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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.
Highlights
Recent advances in genome engineering using Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have facilitated the creation of targeted gene knockout mutations in zebrafish [1,2,3,4,5,6,7,8]
double-strand breaks (DSBs) induced by either ZFNs or TALENs can be repaired by non-homologous end joining (NHEJ) – an error-prone process that results in the creation of insertion or deletion mutations that can shift the translational reading frame and frequently lead to premature termination (Figure 1)
We report higher success rates for TALENs at inducing mutations at any given DNA target site when compared with Context-Dependent Assembly (CoDA) ZFNs
Summary
Recent advances in genome engineering using Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have facilitated the creation of targeted gene knockout mutations in zebrafish [1,2,3,4,5,6,7,8]. ZFNs consist of an engineered array of zinc fingers fused to the non-specific FokI nuclease domain and function as dimers to introduce targeted DNA double-strand breaks (DSBs). TALENs bind to DNA through a highly conserved 33–35 amino acid transcription activator-like (TAL) effector repeat domain found in the plant pathogen Xanthomonas. TAL effector repeats can be fused to the FokI nuclease domain to create TALENs capable of cleaving DNA as a dimer. DSBs induced by either ZFNs or TALENs can be repaired by non-homologous end joining (NHEJ) – an error-prone process that results in the creation of insertion or deletion mutations (indels) that can shift the translational reading frame and frequently lead to premature termination (Figure 1)
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