Induction and Repair of Zinc-Finger Nuclease-Targeted Double-Strand Breaks in Caenorhabditis Elegans Somatic Cells

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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2006-10, Vol.103 (44), p.16370-16375
Main Authors: Morton, Jason, Davis, M. Wayne, Jorgensen, Erik M., Carroll, Dana
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Binding sites
Biological Sciences
Caenorhabditis elegans - enzymology
Caenorhabditis elegans - genetics
DNA
DNA Breaks, Double-Stranded
DNA Ligases - metabolism
DNA repair
DNA Repair - genetics
DNA, Helminth - genetics
Endonucleases - metabolism
Genetic mutation
Genome, Helminth - genetics
Genomics
Germ cells
Germ Cells - metabolism
Ligation
Mutation
Mutation - genetics
Nematodes
Plasmids
Polymerase chain reaction
Proteins
Somatic cells
Zinc
Zinc Fingers
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Summary: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, ≈20% 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0605633103