TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.
TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.
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DOI：
10.1093/nar/gkq704
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发表时间：
2011-01
影响因子：
14.9
通讯作者：
Yang B
中科院分区：
文献类型：
--
作者：
Li T;Huang S;Jiang WZ;Wright D;Spalding MH;Weeks DP;Yang B
DNA双链断裂增强细胞中的同源重组，并已通过使用工程化核酸内切酶用于靶向基因组编辑。在此我们报道了一组罕见切割、位点特异性DNA核酸酶的创建和初步特性，这些核酸酶是通过将限制性内切酶FokI核酸内切酶结构域（FN）与AvrXa7和PthXo1的高特异性DNA结合结构域融合而产生的。AvrXa7和PthXo1是转录激活因子样（TAL）效应子家族的成员，其中心重复单元决定靶DNA识别，并且可以模块化构建以产生新的DNA特异性。杂合的FN - AvrXa7、AvrXa7 - FN和PthXo1 - FN蛋白既保留了对其靶DNA的识别特异性（AvrXa7为26bp序列，PthXo1为24bp），又保留了FokI的双链DNA切割活性，因此被称为TAL核酸酶（TALNs）。在体外的最佳条件下，所有三种TALNs都在TAL结合位点附近切割DNA。当在酵母中表达时，TALNs促进含有成对AvrXa7或不对称AvrXa7/PthXo1靶序列的LacZ基因的DNA同源重组。我们的结果证明了创建一个新型TALNs工具箱的可行性，其在缺乏便捷的靶向基因敲除和同源重组机制的生物体中具有靶向基因组修饰的潜力。
DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.
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