Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.
Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.
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DOI：
10.1093/nar/gkt754
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发表时间：
2013-11
影响因子：
14.9
通讯作者：
Barbas CF 3rd
中科院分区：
文献类型：
--
作者：
Lamb BM;Mercer AC;Barbas CF 3rd
转录激活因子样效应物（TALE）蛋白可以被设计为结合几乎任何DNA序列。设计TALE DNA结合结构域的一般指导原则表明，TALE结合的DNA序列的最5′碱基（N 0碱基）应该是胸腺嘧啶。我们通过分析TALE转录因子（TALE-TF）、TALE重组酶（TALE-R）和TALE核酸酶（TALEN）在该位置处的每个DNA碱基的活性来定量N 0需求。在不存在5′ T的情况下，与含有5′ T的靶序列相比，我们观察到TALE活性降低，TALE-TF活性降低高达>1000倍，TALE-R活性降低高达100倍，TALEN活性降低高达10倍。为了开发识别所有可能的N 0碱基的TALE架构，我们使用结构指导的文库设计结合TALE-R活性选择来进化新的TALE N-末端结构域以适应任何N 0碱基。一个G-选择性结构域和广泛的反应域进行了分离和表征。以TALE-R形式选择的工程化TALE结构域表现出模块性，并且在TALE-TF和TALEN架构中具有活性。进化的N-末端结构域作为TALE结合蛋白和设计酶提供任何DNA序列的有效且不受约束的基于TALE的靶向。
Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5′-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5′ T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5′ T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.
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Tale-DNA相互作用的定量分析表明极性效应。
影响因子：
14.9
作者：
Meckler JF;Bhakta MS;Kim MS;Ovadia R;Habrian CH;Zykovich A;Yu A;Lockwood SH;Morbitzer R;Elsäesser J;Lahaye T;Segal DJ;Baldwin EP
通讯作者：
Baldwin EP
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1073/pnas.040552697
发表时间：
2000-02-15
影响因子：
11.1
作者：
Beerli, RR;Dreier, B;Barbas, CF
通讯作者：
Barbas, CF
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
影响因子：
48
作者：
Gaj, Thomas;Guo, Jing;Kato, Yoshio;Sirk, Shannon J.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III