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.

Figure 1. Genome engineering using ZFNs and TALENs.

ZFNs utilize DNA binding domains that recognize ∼3 bp sequences and are joined together to create arrays that can target specific DNA sequences. TALENs bind DNA using TAL effector repeat domains derived from Xanthomonas that recognize individual nucleotides. These TALE repeats are ligated together to create binding arrays that recognize extended DNA sequences. Each ZFN or TALEN binds to a half-site with dimeric FokI nuclease domains cleaving the DNA within the intervening spacer region. The mechanism responsible for inducing DNA mutations is identical using either methodology, where nuclease-induced double stranded DNA breaks are repaired by error-prone non-homologous end joining (NHEJ) resulting in the creation of insertion or deletion mutations (indels).

Figure 2. CoDA ZFN and TALEN targeting sites used for assessing mutation rates across platforms.

A) Schematic comparing target sites for ZFNs generated using CoDA compared with TALENs. Peptide sequence is represented by brown bar. Arrowheads denote sites used to target ZFNs (blue) compared to TALENs (open white). All potential CoDA sites are shown as blue hashes within the coding sequence. B) Target sequences for ZFNs and TALENs. Blue text indicates DNA binding site of ZFN or TALEN, red text indicates spacer region. The jak3 and myoD targeting sites are overlapping for both ZFNs and TALENs, shown by boxed text within the TALEN sequence. Start codons are shown in boxed text for TALENs designed to bmi1, ikzf1, and phf6. Target sites are denoted by their percentage distance within the coding region (% coding = % peptide sequence).

Figure 3. TALENs exhibit high mutation rates.

A) Table showing somatic mutation rates of ZFNs and TALENs for five genes. Mutation rate was calculated as number of mutant sequences divided by the total number of sequences analyzed for a given target region. Raw sequence scores are shown in parentheses. Each TALEN had a significantly different mutation rate compared to ZFNs as assessed by Fisher Exact Test (p<0.001 denoted by asterisks). B) Plot of somatic mutation rates of CoDA ZFNs and TALENs in zebrafish , , . The average mutation rate of all TALENs was higher than that of CoDA ZFNs. ZFNs with mutation rates of 0% were excluded from this analysis. Datum points of TALENs from different research groups are distinguished by color.

Figure 4. Sequences of somatic zebrafish gene mutations induced by TALENs.

Mutant sequences were aligned to the wild-type sequence. The length and frequency of indels are noted to the right. The target site is shown at top with each TALEN half-site highlighted in yellow and the spacer sequence highlighted in gray. Deletions (Δ) are shown in red with gray highlight, insertions (+) are shown in blue.

Figure 5. TALENs cause a wide diversity of DNA mutations.

Indels were classified according to length. The frequency of different length mutations is shown as a percentage of all sequences.

Figure 6. TALENs and ZFNs exhibit similar toxicity profiles.

The cumulative percentage of embryos that were dead or deformed by 3 days post-fertilization is denoted. CoDA ZFNs from the same genes are shown on the right. The number of fish examined for toxicity is shown below; the mutation rate is derived from a pool of 12 embryos.