| Plant pathogenic bacteria Xanthomonas deliver TALE transcriptional factor proteins intoplant cytoplasma through type III secretion system. TALE protein can reprogram plant cellsby imitating eukaryotic transcriptional factors after entering the cells. There’s a simplerelation between TALE repeats and DNA base which can enable the researchers to predict thebinding sites of natural TALE protein and, on the other hand, make it possible to artificiallychange the repeats for target DNA recognition. However, the challenge to assemble TALErepeat into a specific order has always restricted the wide use of TALE nuclease. What’s more,the average number of amino acids used for recognizing one DNA base is four times largerthan that of ZFN. So, it has significant meaning to reduce some unnecessary part of TALEprotein as long as not lower its cleavage efficiency. In addition, SunnyTALEN backbone canperform7times higher cleavage efficiency than normal backbones in yeast, while more than2.5higher in human genome modification. This backbone has higher efficiency than theothers. Following experiments were carried out according to the information above:1. TALE repeat monomers were PCR amplified and assembled into repeat tetramersthrough Golden Gate cloning method making use of cut-ligation reaction. A library covers256tetramer plasmids were constructed.2. An expression vector coding TALEN that targets mouse Rosa26gene wereconstructed through our novel method fusing PCR mutation and Golden Gate cloning basedon the tetramer library.3. Efficiencies of TALENs with N-terminus of different lengths were tested both in yeastand human293T cells. SunnyTALEN backbone was constructed using our novel method formultiple site-directed mutagenesis.In total, this study has proven the feasibility and applicability of our fast, simple,efficient, tetramer library based method for TALEN construction. We also optimized thelength of N-terminus and successfully constructed SunnyTALEN backbone. |
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