Light-Activated Gene Editing with a Photocaged Zinc-Finger Nuclease
Light-Activated Gene Editing with a Photocaged Zinc-Finger Nuclease
复制标题
DOI：
10.1002/anie.201101157
复制
发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Deiters, Alexander
中科院分区：
文献类型：
--
作者：
Chou, Chungjung;Deiters, Alexander
具有精确外部控制和高空间和时间分辨率的靶向基因修饰的一般方法将极大地推进遗传学、基因治疗和发育生物学的研究。然而，用于基因组DNA序列的引入和缺失的传统方法如同源重组[1]和非同源末端连接[2]通常在体内显示出非常低的效率，从而限制了它们的适用性。最近，这些过程的效率已大大提高了位点特异性引入双链断裂（DSB）到基因组DNA的能力。[3，4]已经开发了一个人工限制酶家族，即锌指核酸酶（ZFN），以序列选择性地实现dsDNA切割。ZFN已经成为重要的和广泛认可的工具，用于细胞、模式生物和可能的人类的遗传修饰，以研究基因功能和治疗遗传疾病。[5 - 9]在结构上，ZFN是含有两个结构域的嵌合蛋白：N-末端锌指结构域和C-末端核酸酶结构域。N-末端锌指结构域通常由三至四个Cys2His2 "指"组成。[10 - 13]每个手指通过DNA大沟中的氢键相互作用识别三个DNA碱基对。这些指状物与某些DNA序列的结合特异性可以通过选择[14]或模块化组装[15]以及随后的体内测试来工程化。[16]C-末端核酸酶结构域由IIS型限制酶FokI [17]进化而来，并与第二个ZFN以尾对尾构象二聚化，以在两个识别位点之间引入DSB。ZFN异二聚体识别24 bp复合DNA位点，其在统计学上保证在靶细胞和生物体的基因组中单一出现。[6]美国
A general approach for targeted gene modification with precise external control and high spatial and temporal resolution will greatly advance investigations in genetics, gene therapy, and developmental biology. However, traditional methods such as homologous recombination [1] and nonhomologous end joining [2] for the introduction and deletion of genomic DNA sequences usually display very low efficiency in vivo, thus limiting their applicability. Recently, the efficiency of these processes has been greatly improved by the ability to site-specifically introduce doublestrand breaks (DSBs) into genomic DNA.[3, 4] A family of artificial restriction enzymes, namely zinc-finger nucleases (ZFN), has been developed to sequence-selectively achieve dsDNA scission. ZFNs have since emerged as important and widely recognized tools for the genetic modification of cells, model organisms, and possibly humans to investigate gene function and to treat genetic disorders.[5–9]Structurally, a ZFN is a chimeric protein containing two domains: an N-terminal zinc-finger domain and a C-terminal nuclease domain. The N-terminal zinc-finger domain usually consists of three to four Cys2His2 “fingers”.[10–13] Each finger recognizes three DNA basepairs through hydrogen-bonding interactions in the major groove of the DNA. The binding specificities of these fingers to certain DNA sequences can be engineered by selection [14] or modular assembly [15] and subsequent in vivo testing.[16] The C-terminal nuclease domain was evolved from the type IIS restriction enzyme FokI [17] and is dimerized in a tail-to-tail conformation with a second ZFN to introduce a DSB between two recognition sites. ZFN heterodimers recognize a 24 bp composite DNA site which statistically guarantees single occurrence in the genome of targeted cells and organisms.[6]