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的能力，开启了故意将遗传特征引入昆虫种群的可能性，作为改变它们繁殖、造成作物损害或传播致病病原体的能力的一种方式。然而，一件事是识别一个人想要引入到改良昆虫中的基因特征；而将引入的特征传播到种群中则完全是另一回事。这很困难的原因是，额外的遗传特征通常不会改善拥有它的昆虫的进化适合度，这意味着它在种群中的代表不太可能一代又一代地增加。事实上，在某些情况下，遗传特征被设计成对种群产生强烈的负面适应效应。在这两种情况下，这意味着需要释放大量人口，通常是数千万人，远远超过当地目标人口的人数，以便对人口产生明显影响。这不仅成本高昂，而且在后勤方面也颇具挑战性。此外，只有当一个人能够继续发布这样的数字时，这种影响才会持续。最近在基因控制方面的创新，如基因驱动，通过确保每一代人都有偏向的遗传，这意味着它在种群中的频率可以相对快速地增加，从而绕过了这个问题。这些类型的方法很有希望，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-而且它们是特定物种的，因为这些特征是通过同一物种的昆虫之间的交配来传递的。这些基因驱动设计中的许多都使用CRISPR等基因组编辑工具作为它们的分子马达，该马达的工作是在昆虫制造并贡献给下一代的精子或卵子中偏向基因驱动元件的遗传。对CRISPR元件在基因驱动中的持续时间和/或时机进行微小的改变，可能会极大地影响其遗传可能性的表现--将其表达仅限于其需要激活的生殖系细胞，可以极大地改善携带驱动元件的昆虫的适合性，从而增加其穿透目标种群的可能性。类似地，许多基因驱动也包含一种旨在对携带该基因的昆虫产生某种预期效果的基因“货物”--例如，激活针对病原体的先天免疫系统或产生干扰寄生虫复制的蛋白质--而在未受病原体感染的昆虫或昆虫组织中表达这些效果的成本可能非常高。因此，在这两种情况下，在昆虫体内微调表达的能力，无论是在时间上还是在空间上，都可以在提高疗效方面发挥很大作用。我们在这里提出的建议是：1)在单细胞水平上剖析卵巢和睾丸中精子和卵子的形成过程，并提取基因组中控制相关细胞表达的遗传开关的DNA序列的信息，以确保基因驱动的偏向遗传。然后我们将测试这些新开关，看看它们是否改善了基因驱动性能；2)我们将通过确保每个开关仅在响应信号(例如来自病原体的RNA)时才有效，从而为基因驱动和/或其载体的表达提供额外水平的精致特异性。这些基于RNA的‘核糖开关’非常新颖，证明它们在这个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，而且在一系列应用中提高基因组编辑的实用性和特异性，包括医疗保健应用，如活体基因组编辑和基于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.