Program in Gene Function and Expression; Department of Biochemistry and Molecular Pharmacology; Program in Molecular Medicine
Zebrafish; Genome; Chromosome Deletion
Genetics and Genomics | Genomics
Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.
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This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.
DOI of Published Version
Genome Res. 2013 Jun;23(6):1008-17. doi: 10.1101/gr.154070.112. Epub 2013 Mar 11. Link to article on publisher's site
Gupta, Ankit; Hall, Victoria L.; Kok, Fatma O.; Shin, Masahiro; McNulty, Joseph C.; Lawson, Nathan D.; and Wolfe, Scot A., "Targeted chromosomal deletions and inversions in zebrafish" (2013). University of Massachusetts Medical School Faculty Publications. 87.