Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

Abstract

Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 39UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 39UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 39UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.