Switchable gene drives

研究和生物技术通常需要在生物体内打开或关闭蛋白质，以了解或操纵它们的功能。在这个提议中，我们描述了如何使用小鼠作为模型系统快速打开蛋白质。作为原理证明，我们选择了基因驱动，其中严格的调控是必不可少的，开关机制将是非常有利的。基因驱动复制基因组的一个片段，无论它们是否赋予任何选择性优势，原则上在任何有性繁殖的物种中起作用，以便所有后代继承作为基因驱动一部分的基因组片段。基因驱动在防治疾病、促进可持续农业和根除入侵害虫方面的潜力已得到广泛认可。然而，考虑到它们的潜在危害以及良好的，基因驱动引起了人们对它们的可逆性和控制的担忧。在这里，我们提出了一种可切换的蛋白质系统，它可以精确地控制小鼠的基因驱动。我们描述了一个可切换的系统，可以通过添加合成的氨基酸，BOC，没有发现在哺乳动物中控制。我们已经设计了这个系统的基础，使其包含非哺乳动物氨酰-tRNA合成酶，以便将BOC添加到非哺乳动物tRNA上。该tRNA识别终止密码子，因此当BOC存在时，它被掺入靶蛋白的一个位置，否则会导致蛋白质合成终止：换句话说，BOC可以打开蛋白质的生产。这种可转换的合成蛋白质系统在培养的哺乳动物细胞中工作良好，但在活的脊椎动物中从未报道过。在一项初步研究中，我们正在应用这个系统来启动小鼠体内荧光蛋白的表达，这样它们只有在食物中含有BOC时才含有荧光细胞。虽然这些实验是初步的，但它们鼓励我们将我们的工作扩展到基因驱动。由于基因驱动工作的主要关注点是遏制，因此基因驱动系统的开发非常适合我们提出的小鼠模型;小鼠最大限度地减少了遏制问题，并将具有广泛和直接的研究，生物医学和农业应用。我们的方法将使用特定的蛋白质来执行基因驱动。使用细胞培养系统，我们将确定突变的蛋白质，保持活性时，他们含有BOC。这些信息也将使我们能够生产含有2或3个BOC残基的活性蛋白质。这个可转换系统所需的基因将被引入小鼠基因组中;该系统将被设置为使基因仅在受精时活跃。我们的目标是通过证明功能性BOC突变蛋白仅在受精时以及BOC存在于饲料或饮用水中时存在来确认系统的可切换性。基于我们的初步研究，我们有信心成功测试使用酪氨酸酶基因Tyr的基因驱动。有一个或两个Tyr基因的小鼠有黑色的毛色;没有Tyr基因的小鼠（例如，因为它们被基因驱动删除）是白色。在我们的实验中，如果饲料中没有BOC（正常情况下），后代是黑色的，但如果包含BOC，它们应该是白色的，这为诱导型基因驱动是否有效提供了明确的测试。拟议的工作符合BBSRC在合成生物学和生物科学技术开发方面的战略重点。该系统的应用不限于给定的蛋白质，并可能导致在病原体研究和人工蛋白质调控发育分析的保障。它构成了一个易于处理的跨代基因组修改系统，有望在植物、昆虫和其他动物中得到应用，包括简化抗病牲畜的生产。

Research and biotechnology often require that proteins are switched on or off within living organisms, to understand or manipulate their function. In this proposal, we describe how proteins can be rapidly switched on using the mouse as a model system. As a proof-of-principle, we have selected the gene drive, where tight regulation is essential and a switching mechanism would be extremely advantageous. Gene drives duplicate a segment of the genome whether or not they confer any selective advantage and in principle work in any sexually reproducing species so that all offspring inherit the genome segment that is part of the gene drive. The potential of gene drives to combat disease, foster sustainable agriculture and eradicate invasive pests has been widely recognised. However, given their potential to do harm as well as good, gene drives have provoked concerns about their reversibility and control.Here, we propose a switchable protein system that gives exquisitely tight control of a gene drive in mice. We describe a switchable system that can be controlled by the addition of a synthetic amino acid, BOC, not found in mammals. We have already engineered the basics of this system to contain a non-mammalian aminoacyl-tRNA synthetase enzyme so that it adds BOC to a non-mammalian tRNA. This tRNA recognises a stop codon, so that when BOC is present it is incorporated into the target protein at a position that otherwise causes protein synthesis to terminate: in other words, BOC switches production of the protein on. The switchable synthetic protein system works well in cultured mammalian cells but has never been reported in living vertebrates.In a pilot study, we are applying this system to switch on the expression of a fluorescent protein in mice so that they contain fluorescent cells only when their food includes BOC. Although these experiments are preliminary, they have encouraged us to extend our work to gene drives. As containment is a major concern in work on gene drives, development of a gene drive system lends itself well to our proposed mouse model; the mouse minimises containment issues and will have broad and direct research, biomedical and agricultural applications.Our approach will use a specific protein to execute the gene drives. Using a cell culture system, we will identify mutants of the protein that remain active when they contain BOC. This information will also allow us to produce active proteins containing 2 or 3 BOC residues. The genes required for this switchable system will then be introduced into the mouse genome; the system will be set up so that the genes are active only at the time of fertilisation. Our goal here will be to confirm the switchability of the system by showing that functional BOC mutant proteins are only present at the time of fertilisation and when BOC is present in feed or drinkwater.Based on our pilot study, we are confident of success that will lead to the test of a gene drive using the tyrosinase gene, Tyr. Mice with one or two Tyr genes have a black coat colour; those with no Tyr genes (for example, because they are removed by a gene drive) are white. In our experiment, if BOC is absent from the feed (as normal), the offspring are black, but if it is included, they should be white, providing a clear test of whether the inducible gene drive worked.The proposed work aligns with BBSRC strategic priorities in synthetic biology and technology development for biosciences. Application of this system is not restricted to given proteins and may lead to safeguards in pathogen research and artificial protein regulation for developmental analysis. It constitutes a tractable system for transgenerational genome modification that promises to have applications in plants, insects and other animals, including streamlining the generation of disease-resistant livestock.