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.

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，为有意将遗传性状引入昆虫种群提供了可能性，以改变它们的繁殖能力、造成作物损害的能力或传播致病病原体的能力。然而，一件事是确定一个人想引入到一个改造昆虫的遗传特征;这是另一回事完全引入性状传播到一个群体。这很困难的原因是，增加的遗传性状通常不会改善那些拥有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，遗传性状被设计成对群体具有强烈的负适应性效应。在这两种情况下，这意味着需要释放大量的地雷，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。这是昂贵的和后勤上的挑战。此外，这种影响只持续到一个人能够继续释放这些数字的时候。最近在遗传控制方面的创新，如“基因驱动”，通过确保每一代都有一个有偏见的遗传修饰来解决这个问题，这意味着它在种群中的频率可以相对迅速地增加。这些类型的方法有很大的希望，因为它们是自我维持的-只有少数昆虫需要被释放，以产生长期的影响-他们是物种特异性的，因为这些特征是通过同一物种的昆虫之间的交配传递。许多这些基因驱动设计使用基因组编辑工具，如CRISPR作为它们的“分子马达”，其作用是使基因驱动元件在昆虫产生的精子或卵子中的遗传偏向，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-将其表达仅限于需要激活的种系细胞可以导致携带驱动元件的昆虫适应性的巨大改善，因此可以增加其穿透目标群体的可能性。类似地，许多基因驱动器还包含遗传“货物”，其被设计为在携带它的昆虫中产生一些预期的效果-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-并且在未被病原体感染的昆虫或其中的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表达的能力可以在提高功效方面具有很大的效果。我们在这里提出的是：1）在单细胞水平上解剖卵巢和睾丸中精子和卵子形成的过程，并提取基因组中控制相关细胞中表达的基因开关的DNA序列信息，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水平的精确特异性，确保每个开关仅在响应信号时才有活性-例如来自病原体的RNA-忠实地发出信号，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，并且证明它们在该系统中工作的能力将具有深远的重要性，不仅在昆虫控制中，而且在改善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，例如体内基因组编辑和基于CRISPR的诊断测定。

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