Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

Abstract

Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function inCiona

“…A TALEN targeting this gene was chosen to act as a control. Potential off-target mutagenic effects or other forms of toxicity are a serious concern when using TALENs (Moore et al, 2012). When mutations are introduced to target Fgf11, no knockout phenotypic effects should be seen.…”

snow white, a Zebrafish Model of Hermansky-Pudlak Syndrome Type 5

“…To this end, several zebrafish mutants, including fading vision (fdv; pmel17), fade out (fad; unknown locus), platinum (plt; vps11), vps18, and leberknodel (lbk; vps39), have already been characterized for LRO dysfunction; Pmel17 is critical for melanosome formation, as described above, and vps11, vps18, and vps39 are part of the HOPS complex required for vesicle fusion and tethering (Schonthaler et al 2005(Schonthaler et al , 2008Bahadori et al 2006;Maldonado et al 2006;Thomas et al 2011;see Navarro et al 2008 for review). Given the ability to perform forward genetic screens in zebrafish, the relative ease of cloning mutations from these screens (Bibliowicz et al 2011;Gestri et al 2012;Obholzer et al 2012), and the ability to generate targeted mutations (Cade et al 2012; Dahlem et al 2012;Moore et al 2012;Blackburn et al 2013;Hwang et al 2013), the identification and generation of additional zebrafish models of human albinism disorders should provide a promising complement through which the molecular and cellular underpinnings of these disorders can be elucidated. 1, 2, 3).…”

A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish

“…More recently, specific genomic DSB has also been successfully generated by using RNA-guided Cas9 nuclease, providing an alternative approach for genome editing [4,5]. To date, genome editing mediated by these engineered nucleases has been successfully implemented in cells and in a number of organisms including Xenopus tropicalis and zebrafish [6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”