Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells
Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells
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DOI：
10.1073/pnas.0605633103
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发表时间：
2006-10-31
影响因子：
11.1
通讯作者：
Carroll, Dana
中科院分区：
文献类型：
--
作者：
Morton, Jason;Davis, M. Wayne;Carroll, Dana
锌指核酸酶是一种嵌合蛋白，由连接到外壳核酸酶结构域的工程化锌指 DNA 结合基序组成。这些蛋白能诱导基因组 DNA 中特定位点的 DNA 双链断裂，然后成为细胞修复机制的底物。在这里，我们证明了工程锌指核酸酶能在线虫的体细胞中有效发挥作用。虽然在我们的试验中基因转换事件与未切割的 DNA 没有区别，但非同源末端连接会导致目标位点发生突变。染色体外阵列上的合成靶点是用一种先前表征过的核酸酶作为靶点，而内源基因组序列则是用一对专门设计的核酸酶作为靶点。在这两种情况下，诱导相应的核酸酶后，约有 20% 的靶位点发生了突变。染色体外靶点的改变主要是末端填充和钝性连接的产物。相比之下，染色体靶点的改变主要是缺失。我们认为这些差异反映了染色体外阵列中存在大量的同源模板，而用于修复染色体断裂的同源模板则很少。此外，我们还发现了易错 DNA 合成参与同源和非同源修复途径的证据。DNA 连接酶 IV 是高效末端连接（尤其是钝端连接）所必需的。如果缺乏这种酶，次要的末端连接途径在产生缺失时就会更多地依赖于微缺失。
Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an enclonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, approximate to 20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.