Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants.
Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants.
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扩展锌指重组酶库：丝氨酸重组酶特异性决定因素的定向进化和突变分析。
DOI：
10.1093/nar/gkt1389
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发表时间：
2014-04
影响因子：
14.9
通讯作者：
Barbas CF 3rd
中科院分区：
文献类型：
--
作者：
Sirk SJ;Gaj T;Jonsson A;Mercer AC;Barbas CF 3rd
丝氨酸重组酶是一个多样化的模块化酶家族，其促进特定靶位点之间的高保真DNA重排。用定制设计的Cys 2-His 2锌指蛋白替换其天然DNA结合结构域导致能够实现靶向遗传修饰的工程化锌指重组酶（ZFR）的产生。由锌指结构域提供的灵活性使得能够设计识别多种潜在靶位点的杂交重组酶;然而，该技术仍然受到由ZFR催化结构域施加的严格识别特异性的限制。特别是，完全重编程丝氨酸重组酶催化特异性的能力受到每个重组酶靶位点内保守碱基要求和对DNA识别因素的不完全理解的阻碍。在这里，我们描述了一种补充ZFR的靶向能力的方法。使用定向进化，我们分离了特异性识别先前在ZFR范围之外的靶位点的β和Sin重组酶的突变体。此外，我们开发了一种遗传筛选，以确定位点特异性重组的特定碱基要求，并表明特异性分析能够发现独特的基因组ZFR底物。最后，我们对丝氨酸重组酶DNA结合臂区域进行了广泛的全家族突变分析，并发现了赋予靶特异性的多种残基网络。这些结果表明，ZFR库是可扩展的，并突出了ZFR作为一类灵活的工具，用于靶向基因组工程的潜力。
The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys2–His2 zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-finger domains enables the design of hybrid recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains. In particular, the ability to fully reprogram serine recombinase catalytic specificity has been impeded by conserved base requirements within each recombinase target site and an incomplete understanding of the factors governing DNA recognition. Here we describe an approach to complement the targeting capacity of ZFRs. Using directed evolution, we isolated mutants of the β and Sin recombinases that specifically recognize target sites previously outside the scope of ZFRs. Additionally, we developed a genetic screen to determine the specific base requirements for site-specific recombination and showed that specificity profiling enables the discovery of unique genomic ZFR substrates. Finally, we conducted an extensive and family-wide mutational analysis of the serine recombinase DNA-binding arm region and uncovered a diverse network of residues that confer target specificity. These results demonstrate that the ZFR repertoire is extensible and highlights the potential of ZFRs as a class of flexible tools for targeted genome engineering.
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影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
4.8
作者：
Díaz, V;Rojo, F;Bernad, A
通讯作者：
Bernad, A
DOI：
10.1073/pnas.1533177100
发表时间：
2003-07-22
影响因子：
11.1
作者：
Akopian, A;He, JY;Stark, WM
通讯作者：
Stark, WM
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
影响因子：
56.9
作者：
Bibikova, M;Beumer, K;Carroll, D
通讯作者：
Carroll, D