Targeted chromosomal deletions and inversions in zebrafish

Sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), provide effective tools for engineering the genomes of plants and animals (Urnov et al. 2010; Carroll 2011; Joung and Sander 2013). These reagents are revolutionizing the application of reverse genetic approaches in a broad array of organisms by facilitating the generation of targeted double-strand breaks (DSBs) within the genome. These DSBs can disrupt gene function through imprecise break repair or promote tailor-made alterations to the genome through homologous recombination from an exogenously supplied donor DNA (Urnov et al. 2010; Carroll 2011; Joung and Sander 2013). Each SSN comprises a pair of programmable DNA-binding domains recognizing neighboring genomic sequences fused to the nuclease domain from FokI. Upon binding of both partners to the target locus, the appended FokI domains dimerize and generate a DSB in the spacer between the monomer binding sites. The creation of two DSBs within the genome can stimulate the formation of larger genomic aberrations. In cultured cells, two nucleases that target nearby regions within the same chromosome can generate deletions of the intervening genomic segment (∼15 kb) with efficiencies approaching 10% of the treated genomes (Lee et al. 2010; Carlson et al. 2012; Kim et al. 2012). In addition, these tandem DSBs can generate segmental inversions, as well as segmental duplications through unequal exchange between homologous/sister chromosomes (Carlson et al. 2012; Lee et al. 2012). Likewise, the generation of DSBs on disparate chromosomes can promote translocations between the cleaved segments albeit at low frequency (Brunet et al. 2009; Simsek et al. 2011). Targeted genomic deletions have also been generated in invertebrate animals using two SSNs (Ma et al. 2012). In vertebrates, the description of segmental deletions using two SSNs has been limited to a single founder in rats harboring a 2.5-kb deletion (Menoret et al. 2010). However, this study lacks a description of the deletion rates induced in treated embryos and the transmission frequency of the associated deletion, which leaves undefined the efficiency of these processes in a complex vertebrate organism. The ability to generate large segmental deletions reliably within a vertebrate genome would provide an avenue for the functional interrogation of noncoding elements, such as intergenic or intronic cis-regulatory modules, miRNA clusters, or lincRNAs. Due to the paucity of described functional sequences or conservation in such elements, they may be challenging to disrupt using a single nuclease. Targeted deletions would also facilitate the removal of alternately spliced exons to specifically disable the function of a single protein isoform. Here we examine the feasibility of creating segmental deletions and inversions within the zebrafish genome using two SSNs targeting three different protein-coding loci (apoea, flt4, slc24a5-lepa) and two noncoding sequences (the linc-birc6 [megamind] lincRNA gene and the globin locus control region). We find that two highly active SSNs can efficiently create segmental deletions of up to 69 kb at frequencies between 1% and 15% in somatic cells. Importantly, we demonstrate that these deletions can be transmitted through the germline of treated animals with comparable frequencies. Local segmental inversions can also be created through this approach, although these aberrations occur at a lower frequency than simple deletions. Given the simplicity with which SSNs can be constructed or purchased for any desired target sequence (Urnov et al. 2010; Carroll 2011; Joung and Sander 2013), large targeted deletions can be generated throughout the genome in vertebrates, allowing the functional analysis of noncoding sequence elements during development or in disease progression.