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Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.
Fig. 3. Frequencies of mutations and abnormal embryos induced by the indicated TALENs or ZFNs in Xenopus. Mutation frequencies were assayed as shown in Fig. 2. Only the mutation ratios induced by lower doses of ZFNs are shown because higher doses (200, 500, and 800 pg) resulted in dead or malformed embryos. (A) Frequencies of targeted mutagenesis induced by TALENs or ZFNs for the genes and at the doses indicated in the panels. All data refer to X. tropicalis except in C where X. laevis results are also shown. (D) Percentage of normal, abnormal, and dead X. tropicalis embryos injected with the indicated doses of TALEN or ZFN mRNAs. The injected embryos were inspected at 48 h postfertilization (about stage 41). (G) Overall morphology of X. tropicalis embryos injected with TALEN mRNAs directed against the indicated gene at stage 41. Curled axis, repression of head structures including eyes, and loss of pigments were observed. Such abnormal tadpoles usually could not complete metamorphosis.
Fig. 4. Phenotypes of ptf1a/p48-TALEN targeted X. tropicalis. (A and B) Anatomical analysis revealed visceral abnormalities such as much reduced pancreas in ptf1a/p48-TALEN-injected G0 froglets. The pancreas is outlined by dashed lines. (C) DNA sequencing of genomic DNA extracted from hindlimbtissue dissected from a froglet showing a phenotype similar to that in B confirmed the gene disruption at the ptf1a/p48 locus (20/23) (du, duodenum; st, stomach). (D) Whole-mount in situ hybridization of pancreas marker pdip in X. tropicalis tadpoles injected with the indicated TALEN mRNAs. ptf1a/p48-TALENs, but not ets1-TALENs, induced repression of pancreas bud formation. (D) Uninjected control embryos, (E) ptf1a/p48-TALEN- injected embryos (800 pg), and (F) ets1-TALEN-injected embryos (800 pg). (G) Summary of the phenotypes shown in D. The TALEN mRNAs were injected into the animal pole region at the one-cell stage; embryos were analyzed at stage 40.
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