Applying synthetic biology to the development of in vivo technologies for the monitoring and control of vector-borne diseases.

对具有医学和农业重要性的昆虫进行遗传修饰的能力开辟了有意将遗传性状引入昆虫种群的可能性。这样做可以改变它们的繁殖能力、造成作物损害或传播致病病原体。然而，要使引入的性状在整个种群中传播存在挑战。通常，添加的遗传性状不会改善携带它的昆虫的适应性，有时甚至会产生负面影响。这意味着需要释放大量的转基因昆虫，通常是数千万只，才能对种群产生重大影响。这一过程成本高昂，而且在后勤上具有挑战性，而且效果只能持续到大量转基因昆虫的持续释放。最近在遗传控制方面的进展，如“基因驱动”，通过使每一代的转基因遗传偏向来解决这个问题。这意味着，在群体中，修饰性状的频率可以迅速增加。这些方法显示出希望，因为它们是自我维持的，只需要少数释放的昆虫就能产生长期影响，而且它们是物种特异性的，因为修改后的性状通过同一物种昆虫之间的交配传递。许多基因驱动设计利用CRISPR等基因组编辑工具来偏向精子或卵子中基因驱动元件的遗传，然后将其传递给下一代。通过对CRISPR元件进行微小的改变，例如其DNA切割活性的持续时间和时间，其在遗传方面的表现可能会受到极大的影响。将基因驱动元件的表达限制在需要其具有活性的生殖系细胞中，可以大大增强携带基因驱动的昆虫的适应性，并增加其在目标种群中传播的机会。此外，许多基因驱动器包含遗传“货物”，其在携带它的昆虫中产生期望的效果，例如激活免疫系统对抗病原体或干扰寄生虫复制。仅在未感染的昆虫或特定组织中表达这些效果可能是昂贵的。具有微调昆虫内基因驱动及其货物的表达的能力，无论是在时间还是位置方面，都可以通过有效地最小化不同元件仅在细胞或条件下的表达来显着提高它们的有效性，其中它们起作用是必要的。完成在卵巢和睾丸单细胞水平上描述基因表达的过程。我们将提取有关基因开关的DNA序列的信息，这些基因开关控制相关细胞中的表达，可以确保基因驱动器的偏向遗传，并且具有最小或没有不必要的影响。我们将设计一种基于细胞的方法来有效地测试不同的控制序列，然后在实验室修饰蚊子种群中评估最有前途的组合。增强基因驱动表达和/或其货物的特异性，确保它们仅在响应特定信号时才有活性，例如来自病原体的特定RNA序列。这些基于RNA的“核糖开关”是创新的，并且证明它们在该系统中的有效性将具有广泛的意义，不仅在昆虫控制中，而且在各种应用中提高基因组编辑的精确性和特异性，包括医疗保健应用，如体内基因组编辑和基于CRISPR的诊断测定。

The ability to genetically modify insects of medical and agricultural importance has opened up the potential for intentionally introducing genetic traits into insect populations. This can be done to alter their ability to reproduce, cause crop damage, or transmit disease-causing pathogens. However, there are challenges in getting the introduced traits to spread throughout the population. Usually, the added genetic trait does not improve the fitness of the insects carrying it, and sometimes it even has a negative effect. This means that a large number of modified insects, often tens of millions, need to be released to have a significant impact on the population. This process is costly and logistically challenging, and the effects only last as long as the continuous release of large numbers of modified insects.Recent advancements in genetic control, such as "gene drive," address this problem by biasing the inheritance of the modification in each generation. This means that the frequency of the modified trait can increase rapidly in the population. These approaches show promise because they are self-sustaining, requiring only a few released insects to have a long-term effect, and they are species-specific since the modified traits are passed on through mating between insects of the same species. Many gene drive designs utilize genome editing tools like CRISPR to bias the inheritance of the gene drive element in sperm or eggs, which are then passed on to the next generation. By making small changes to the CRISPR element, such as its duration and timing of DNA cleavage activity, its performance in terms of inheritance can be drastically affected. Limiting the expression of the gene drive element to the germline cells, where it needs to be active, can greatly enhance the fitness of insects carrying the gene drive and increase its chances of spreading through the target population. Additionally, many gene drives contain a genetic "cargo" that produces a desired effect in the insects carrying it, such as activating the immune system against a pathogen or interfering with parasite replication. Expressing these effects only in non-infected insects or specific tissues can be costly. Having the ability to fine-tune the expression of the gene drive and its cargo within the insect, both in terms of timing and location, can significantly improve their effectiveness by effectively minimising expression of the different elements to only the cells or conditions where it is essential that they act.In this proposal, we aim to:1. Complete the process of describing gene expression at the single-cell level in the ovary and testis. We will extract information about the DNA sequences of the genetic switches that control expression in the relevant cells that can ensure biased inheritance of the gene drive with minimal or no unwanted effects. We will design a cell-based approach to test different control sequences efficiently and then evaluate the most promising combinations in laboratory populations of modified mosquitoes.2. Enhance the specificity of gene drive expression and/or its cargo by making sure they are only active in response to specific signals, such as specific RNA sequences from the pathogen. These RNA-based "riboswitches" are innovative, and demonstrating their effectiveness in this system would have wide-ranging implications, not just in insect control but also in improving the precision and specificity of genome editing in various applications, including healthcare applications like in vivo genome editing and CRISPR-based diagnostic assays.