Applying synthetic biology to the improved control of insect disease vectors

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.

Selfish genetic elements such as transposable elements and gene drives replicate in their host genome, biasing their inheritance to offspring, even at a cost to the host organism. Similarly, some intracytoplasmic bacteria, such as Wolbachia, bias their own inheritance by imposing a fitness cost on offspring that do not inherit them.
Both types of elements exist naturally in several insect species, are active in the germline and have shaped the evolutionary history of these species through past invasions of populations. Recent attempts to re-create, synthetically, these selfish genetic elements, and to re-purpose these into forms of genetic population control, have shown much promise. Such control approaches can include the release of selfish synthetic genetic elements designed to reduce the reproductive potential of the population or to affect its intrinsic ability to harbour pathogens for transmission. However, absolutely critical for these approaches is the precise temporal and spatial control to allow maximal intended effect of the introduced genetic element on the population while minimising undesired fitness effects caused by expression in tissues or life stages not necessary for its efficacy. We plan to first use a single cell transcriptomics approach to dissect gametogenesis, in order to identify suites of promoters that can be used to fine tune expression of transgenic selfish elements to various stages to maximise their transmission. In combination we also plan to develop riboregulators that add additional control by responding to endogenous mRNAs that can serve as faithful 'triggers' to ensure expression of the synthetic element only in the correct physiological context.
We will use the mosquito as the organism of choice, given the pressing need for new control tools for this insect. However, our research pipeline and the tools established herein will be applicable across a range of insects of agricultural and medical importance.