Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting
Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting
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DOI：
10.1093/nar/gkr739
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发表时间：
2011-09-16
影响因子：
14.9
通讯作者：
Voytas DF
中科院分区：
文献类型：
--
作者：
Cermak T;Doyle EL;Christian M;Wang L;Zhang Y;Schmidt C;Baller JA;Somia NV;Bogdanove AJ;Voytas DF
TALEN是基因组工程的重要新工具。植物病原性黄单胞菌类转录激活因子（TAL）效应子的融合体FokI核酸酶TALENs成对结合和切割DNA. spp.to结合特异性由TAL效应物中多态性氨基酸重复序列的可定制阵列确定。我们提出了一种用于有效组装TALEN构建体与定制重复序列阵列的方法和试剂。我们还描述了基于天然TAL效应子及其结合位点的设计指南。使用应用这些指南的软件，在来自植物、动物和原生生物的9个基因中，我们平均每35个碱基对就发现了候选切割位点。从该组中选择的15个位点中的每一个在基于酵母的测定中用我们的试剂构建的TALEN对切割。我们用两对TALEN突变人类细胞中的HPRT 1和拟南芥原生质体中的ADH 1。我们的试剂包括用于制备定制TAL效应子的质粒构建体和用于TAL效应子融合到其他感兴趣的蛋白质的质粒构建体。利用前者，我们构建了一个新的加氏黄单胞菌AvrHah1的功能类似物。完整的质粒集可通过非营利知识库AddGene获得，我们的软件的网络版本可在线免费访问。
TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenicXanthomonas spp.to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutateHPRT1in human cells andADH1inArabidopsis thalianaprotoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructedde novoa functional analog of AvrHah1 ofXanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
高效设计和组装定制 TALEN 和其他基于 TAL 效应器的 DNA 靶向构建体
影响因子：
14.9
作者：
Cermak T;Doyle EL;Christian M;Wang L;Zhang Y;Schmidt C;Baller JA;Somia NV;Bogdanove AJ;Voytas DF
通讯作者：
Voytas DF