Enhancing gene targeting with designed zinc finger nucleases
Enhancing gene targeting with designed zinc finger nucleases
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DOI：
10.1126/science.1079512
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发表时间：
2003-05-02
期刊：
影响因子：
56.9
通讯作者：
Carroll, D
中科院分区：
文献类型：
--
作者：
Bibikova, M;Beumer, K;Carroll, D
基因靶向-通过同源重组进行基因置换的过程-是一种非常有用但通常效率低下的技术（1）。提高基因靶向效率的一般方法在许多情况下都是有价值的，将这种遗传工具扩展到更广泛的生物体也是如此。模型实验已经证明，染色体靶标中的双链断裂（DSB）极大地提高了局部重组事件的频率（2-6）。我们一直在开发嵌合锌指核酸酶（ZFN）作为基因靶向的潜在刺激物（7，8）。ZFN具有由三个Cys 2 His 2锌指连接至非特异性DNA切割结构域组成的DNA识别结构域（9）。切割结构域必须二聚化以充当核酸酶（7），并且这对于ZFN而言最容易通过为两组紧密接近且处于适当取向的锌指提供结合位点来实现（7，8）。控制锌指的识别特异性的能力（10，11）打开了将切割引导至任意选择的染色体位点而无需预先操纵靶的前景。我们设计并构建了一对ZFN（yA，yB），用于果蝇X染色体上黄色（y）基因第二外显子中的位点（12），方法是将其他研究中显示的锌指结合靶序列的组成三联体（13）。每个三指DNA结合结构域识别9个碱基对（bp）。二聚化的要求指定了基因组中唯一的18-bp识别位点。先前，我们表明，由于切割和非同源末端连接（NHEJ），这些ZFN在幼虫中的表达导致体细胞和种系突变的产生（12）。在这里，我们测试了当提供同源供体DNA时ZFN诱导的断裂刺激基因靶向的能力。构建突变体供体（yM），其携带8 kb的y同源性，其中ZFN识别位点被两个框内终止密码子和Xho I位点取代。将该突变体插入到P元件载体中，所述P元件载体的侧翼为FLP重组酶和大范围核酸酶I-Sce I的识别位点以允许供体的切除和线性化，并将其引入到果蝇基因组中。通过这种方式原位产生线性染色体外供体DNA已显示出增强其重组的有效性（14，15）。供体通过与yA和yB核酸酶的转基因以及FLP和/或I-Sce I的转基因进行适当的杂交来组合，所有这些都在果蝇HSP 70启动子的控制下。携带这些组件的幼虫进行处理的热休克协议在35 0至4天后产卵。实验以五种形式进行：ND，无供体：仅yA yB; ID，整合供体：yA yB供体，无FLP或I-Sce I; CD，环状染色体外供体：yA yB FLP供体; LD，线性染色体外供体：yA yB FLP I-Sce I供体; DO，仅线性供体：FLP I-Sce I供体，但无ZFN。从热休克方案中出现的成年人被交叉以揭示生殖系y突变（12）。在雄性和雌性中，存在供体时突变频率升高，并随着其变为染色体外和线性而进一步升高（表S1）。在最好的情况下，近20%的雄性和14%的雌性产生了至少一个突变后代。从突变体中回收染色体DNA，并通过聚合酶链反应（PCR）、Xho I消化和序列测定进行分析（12）。鉴定了供体的Homoprotein替代物和NHEJ产物。生殖系突变体的恢复占所有后代的百分比...
Gene targeting—the process of gene replacement by homologous recombination—is a very useful but typically inefficient technique (1). A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would the extension of this genetic tool to a broader range of organisms. Model experiments have demonstrated that a double-strand break (DSB) in the chromosomal target greatly enhances the frequency of localized recombination events (2–6). We have been developing chimeric zinc finger nucleases (ZFNs) as potential stimulators of gene targeting (7, 8). The ZFNs have a DNA recognition domain composed of three Cys2His2 zinc fingers linked to a nonspecific DNA cleavage domain (9). The cleavage domain must dimerize to act as a nuclease (7), and this is most readily achieved for the ZFNs by providing binding sites for two sets of zinc fingers in close proximity and in the appropriate orientations (7, 8). The ability to control the recognition specificity of zinc fingers (10, 11) opens the prospect of directing cleavage to arbitrarily chosen chromosomal sites, without prior manipulation of the target. We designed and constructed a pair of ZFNs (yA, yB) for a site in the second exon of the yellow (y) gene on the X chromosome of Drosophila melanogaster (12) by combining zinc fingers that had been shown in other studies to bind the component triplets of the target sequence (13). Each three-finger DNA binding domain recognizes 9 base pairs (bp). The requirement for dimerization specifies an 18-bp recognition site that is unique in the genome. Previously, we showed that expression of these ZFNs in larvae led to the production of both somatic and germline mutations, due to cleavage and nonhomologous end joining (NHEJ)(12). Here, we tested the ability of ZFN-induced breaks to stimulate gene targeting when a homologous donor DNA is provided. A mutant donor (yM) was constructed, which carries 8 kb of y homology, into which the ZFN recognition site was replaced with two in-frame stop codons and an Xho I site. This mutant was inserted into a P-element vector, flanked by recognition sites for the FLP recombinase and the meganuclease I–Sce I to permit excision and linearization of the donor, and was introduced into the fly genome. Generating a linear extrachromosomal donor DNA in situ by this means has been shown to enhance its effectiveness in recombination (14, 15). The donor was combined by appropriate crosses with transgenes for the yA and yB nucleases and those for FLP and/or I–Sce I, all under the control of the Drosophila HSP70 promoter. Larvae carrying these components were treated by a heat-shock protocol at 35 0 to 4 days after egg laying. The experiment was performed in five versions: ND, no donor: yA yB only; ID, integrated donor: yA yB donor, no FLP or I–Sce I; CD, circular extrachromosomal donor: yA yB FLP donor; LD, linear extrachromosomal donor: yA yB FLP I–Sce I donor; DO, linear donor only: FLP I–Sce I donor, but no ZFNs. Adults emerging from the heat-shock protocol were crossed to reveal germline y mutations (12). In both males and females, the frequencies of mutations rose in the presence of the donor and increased further as it became extrachromosomal and linear (table S1). In the best cases, nearly 20% of males and 14% of females yielded at least one mutant offspring. Chromosomal DNA was recovered from the mutants and analyzed by polymerase chain reaction (PCR), Xho I digestion, and sequence determination (12). Homologous replacements with the donor and the products of NHEJ were identified. The recovery of germline mutants as percent of all offspring …