Applying synthetic biology to the improved control of insect disease vectors

应用合成生物学改善昆虫病媒控制

• 批准号: BB/W014661/1
• 负责人: Tony Nolan
• 金额: $ 76.99万
• 依托单位: Liverpool School of Tropical Medicine
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW014661%2F1
• 关键词: Applying; synthetic; biology; improved; control

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.

遗传培训医学和农业重要性的昆虫的能力使故意将遗传特征引入昆虫种群的可能性是改变其繁殖能力，造成农作物损害或引起疾病的载体病原体的一种方式。但是，有一件事是确定人们想将其引入改良昆虫的遗传特征。这是另一件事是使引入特征扩散到人群中是另一回事。之所以困难的原因是，附加的遗传特征通常不会改善那些藏有昆虫的昆虫的进化适应性，这意味着它在人群中的代表性不太可能增加产生的产生。实际上，在某些情况下，遗传特征旨在对人群产生强大的负适应性影响。在这两种情况下，这意味着需要释放大量数千万，远远超过当地目标人群中的数量，以便对人群产生可观的影响。这是昂贵且逻辑上具有挑战性的。此外，只要可以继续发布此类数字，效果就会持续。遗传控制中的最新创新（例如“基因驱动”）通过确保每一代人的修饰具有偏见​​的遗传来解决这个问题，这意味着其在人群中的频率可以相对较快地增加。这些类型的方法具有很大的希望，因为它们是自我维持的 - 只有少数昆虫需要释放长期效应 - 它们是特定于物种的，因为这些特征是通过同一物种的昆虫之间的交配而传播的。这些基因驱动器设计中的许多使用基因组编辑工具（例如CRISPR）为“分子运动”，可将基因驱动元件的遗传偏置在昆虫产生和贡献下一代的精子或卵中的基因驱动元件的继承。对基因驱动器中CRISPR元件的持续时间和/或时间的持续时间和/或时机进行小更改可能会大大影响其遗传的可能性 - 仅将其表达限制在需要活跃的种系细胞上，因此可以使携带驱动器元素的昆虫的适应性大大改善，因此可以增加其穿透目标人群的可能性。同样，许多基因驱动器还包含一种遗传“货物”，旨在在携带它的昆虫中产生一些预期的作用 - 例如，天生免疫系统针对病原体或蛋白质的产生，这些蛋白质会干扰寄生虫复制 - 以及这些作用在昆虫或组织中不受病原体感染的昆虫表达的表达，可能是成本成本的。因此，在这两种情况下，在时间和空间中都可以在昆虫中微调表达的能力在提高疗效方面具有很大的影响。我们在这里提出的是：1）剖析卵巢和睾丸中的精子和卵形成的过程，至单细胞水平，并提取有关基因组中遗传开关的DNA序列的信息，这些信息可以控制基因驱动的偏置遗传所必需的相关细胞中表达的表达。然后，我们将测试这些新开关，以查看它们是否改善了基因驱动性能； 2）我们将通过确保每个人仅对信号（例如病原体的RNA）有效，为基因驱动器的表达和/或其货物的表达提供额外的精美特异性，以表明表达在该细胞类型中应发生。这些基于RNA的“核糖开关”非常新颖，并且证明其在该系统中工作的能力的证明将具有很大的重要性，不仅在昆虫控制方面，而且在改善基因组编辑的效用和特异性方面，包括医疗保健应用，包括体内基因组编辑和基于CRISPR的诊断。

项目成果

CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits.

CRISPR 介导的盒交换 (CriMCE)：一种引入和隔离精确无标记编辑的方法。

• DOI: 10.1089/crispr.2022.0026
• 发表时间: 2022
• 期刊: The CRISPR journal
• 作者: Morianou I

Single-cell profiling of Anopheles gambiae spermatogenesis defines the onset of meiotic silencing and premeiotic overexpression of the X chromosome.

• DOI: 10.1038/s42003-023-05224-z
• 发表时间: 2023-08-15
• 期刊: COMMUNICATIONS BIOLOGY
• 影响因子: 5.9
• 作者: Page, Nicole;Taxiarchi, Chrysanthi;Tonge, Daniel;Kuburic, Jasmina;Chesters, Emily;Kriezis, Antonios;Kyrou, Kyros;Game, Laurence;Nolan, Tony;Galizi, Roberto
• 通讯作者: Galizi, Roberto