Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign.
Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign.
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通过二聚体界面重新设计增强重组酶介导的基因组工程的特异性。
DOI：
10.1021/ja4130059
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发表时间：
2014-04-02
影响因子：
15
通讯作者：
Barbas CF 3rd
中科院分区：
文献类型：
--
作者：
Gaj T;Sirk SJ;Tingle RD;Mercer AC;Wallen MC;Barbas CF 3rd
尽管位点特异性内切核酸酶的出现使基因组工程取得了近期进展，但仍然需要能够将遗传有效载荷特异性递送至人类基因组的工具。基于来自丝氨酸重组酶的解离酶/ invertase家族的活化催化结构域且与Cys2 - His2锌指或TAL效应物DNA结合结构域融合的杂合重组酶是一类能够实现此目的的试剂。然而，这些酶的效用受到其总体靶向特异性低的限制，这在很大程度上是由于能够诱导脱靶修饰的副产物同二聚体的形成。在此，我们结合合理设计和定向进化来重新设计丝氨酸重组酶二聚化界面，并产生一种重组酶结构，使这些不良同二聚体的形成减少>500倍。我们表明，这些增强的重组酶在哺乳动物细胞中显示出显著提高的靶向特异性，并实现了与先前报道的位点特异性核酸酶相似的位点特异性整合率。此外，我们表明增强的重组酶毒性低，并以高特异性促进将人凝血因子IX和α - 半乳糖苷酶基因递送至内源性基因组位点。这些结果为通过蛋白质工程提高杂合重组酶特异性提供了一种通用方法，并说明了这些酶在基础研究和治疗应用中的潜力。
Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications. Here, we combine rational design and directed evolution to re-engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by >500-fold. We show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site-specific integration similar to those previously reported for site-specific nucleases. Additionally, we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor IX and α-galactosidase genes into endogenous genomic loci with high specificity. These results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications.
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使用单个 AAV 载体转导和食品和药物管理局批准的药物增强的锌指核酸酶介导的基因校正。
影响因子：
5.1
作者：
Ellis BL;Hirsch ML;Porter SN;Samulski RJ;Porteus MH
通讯作者：
Porteus MH
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
DOI：
10.1007/978-1-60761-753-2_14
发表时间：
2010-01-01
期刊：
ENGINEERED ZINC FINGER PROTEINS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Cornu, Tatjana I.;Cathomen, Toni
通讯作者：
Cathomen, Toni
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：
64.8
作者：
Ashworth, Justin;Havranek, James J.;Baker, David
通讯作者：
Baker, David