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TALENs Leapfrog ZFNs in Xenopus

10/11/2012
Megan Scudellari

Slicing, dicing nucleases are much-needed tools for genetic engineering in Xenopus. TALENs trump ZFNs for the job.


Xenopus laevis and Xenopus tropicalis may be slimy and strangely egg-shaped, but these aquatic frogs are also classical animal models for embryonic development thanks to the easy availability of fertilized eggs and the large size and rapid development of their embryos. But thespecies have fallen to the wayside during the recent push toward genetics and genetic engineering due to a lack of technologies to perform gene editing in Xenopus embryos.

Now, a collaboration of researchers in China and the United States has applied an old technique in a new way to successfully induce genetic mutations in Xenopus embryos. The results, published online this month in the Proceedings of the National Academy of Sciences (1), demonstrate that transcription activator-like effector nucleases, or TALENs, are robust tools for gene disruption in Xenopus. The new tool could put this popular animal model back on the genetics map.

Xenopus laevis may be slimy and strangely egg-shaped, but these aquatic frogs are also classical animal models for embryonic development thanks to the easy availability of fertilized eggs and the large size and rapid development of their embryos. Source: Tim Vickers





TALENs are engineered DNA nucleases that consist of two domains: a custom-designed DNA-binding domain that attaches to the chosen site in the genome, and a nonspecific nuclease domain to do the cutting. TALENs have been effective tools for gene editing in rat, zebrafish, and Caenorhabditis elegans, but this is the first time they have been applied to Xenopus.

Hui Zhao, a developmental biologist at the Chinese University of Hong Kong, and colleagues constructed eight pairs of TALENs to target eight Xenopus genes. Binding a TALEN at either side of an identified gene causes double-strand breaks at the predetermined sites. Next, DNA repair re-joins the cut ends of DNA, effectively slicing out the gene of choice. In the eight experiments, gene editing was up to 95.7% efficient, and the resulting mutations were successfully passed through the germ line to the next generation of frogs.

“It seems that we could disrupt all genes by using this technology,” said Zhao in an email to BioTechniques. In many embryos, both alleles of a targeted gene were knocked out, says Zhao, demonstrating just how effective the procedure could be.

The team also compared TALENs to another gene-editing technology, zinc finger nucleases (ZFNs). “We had very difficult experience with ZFNs,” says Zhao. “When we shifted to TALENs, we were surprised at how good [they] were.”

Zhao and his team, who study germ layer formation and tissue repair in Xenopus, plan to use TALENs to establish knockout lines for their work, and even hope to use the technology to make gene knock-ins. “Application of this technology will help pave the way for more comprehensive genetic studies using this excellent research organism,” he concluded.

References

1. Lei, Y., et al. 2012. Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). PNAS, doi:10.1073/pnas.1215421109.