Efficient targeting of FATS at a common fragile site in mice through TALEN-mediated double-hit genome modification
Efficient targeting of FATS at a common fragile site in mice through TALEN-mediated double-hit genome modification
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DOI：
10.1007/s10529-013-1387-z
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发表时间：
2014-03-01
影响因子：
2.7
通讯作者：
Li, Zheng
中科院分区：
文献类型：
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作者：
Ma, Ke;Wang, Jianying;Li, Zheng
转录激活剂样效应核酸酶（TALEN）已成为一种新开发的基因组编辑方法。然而，其在针对易受 DNA 损伤的特定基因组位点的应用仍不清楚。在这里，我们报告了一种基于 TALEN 靶向 FATS 的改良方法，FATS 是一种脆弱位点基因，其主要内含子具有富含 AT 的序列和二核苷酸重复。两对 FATS-TALEN 被设计用于专门切割 FATS 编码外显子的两个位点。体外转录后，FATS-TALEN 对的 mRNA 被显微注射到小鼠受精卵中。两对 FATS-TALEN 对的体内靶向效率比一对 FATS-TALEN 对的体内靶向效率高出三倍多。此外，还检测到大尺寸DNA缺失，这些缺失是可遗传的并且很容易通过PCR检测到。我们的研究表明，双击TALEN方法提高了体内靶向效率，并为通过PCR监测突变的种系传递提供了便利，这将有助于脆弱位点基因的功能研究。
Transcription activator-like effector nucleases (TALENs) have emerged as a newly developed approach for genome editing. However, its application in targeting specific genomic loci susceptible to DNA damage remains obscure. Here, we report a modified approach for TALENs-based targeting of FATS, a fragile-site gene whose major introns have AT-rich sequence and di-nucleotide repeats. Two pairs of FATS-TALENs were designed to cleave two sites specifically at a coding exon of FATS. After in vitro transcription, the mRNA from FATS-TALEN pairs was microinjected into mouse zygotes. The targeting efficiency of two FATS-TALEN pairs in vivo was more than threefold higher than that of one FATS-TALEN pair. Moreover, large-size DNA deletions were detected, which were heritable and easily detectable by PCR. Our study indicates that the double-hit TALEN approach enhances targeting efficiency in vivo and provides convenience for monitoring germline transmission of mutations by PCR, which will facilitate the functional research on fragile-site genes.