Site‐Specific Gene Targeting Using Transcription Activator‐Like Effector (TALE)‐Based Nuclease in Brassica oleracea

Abstract

Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.

TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna

“…In knock-in experiments, HR repair has been mainly used for the precise integration of foreign genes via homologous recombination between the genome and donor DNA, which contains the gene(s) of interest. These characteristics have enabled the precise manipulation of the genome in various organisms such as silkworm13, cattle14, Brassica oleracea 15, Anopheles gambiae 16, Aedes aegypti 17, medaka18, liverwort19, and wheat2021.…”

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice

“…(Shan et al, 2013), barley (Hordeum vulgare; Wendt et al, 2013), wheat (Triticum aestivum; Wang et al, 2014) Brassica oleracea (Sun et al, 2013), potato (Solanum tuberosum; Sawai et al, 2014;, soybean (Glycine max; Haun et al, 2014), maize (Zea mays; Char et al, 2015), and tomato (Solanum lycopersicum; Lor et al, 2014). However, there are only a few reports of TALEN-mediated mutations being transmitted to the next generation: in Arabidopsis Forner et al, 2015), rice (Li et al, 2012;Shan et al, 2015;Wang et al, 2015;, wheat (Wang et al, 2014), soybean (Haun et al, 2014), barley (Gurushidze et al, 2014), and maize (Char et al, 2015).…”

Site-Directed Mutagenesis in Barley by Expression of TALE Nuclease in Embryogenic Pollen

“…Therefore, TALENs were successfully applied for site-directed mutagenesis in a number of model and crop plants like Arabidopsis , tobacco , Nicotiana benthamiana (Mahfouz et al 2011), Brachypodium distachyon (Shan et al 2013), rice (Li et al 2012), Brassica oleracea (Sun et al 2013), soybean (Haun et al 2014), wheat (Wang et al 2014), maize (Liang et al 2014), and barley (Wendt et al 2013;Gurushidze et al 2014). …”