Abstract
ABSTRACTZinc-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 3′UTR 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 3′UTR 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 3′UTR 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.
Highlights
Targeted gene disruption using either transcription activator-like effector nucleases (TALENs) (Christian et al, 2010; Li et al, 2011) or the CRISPR/Cas system (Jinek et al, 2012) is a powerful method for determining the function of a specific gene
We expected that the injected TALEN mRNAs would be translated in the oocytes and that the TALEN proteins would digest the genomic DNA before and after fertilization
In the CRISPR/Cas system, a single synthetic guide RNA is injected into embryos with Cas9 mRNA, which binds to the target site and recruits the Cas9 protein to produce double-strand breaks
Summary
Targeted gene disruption using either transcription activator-like effector nucleases (TALENs) (Christian et al, 2010; Li et al, 2011) or the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system (Jinek et al, 2012) is a powerful method for determining the function of a specific gene. TALENs are composed of a nuclear localization signal, an N-terminal domain, a target DNA-binding domain, a Cterminal domain and the nuclease domain of FokI, and are nuclear proteins that bind to target DNA to form dimers in the nuclease domain, inducing double strand cleavage. Division of Embryology and Genetics, Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima 739-8526, Japan
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