand breaks (DSB) in the genome that can lead to a functional knockout of the targeted gene or used to integrate a DNA sequence at a specific locus in the genome. Especially in farm animal species from which true pluripotent embryonic stem cells have not been established, these molecular scissors are a new option for engineering the genome in a way that was not feasible before. Meganucleases (also called homing nucleases) are natural proteins found in many single-cell organisms that are mainly involved in the cell’s repair mechanism after a strand break occurs. They are capable of recognising their binding site by identifying a sequence containing between 12 and >30 base pairs. The prototype enzyme for demonstrating DSB stimulation of gene targeting was I-SceI, which has a long recognition site (I-SceI 18 bp). The recognition specificity of enzymes such as I-SceI can be modified to be specific for a desired sequence within the genome. The use of meganucleases to genetically modify organisms has proved very successful in several species, including frog, fly, fish, plants, and human cells, but the intimate connection between the recognition and cleavage elements in the protein structure makes it difficult to alter one without affecting the other. The class of targeting reagents that has proved the most versatile and effective in recent years is that of ZFN. The ZFN possess separate DNA-binding and cleavage domains, which facilitate design according to the desired target. These molecules originate from the natural type IIS restriction enzyme FokI (Li et al. 1992 Proc. Natl. Acad. Sci. USA 89, 4275–4279). The cleavage domain has no sequence specificity and the binding domain can be used to make ZFN specific to a targeted sequence. The requirement for dimerisation of the FokI makes ZFN even more specific and avoids off-target events, as a monomeric cleavage does not occur at single binding sites. One zinc-finger molecule is specific for a base triplet; joining several zinc-finger molecules is sufficient to pick out a single target in a complex genome. ZFN have been used to modify the genome of several species as Xenopus, drosophila, C. elegans, zebrafish, rat, mouse, human cells, hamster cells, rabbit, pigs, and cattle. Different methods have been used to alter the host genomes either by ZFN mRNA or DNA injection into zygotes or by transfection of somatic cells followed by somatic cell nuclear transfer. Even a direct delivery of ZFN proteins can generate a targeted mutation (Gaj et al. 2012 Nat. Methods 9, 805–807). The efficiency of ZFN-mediated knockout was increased up to 10,000-fold compared with traditional gene knockout by homologous recombination. Rarely, off-target events were described but most were located in an intergenic or intronic region of the genome. Transcription activator-like effectors are a family of virulence factors produced by a genus of plant pathogens, Xanthomonas spp. The proteins naturally comprise 17 to 18 repeats of 34 amino acids. The binding specificity is determined by the amino acids at positions 12 and 13 within each repeat. Combined with an endonuclease, TALEs (referred to as TALENs) can be used to specifically target almost any known genomic sequence. The main difference between ZFNs and TALENs is the recognition of the DNA sequence. While ZFNs recognise nucleotide triplets, TALENs recognise single nucleotides, rendering TALENs, in theory, adjustable to any given sequence in a genome while ZFNs need defined prerequisites to be specific. TALENs have already been used to alter the genomes of rats, zebrafish, human iPSCs, and pigs (personal communication). Molecular scissors open a wide range of new applications for modifying the genome of different species or cells with which it has remained very difficult to work. Breeding for agricultural purposes and biomedicine, including the development of large animal models for human diseases and xenotransplantation, will greatly benefit from these new tools. With the advent of ZFN- and TALEN-mediated gene knockouts, mammalian transgenesis has taken a major leap forward as a straightforward technology for gene knockout and knock-in.
Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option