Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Abstract

Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, Ϸ20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.DNA repair ͉ gene targeting ͉ nematodes ͉ nonhomologous end joining T he ability to make targeted double-strand breaks in chromosomal DNA has several important uses. It allows the detailed study of DNA repair mechanisms; it leads to localized mutagenesis at the break site; and it enhances the efficiency of targeted gene replacement through homologous recombination. We have been exploring the capabilities of one class of targetable cleavage reagents, the zinc-finger nucleases (ZFNs).ZFNs are chimeric proteins composed of DNA-binding Cys 2 His 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme FokI (1). Each finger makes contact primarily with a separate DNA triplet (2, 3). Natural and artificial zinc fingers have been characterized that bind to all 5Ј-GNN-3Ј, many ANN and CNN, and some TNN triplets (4-7). Furthermore, the modular nature of the zinc fingers allows them to be joined in essentially arbitrary combinations. Typically, three zinc fingers are combined to bind to a specific 9-bp DNA sequence with the nanomolar affinity required to be biologically useful, but additional fingers can be incorporated to confer increased specificity (8-11). Zinc-finger fusions to various functional domains have been used to create artificial transcription factors and DNA-modifying proteins (12,13).When attached to the FokI nuclease domain, zinc fingers can direct cleavage to specific DNA sequences. The nuclease domain must dimerize to cleave DNA (14), and because the di...

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…In previous studies, HR-mediated gene-replacement strategies have been the preferred mode of gene replacement in plants (Wright et al, 2005;Shukla et al, 2009;Townsend et al, 2009;de Pater et al, 2013), while NHEJ-mediated repair of ZFN-induced DSBs has been limited to targeted mutagenesis and transgene removal (Lloyd et al, 2005;Morton et al, 2006;Maeder et al, 2008;de Pater et al, 2009;Tovkach et al, 2009;Osakabe et al, 2010;Petolino et al, 2010;Zhang et al, 2010). Our findings show that the NHEJ DNA-repair pathway can be harnessed for gene replacement in plant species.…”

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

“…In such instances, rapid and permanent gene knockout, using ZFNs, can offer distinct advantages. Moreover, the growing number of reports, using ZFNs across different species (11)(12)(13)16), suggest that ZFN-mediated gene disruption may be a robust and general method for targeted gene knockout. There are several other critical differences between the present approach and existing methods that offer further advantages.…”

“…In mammalian systems, such as Chinese hamster ovary (CHO) cells, the ratio of HDR to NHEJ-based repair has been found to be 9:13 (10). Studies in Drosophila (11) and later in both plants and worms (12,13) showed that DSBs generated by site-specific zinc-finger nucleases (ZFNs) resulted in targeted mutagenesis consistent with repair by NHEJ. In this article, we now extend the ZFN approach to mammalian systems.…”

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

“…For example, it may be useful for elucidating the transfating mechanisms of presumptive blastocoelar cells to PMCs in embryos whose PMCs were depleted at the mesenchyme stage (31). The combined approach described here using sea urchin embryos might be extended to other multicellular model systems, such as nematodes and zebrafish, in which not only fluorescence imaging techniques but also ZFN-mediated genome modification techniques are available (9,10,32).…”