Gupta, A., Hall, V.L., Kok, F.O., Shin, M., McNulty, J.C., Lawson, N.D., and Wolfe, S.A. (2013) Targeted Chromosomal Deletions and Inversions in Zebrafish. Genome research. 23(6):1008-17.
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 non-coding 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 non-coding 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 to 15%), and have excised an entire lincRNA 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 non-coding elements on an organismic