利用TALEN进行果蝇基因组大片段DNA knock-out与knock-in新技术的建立及其应用

TALE（transcription activator-like effector）是最初发现于植物病原菌Xanthomonas sp. 中的一类DNA结合蛋白，具有高度保守的DNA结合结构域。每个DNA结合结构域通常包含17.5个（共4种，分别识别A、T、C、G）串联排列的重复单元。这些重复单元的不同排列组合决定了TALE蛋白的DNA结合特异性。TALEN（TALE nuclease ）是TALE DNA结合结构域与核酸内切酶FokI功能结构域的融合蛋白，兼具DNA结合特异性和核酸内切酶活性，人工设计TALEN几乎可以使得在果蝇基因组任意位点产生DNA双链断裂成为可能。本项目探索建立利用TALEN在果蝇体内实现DNA大片段knock-out与knock-in的新技术,并以此为基础研究DNA重复序列与异染色质形成及维持的关系。

The TALEs (transcription activator-like effectors) are a family of proteins that were first discovered in the plant pathogen Xanthomonas sp., containing a central conserved, but sequence specific DNA binding domain, which usually consists of 17.5 tandem repeats of a 34 amino acids monomer. The tadem repeats specifies the DNA binding specificity by its 12th and 13th repeat variable di-residues (RVDs). Upon the fusion of the customized DNA binding domain of　a TALE and the enzymatic active domain of the FokI nuclease, TALEN can be directed to any selected target of the genome to yield a double-strand break (DSB). Using these TALENs, we have for the first time targeted the yellow and the fio genes in Drosophila with high efficiency and specificity. We aim to establish a new technique by which we can use TALEN to efficiently carry out the knock-out and knock-in of the large fragment DNA in Drosophila genome. With the established techniques, we are going to study the mechanistic relationship between repetitive DNA sequence and heterochromatin formation/maintenance.