Applying synthetic biology to the development of in vivo technologies for the monitoring and control of vector-borne diseases.
Lead Research Organisation:
Liverpool School of Tropical Medicine
Department Name: Vector Biology
Abstract
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.
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.
Technical Summary
Our research aims to enhance the control of gene expression in mosquito germline cells through transcriptomic analysis and regulatory element identification. We seek to develop posttranscriptional switches for precise activation of CRISPR editing in specific tissues or in response to pathogen-related signals. By employing computational design, in vitro testing, and validation in mosquito models, we aim to advance the development of genetic control technologies for mosquito-borne diseases.
Objective 1: Through dissecting the transcriptomic profiles of individual cell types in male and female gonads, we can discern genes expressed at specific timepoints during gamete development. From these genes, we will extract putative regulatory sequences active in distinct developmental stages (premeiotic, meiotic, and postmeiotic germline clusters). We will test their ability to recapitulate reporter gene expression in different spatiotemporal patterns within the germline tissue.
Objective 2: We aim to automate computational design of RNA sequences for gRNA activation upon interaction with mosquito or pathogen-specific RNAs. Validated designs will provide a repertoire of switchable gRNAs. Testing with bacterial-based and mosquito cell extracts will enhance predictability and scalability.
Objective 3: We will test validated control switches in transgenic mosquito models, focusing on nuclease-based gene drive efficacy in germline tissues. We will expand switchable gRNAs to include triggers from key arbovirus pathogens, benefitting the research community.
Objective 1: Through dissecting the transcriptomic profiles of individual cell types in male and female gonads, we can discern genes expressed at specific timepoints during gamete development. From these genes, we will extract putative regulatory sequences active in distinct developmental stages (premeiotic, meiotic, and postmeiotic germline clusters). We will test their ability to recapitulate reporter gene expression in different spatiotemporal patterns within the germline tissue.
Objective 2: We aim to automate computational design of RNA sequences for gRNA activation upon interaction with mosquito or pathogen-specific RNAs. Validated designs will provide a repertoire of switchable gRNAs. Testing with bacterial-based and mosquito cell extracts will enhance predictability and scalability.
Objective 3: We will test validated control switches in transgenic mosquito models, focusing on nuclease-based gene drive efficacy in germline tissues. We will expand switchable gRNAs to include triggers from key arbovirus pathogens, benefitting the research community.
