Chimaeric site-specific recombinases for 'genomic surgery'

所有生物体都含有极长的双螺旋DNA分子，这些分子携带着每个细胞生长和繁殖所需的信息。这些信息被编码在称为碱基的DNA构建块的序列中，碱基串编码称为基因的信息。细胞机器将代码翻译成有用的分子，如蛋白质。有时我们想“编辑”DNA密码;例如，通过删除导致疾病的“坏”基因，或通过插入一个新的基因来治愈疾病或使生物体产生有用的物质，如抗体。我们正试图开发进行这种DNA编辑或“基因组手术”的工具，通过修饰一类称为位点特异性重组酶的天然蛋白质，这种蛋白质已经可以在细菌中进行这样的反应。然而，细菌酶只对它们进化识别的细菌DNA序列起作用。为了使它们更普遍地有用，我们必须找到一种转换它们的方法，以便它们能够识别并“剪切和粘贴”我们选择的任何DNA序列。我们已经证明了一种称为解离酶的位点特异性重组酶可以被修饰，使其作用于一个新的序列。为了做到这一点，我们用另一种叫做Zif 268的蛋白质的DNA识别模块替换了解离酶蛋白中用于识别DNA碱基序列的部分。我们称这种杂合蛋白为Z-解离酶。之所以使用Zif 268模块，是因为其他研究小组已经研究出如何改变它，使其能够识别几乎任何选择的约9个碱基的DNA序列。因此，我们可以潜在地制造Z-分解酶，其可以使用其连接的Zif 268模块放置在任何DNA靶标上。然而，在Z-分解酶可以用于非常苛刻的应用之前，还有很多工作要做，例如治疗人类疾病，其中任何错误的DNA序列的编辑都可能是灾难性的。在这个项目中，我们希望开发Z-解离酶，使它们能够以生物技术和基因治疗中真实的应用所需的效率和精度作用于几乎任何选定的DNA序列。为了达到这个目标，我们必须优化Z-解离酶的性质。我们需要修改Z-解离酶中负责断裂和重新连接DNA链的部分，这样它就可以处理它所处的任何DNA序列，我们必须确保我们的Z-解离酶蛋白质对所选序列非常特异，并且不会破坏其他地方的DNA。我们希望能够控制Z-解离酶带来的DNA变化类型;例如，确保它切下一个部分而不会将其放回。最后，我们必须确保Z-分解酶在人类和其他可能使用它们的物种中有效地发挥作用。为了测试我们是否已经实现了这些目标，我们的目标是证明我们可以使用Z-分解酶从细胞的DNA中切割出编码HIV（导致人类艾滋病的病毒）的DNA片段，从而防止它产生更多的病毒拷贝。

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.