Chimaeric site-specific recombinases for 'genomic surgery'

用于“基因组手术”的嵌合位点特异性重组酶

• 批准号: BB/F021593/1
• 负责人: Marshall Stark
• 金额: $ 44.55万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF021593%2F1
• 关键词: Chimaeric; site; specific; recombinases; genomic

All living organisms contain immensely long double-helical DNA molecules that carry the information each cell needs to grow and multiply. This information is encoded in the sequence of the DNA building blocks called bases, strings of which encode messages known as genes. Cellular machines translate the code into useful molecules such as proteins. Sometimes we would like to 'edit' the DNA code; for example, by deleting a 'bad' gene that causes a disease, or by inserting a new gene that cures a disease or that makes an organism produce a useful substance such as an antibody. We are trying to develop tools for doing this DNA editing or 'genomic surgery', by modifying a class of natural proteins called site-specific recombinases which can already do reactions like this in bacteria. However, the bacterial enzymes only work at bacterial DNA sequences that they have evolved to recognize. In order to make them more generally useful, we must find a way of converting them so that they can recognize and 'cut and paste' at any DNA sequences that we choose. We have already demonstrated that one type of site-specific recombinase called resolvase can be modified so that it will act at a new sequence. To do this we replace the part of the resolvase protein that is designed to recognize the sequence of DNA bases with a DNA-recognizing module from another protein called Zif268. We call this hybrid protein a Z-resolvase. The Zif268 module is used because other research groups have worked out how to change it so that it can recognize almost any chosen DNA sequence of about 9 bases. We can therefore potentially make Z-resolvases that can be placed on any DNA target using their attached Zif268 module. However, there is still a lot to do before Z-resolvases can be used for very demanding applications, like curing human diseases, where any editing of the wrong DNA sequences could be disastrous. In this project, we want to develop Z-resolvases so that they work on almost any chosen DNA sequence with the efficiency and precision necessary for real uses in biotechnology and gene therapy. To reach this goal we will have to optimize the properties of Z-resolvase. We need to modify the part of Z-resolvase that actually breaks and rejoins DNA strands, so that it can deal with any DNA sequence that it is placed on. We must ensure that our Z-resolvase proteins are very specific for the chosen sequence, and do not damage the DNA elsewhere. We want to be able to control the type of changes in the DNA that a Z-resolvase brings about; for example, to make sure that it cuts a section out and does not put it back in. Finally, we must make sure that Z-resolvases work efficiently in humans and other species where they might be used. To test whether we have achieved these objectives, we aim to demonstrate that we can use Z-resolvases to cut out the piece of DNA encoding HIV (the virus that causes AIDS in humans) from a cell's DNA, thus preventing it from making more virus copies.

所有生物体都包含非常长的双螺旋DNA分子，这些DNA分子带有每个细胞生长和繁殖所需的信息。此信息是按照称为碱基的DNA构建块的序列编码的，其字符串编码称为基因的字符串。蜂窝机将代码转化为有用的分子，例如蛋白质。有时我们想“编辑” DNA代码；例如，通过删除引起疾病的“不良”基因，或插入治愈疾病或使生物体产生有用物质（例如抗体）的新基因。我们正在尝试通过修改一类称为位点特异性重物组织酶的天然蛋白质来开发用于进行DNA编辑或“基因组手术”的工具，这些蛋白质已经可以在细菌中进行类似的反应。然而，细菌酶仅在其演变为识别的细菌DNA序列上起作用。为了使它们更普遍有用，我们必须找到一种转换它们的方法，以便它们可以在我们选择的任何DNA序列上识别并“切割和粘贴”。我们已经证明，一种称为Resolvase的一种类型的位点特异性重组酶可以修改，以便它以新的序列作用。为此，我们取代了分解蛋白的一部分，该部分旨在用另一种称为ZIF268的蛋白质的DNA识别模块识别DNA碱基的序列。我们称这种杂种蛋白为z溶剂箱。使用ZIF268模块是因为其他研究组已经弄清了如何更改它，以便它几乎可以识别出约9个基础的所有选择的DNA序列。因此，我们可以使用其连接的ZIF268模块将可以放置在任何DNA靶标上的Z-分子。但是，在Z-分子可用于非常苛刻的应用（例如治愈人类疾病）之前，仍然有很多事情要做，因为对错误的DNA序列进行任何编辑都可能是灾难性的。在这个项目中，我们希望开发Z分子，以便它们几乎可以使用任何选择的DNA序列，具有生物技术和基因治疗中实际用途所必需的效率和精确度。为了实现此目标，我们将不得不优化Z分子的属性。我们需要修改Z分子的一部分，该部分实际上会破坏并重新加入DNA链，以便它可以处理其放置在上面的任何DNA序列。我们必须确保我们的Z-分子蛋白对所选序列非常特异，并且不会损坏其他地方的DNA。我们希望能够控制z旋转带来的DNA中变化的类型。例如，为了确保将其切开并不会放回其中。最后，我们必须确保Z分子在人类和其他可能使用的物种中有效地工作。为了测试我们是否实现了这些目标，我们旨在证明我们可以使用z溶瘤来切除细胞DNA中编码HIV（引起人类有助于人类的病毒）的DNA部分副本。

项目成果

Zinc finger recombinases with adaptable DNA sequence specificity.

• DOI: 10.1371/journal.pone.0019537
• 发表时间: 2011-04-29
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Proudfoot C;McPherson AL;Kolb AF;Stark WM
• 通讯作者: Stark WM

Mobile DNA III

• DOI: 10.1128/9781555817954
• 发表时间: 2002
• 作者: N. Craig;M. Chandler;M. Gellert;A. Lambowitz;P. Rice;S. Sandmeyer

Zinc-finger recombinase activities in vitro.

• DOI: 10.1093/nar/gkr652
• 发表时间: 2011-11
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Prorocic MM;Wenlong D;Olorunniji FJ;Akopian A;Schloetel JG;Hannigan A;McPherson AL;Stark WM
• 通讯作者: Stark WM