Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues.
Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues.
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用 TAL 效应器靶向 G：用 NN 和 NK 重复可变二残基构建的 TALEN 的活性比较
DOI：
10.1371/journal.pone.0045383
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Voytas DF
中科院分区：
文献类型：
--
作者：
Christian ML;Demorest ZL;Starker CG;Osborn MJ;Nyquist MD;Zhang Y;Carlson DF;Bradley P;Bogdanove AJ;Voytas DF
转录激活因子样（TAL）效应物的DNA结合域可以很容易地被设计成具有新的DNA序列特异性。因此，工程TAL效应蛋白已成为操纵体内基因组的重要试剂。TAL效应物的DNA结合是由34个氨基酸重复序列阵列介导的。在每次重复中，两个氨基酸（重复可变二残基，RVDs）中的一个与DNA靶中的碱基接触。具有C、T和A特异性的RVDs已被描述；然而，在靶向G的RVD中，RVD神经网络也结合A， NK在天然存在的TAL效应物中是罕见的。在这里，我们发现用NK合成的用于指定G的TAL效应核酸酶（TALENs）的活性低于含有NN的同类酶：在酵母重组实验中，用NN合成的15对TALENs中有14对比用NK合成的相同TALENs显示出更高的活性。在人细胞中测定了其中三对TALEN的活性，结果与酵母的数据相似。体内数据可以通过结合亲和力的体外测量来解释，表明含有nk的TAL效应物对含有G的靶标的亲和力低于含有nn的效应物。在G被A取代的靶标上，观察到含有nk的TALENs具有更高的G特异性。具有不同N端和c端截断的TALENs也在两个TALEN结合位点之间间隔物长度不同的靶标上进行了测试。TALENs的c端长度为63或231个氨基酸，在重复序列上切割间隔长度从14到33 bp不等的靶标。然而，重复阵列后只有18 aa的TALENs显示出13至16 bp的间隔物的明显最佳。本文提供的数据为提高工程TAL效应蛋白的特异性和活性提供了有用的指导。
The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths – from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.
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影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1073/pnas.1019533108
发表时间：
2011-02-08
影响因子：
11.1
作者：
Mahfouz, Magdy M.;Li, Lixin;Zhu, Jian-Kang
通讯作者：
Zhu, Jian-Kang
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
影响因子：
46.9
作者：
Miller, Jeffrey C.;Tan, Siyuan;Rebar, Edward J.
通讯作者：
Rebar, Edward J.
影响因子：
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作者：
Sun, Ning;Liang, Jing;Zhao, Huimin
通讯作者：
Zhao, Huimin