Abstract
SummaryTranscription activator-like effector nucleases (TALENs) are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using Xenopus tropicalis (X. tropicalis), we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.
Highlights
Gene knockout is an important method used to demonstrate the function of a specific gene
Summary Transcription activator-like effector nucleases (TALENs) are facile and potent tools used to modify a gene of interest for targeted gene knockout
TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI
Summary
Gene knockout is an important method used to demonstrate the function of a specific gene. TALENs are becoming powerful molecular tools because of their simple design (Boch et al, 2009; Moscou and Bogdanove, 2009) and rapid assembly (Cermak et al, 2011), and they have been successfully used for targeted mutagenesis in several species (Wood et al, 2011; Cade et al, 2012; Lei et al, 2012; Liu et al, 2012; Wang et al, 2013) Both ZFNs and TALENs work as dimers to cleave the target DNA sequence, which is repaired by homologous recombination (Bibikova et al, 2001) or modified by non-homologous end-joining (Lukacsovich et al, 1994). The latter results in nucleotide insertion and/or deletion at the cleavage sites, frequently leading to a loss of gene function
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