piggyBac transposase tools for genome engineering
piggyBac transposase tools for genome engineering
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DOI：
10.1073/pnas.1305987110
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发表时间：
2013-06-18
影响因子：
11.1
通讯作者：
Craig, Nancy L.
中科院分区：
文献类型：
--
作者：
Li, Xianghong;Burnight, Erin R.;Craig, Nancy L.
转座子piggyBac越来越多地用于遗传研究。在这里，我们描述了piggyBac转座酶的修改版本，具有潜在的广泛的应用，如可逆转基因和修改的插入靶向。piggyBac的特点是能够精确切除，将供体位点恢复到其前转座子状态。这一特性使得piggyBac可用于可逆转基因，当产生诱导多能干细胞而不永久改变基因组序列时，这是一个潜在的有价值的特征。为了避免piggyBac切除后通过重组的进一步基因组修饰，我们产生了切除能力/整合缺陷型（Exc（+）Int（-））转座酶。我们的研究结果还表明了靶DNA-转座酶相互作用的位置。基因组工程的另一个目标是开发可以将转基因引导到优选基因组区域的试剂。其他人已经表明，piggyBac转座酶在融合到异源DNA结合结构域时可以是有活性的。固有靶向有缺陷的Exc（+）Int（-）转座酶也可能是产生转座酶的有用中间体，所述转座酶的整合活性可以通过融合到位点特异性DNA结合结构域而被拯救和重定向。我们发现，融合到两个设计的锌指蛋白拯救了Int（-）表型。成功地引导转基因整合到基因组DNA中将在基因治疗和分子遗传学中具有广泛的应用。因此，Exc（+）Int（-）转座酶是基因组工程的潜在有用试剂，并提供了对转座酶-靶DNA相互作用机制的深入了解。
The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc(+)Int(-)) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc(+)Int(-) transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int(-) phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc(+)Int(-) transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.