Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.
Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.
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DOI：
10.1021/ar500039w
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发表时间：
2014-08-19
影响因子：
18.3
通讯作者：
Barbas, Carlos F., III
中科院分区：
文献类型：
--
作者：
Gersbach, Charles A.;Gaj, Thomas;Barbas, Carlos F., III
20世纪末，对基因调控以及人类基因组结构和功能的认识有了巨大提高。然而，用于操纵基因组的技术发展较为缓慢。例如，基因治疗领域40多年来一直专注于纠正遗传疾病和促进组织修复。然而，除了少数效率极低的方法外，传统的基因工程方法只能向细胞中添加辅助基因。这一直是基因治疗在临床上取得成功的重大障碍，并且在一些案例中还导致了严重的意外后果。因此，促进细胞基因组精确修饰的技术在研究的许多方面具有多样且重大的意义，对于将基因组革命的成果转化为医学和生物技术的切实利益至关重要。为了满足这一需求，在20世纪90年代，我们开始致力于开发用于设计蛋白质 - DNA相互作用的技术，目的是创建能够靶向任何DNA序列的定制工具。我们的目标是让研究人员能够深入基因组，特异性地调控、敲除或替换任何基因。为了实现这些目标，我们最初专注于理解和操纵锌指蛋白。特别是，我们试图创建一种简单直接的方法，使非专业实验室仅使用特定的模块组件、一个基于网络的工具以及标准的重组DNA技术就能设计定制的DNA修饰蛋白。我们面临的两个重大挑战是：（i）开发能够靶向天然存在的锌指蛋白无法识别的序列的锌指结构域；（ii）确定如何将单个锌指结构域连接在一起形成多指蛋白，以识别复杂基因组内的独特位置。从那以后，我们和其他人使用这种模块组装方法设计了人工蛋白和酶，这些蛋白和酶能够激活、抑制或对人类细胞、植物和其他生物体中用户指定的基因产生特定的变化。我们还设计了用于外部控制蛋白质活性和传递的新方法，以及开发了用于蛋白质和酶功能定向进化的新策略。本报告总结了我们在这些领域的工作，并重点介绍了成功使用模块组装方法创建具有新功能蛋白质的独立研究。我们还讨论了新兴的基因组靶向替代方法，包括转录激活因子样效应物（TALEs）和CRISPR/Cas系统，以及它们如何补充合成锌指蛋白技术。
The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein–DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular assembly method to engineer artificial proteins and enzymes that activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms. We have also engineered novel methods for externally controlling protein activity and delivery, as well as developed new strategies for the directed evolution of protein and enzyme function. This Account summarizes our work in these areas and highlights independent studies that have successfully used the modular assembly approach to create proteins with novel function. We also discuss emerging alternative methods for genomic targeting, including transcription activator-like effectors (TALEs) and CRISPR/Cas systems, and how they complement the synthetic zinc finger protein technology.
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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。
影响因子：
15
作者：
Gaj T;Sirk SJ;Tingle RD;Mercer AC;Wallen MC;Barbas CF 3rd
通讯作者：
Barbas CF 3rd
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：
12.4
作者：
Alwin, S;Gere, MB;Cathomen, T
通讯作者：
Cathomen, T
影响因子：
5.4
作者：
Eberhardy, SR;Goncalves, J;Barbas, CF
通讯作者：
Barbas, CF
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