Applying synthetic biology to the improved control of insect disease vectors

对具有医学和农业重要性的昆虫进行基因工程改造的能力，使人们有可能故意将遗传性状引入昆虫种群，以此改变它们繁殖、造成作物损害或导致疾病的媒介病原体的能力。然而，有一件事是确定人们想要引入转基因昆虫的遗传特征；将引进的性状传播到种群中完全是另一回事。这很困难的原因是，增加的遗传特征通常不会提高那些携带它的昆虫的进化适应性，这意味着它在种群中的代表性不太可能一代一代地增加。事实上，在某些情况下，这种遗传特征被设计成对种群有很强的负适应性影响。在这两种情况下，这都意味着需要释放大量的人口，通常是数千万，远远超过当地目标人口的数量，以便对人口产生明显的影响。这是昂贵的和后勤挑战。此外，只要人们能够继续发布这样的数据，这种影响就会持续下去。最近在基因控制方面的创新，如“基因驱动”，通过确保每一代基因修饰都有一个偏遗传来解决这个问题，这意味着它在种群中的频率可以相对快速地增加。这类方法很有希望，因为它们是自我维持的——只需要释放少数昆虫就能产生长期效果——而且它们是物种特异性的，因为这些特征是通过同一物种的昆虫之间的交配传递的。许多基因驱动设计使用CRISPR等基因组编辑工具作为它们的“分子马达”，这些工具可以在昆虫制造的精子或卵子中偏爱基因驱动元素的遗传，并为下一代做出贡献。对基因驱动中CRISPR元件的持续时间和/或时间进行微小的改变，可以极大地影响其遗传可能性的表现——将其表达限制在需要活跃的生殖细胞中，可以极大地改善携带驱动元件的昆虫的适应性，因此可以增加其穿透目标种群的可能性。同样，许多基因驱动也包含遗传“货物”，旨在对携带它的昆虫产生一些预期的效果——例如，激活先天免疫系统来对抗病原体或产生干扰寄生虫复制的蛋白质——并且在未被病原体感染的昆虫或其组织中表达这些效果可能非常昂贵。在这两种情况下，在时间和空间上微调昆虫内部表达的能力，可以对提高功效产生很大的影响。我们在这里提出的建议是：1)解剖卵巢和睾丸中精子和卵子的形成过程，到单细胞水平，并提取基因组中控制相关细胞表达的基因开关的DNA序列信息，这些基因开关是确保基因驱动的偏遗传所必需的。然后，我们将测试这些新开关，看看它们是否能提高基因驱动的性能；2)我们将为基因驱动和/或其货物的表达提供额外的精致特异性，确保每一种基因驱动和/或其货物仅在响应信号（如来自病原体的RNA）时才有活性，这些信号忠实地表明表达应该发生在该细胞类型中。这些基于rna的“核糖开关”是非常新颖的，证明它们在该系统中工作的能力将具有深远的重要性，不仅在昆虫控制方面，而且在提高基因组编辑在一系列应用中的实用性和特异性方面，包括医疗保健应用，如体内基因组编辑和基于crispr的诊断分析。

The ability to genetically engineer insects of medical and agricultural importance has opened the possibility of deliberately introducing genetic traits into insect populations as a way to alter their ability to either reproduce, to cause crop damage or to vector pathogens that cause disease. However, one thing is identifying the genetic trait that one would like to introduce into a modified insect; it is another thing completely to get that introduced trait to spread into a population. The reason this is difficult is that the added genetic trait usually does not improve the evolutionary fitness of those insects that harbour it, meaning that its representation in the population is unlikely to increase generation upon generation. In fact in some cases the genetic trait is designed to have a strong negative fitness effect on the population. In either of these scenarios this means that huge numbers, usually tens of millions and far in excess of the numbers in the local target population, need to be released in order to have an appreciable effect on the population. This is expensive and logistically challenging. Moreover, the effect lasts only as long as one can continue to release such numbers. Recent innovations in genetic control, such as 'gene drive', get round this problem by ensuring that there is a biased inheritance of the modification each generation, meaning that its frequency in the population can increase relatively rapidly. These types of approaches hold much promise because they are self-sustaining - only a few insects need to be released to have a long term effect - and they are species-specific because the traits are passed on by mating between insects of the same species. Many of these gene drive designs use genome editing tools such as CRISPR as their 'molecular motor' that works to bias the inheritance of the gene drive element among the sperm or eggs that an insect makes and contributes to the next generation. Making small changes to the duration and/or timing of the CRISPR element in the gene drive can drastically affect its performance in how likely it is to be inherited - limiting its expression only to the germline cells where it needs to be active can cause huge improvements in the fitness of insects carrying the drive element and therefore can increase its likelihood of penetrating a target population. Similarly, many gene drives contain also a genetic 'cargo', designed to produce some intended effect in insects carrying it - for example, activation of innate immune system against a pathogen or the production of proteins that interfere with parasite replication - and expression of these effects in insects, or tissues therein, not infected by the pathogen can be very costly. In both cases then, an ability to fine tune expression within the insect, in both time and space, can have a large effect in improving the efficacy. What we are proposing here is to: 1) dissect the process of sperm and egg formation in the ovary and testis, to the single cell level, and extract information on the DNA sequence of the genetic switches in the genome that control expression in the relevant cells necessary to ensure biased inheritance of the gene drive. We will then test these new switches to see if they improve the gene drive performance; 2) We will provide an additional level of exquisite specificity to the expression of the gene drive and/or its cargo by ensuring that each is only active in response to signals - such as RNA from the pathogen - that faithfully signal that expression should occur in that cell type. These RNA-based 'riboswitches' are very novel and proof of their ability to work in this system would have far reaching importance, not just in insect control but in improving the utility and specificity of genome editing in a range of applications including healthcare applications such as in vivo genome editing and CRISPR-based diagnostic assays.