The universal X-shredder: Enabling genetic control by sex ratio distortion in agricultural pests and disease vectors

Lead Research Organisation: Imperial College London
Department Name: Life Sciences


Insects transmit a great number of diseases to humans, animals and plants. They also directly feed on agricultural plants, reducing agricultural production and causing empty stomachs for the poorest and huge economic losses for farmers. The world's climate is changing allowing insects to invade new regions and reproduce faster. A growing human population puts a rising strain on the worlds agricultural systems while at the same time insects are becoming increasingly resistant to our most powerful weapon - insecticides. The global burden insects pose on human health and society is therefore expected to increase. These challenges are forcing us to envision novel ways in which we can sustainably control insects.

Genetic control of insects is a form of biological control where the release of modified insects reduces or eliminates the potential of wild insect populations to do harm. For example the mass-release of sterilized males has been used to reduce the population size of many insect pests for many decades.

One such genetic control strategy is to strongly bias the sex ratio of a population towards males so that the population, due to a lack of females, decreases in size or is eliminated altogether. Normally half of a male's sperm carry the Y-chromosome and go on to make males whereas the other half carries the X chromosome and go on to make females. A gene that biases the production of sperm towards those that carry the Y-chromosome is refereed to as a sex ratio distorter. We have, for the first time, generated such a distorter gene which destroys the X-chromosome by cutting its DNA in many places and successfully tested it in the malaria mosquito. The biology of the mosquito allowed us take a shortcut, as "molecular scissors" that could cut the DNA of the mosquito's X-chromosome were already available and could be used with little modification.

In this project we want to explore whether similar distorter genes can also be created for many other insect pest or vector species and to destroy their respective X-chromosomes. First we will develop a bioinformatic algorithm that is able to identify, using DNA sequences from male and female insects, positions where the X-chromosome can be cut. We will then use the novel CRISPR "molecular scissor" technology to cut the X-chromosome when sperm are made and study the effect on the progeny. If it works and only sperm carrying the Y survive we will find that these insects now produce mostly male offspring.

We will first test this approach in the fruit fly Drosophila, as the fly is a genetic powerhouse allowing many genetic experiments to be concluded quickly and efficiently. If successful we will then test this technology in one well studied insect pest species for example Ceratitis capitata, the Mediterranean fruit fly capable of damaging many fruit crops. We will show that male flies carrying the distorter gene when placed in a cage with a population of normal flies will reduce that population or even eliminate it.

If it works our control strategy can in the future be applied to protect food crops from insect pests or humans and animals from insect-borne diseases.

Technical Summary

We seek to establish sex-ratio distortion by X-chromosome shredding as a new genetic control strategy that can be applied to populations of a wide variety of insect pests of agriculture or vectors of human and animal diseases.

It has long been theorized that inducing extreme reproductive sex ratios could be a powerful genetic method to eliminate insect pest populations. We have recently generated the first synthetic sex distortion system in the malaria mosquito. We showed that this system was powerful enough to eliminate caged populations of this important vector via the mass release of transgenic males. However, to establish this system we exploited the peculiar genetic makeup of A. gambiae namely that the mosquito's rDNA repeats are located exclusively on the X-chromosome. Using an endonuclease known to cut a conserved sequence within the rDNA destroyed the paternal X-chromosome during spermatogenesis, a process termed X-shredding, preventing it's transmission to the progeny and resulting in >95% male offspring.

To render this technology more broadly applicable to any pest or vector species with suitable sex chromosomes we are developing a bioinformatic pipeline that is able to identify sequences that are present exclusively and abundantly on an organism's X-chromosome. We have validated a prototype of this tool that takes as an input only unassembled long and short genome sequencing reads. We are using the Drosophila model to target a number of such sequence repeats and to establish the criteria of functionality for CRISPR based X-shredders. We are performing short and long read genome sequencing on a number of prominent pest and vector species to determine their suitability for this approach and we finally seek to establish a fully functioning X-shredder transgene in one chosen target species. The ability of CRISPR-based X-shredders to bias the sex ratio towards males would constitute a powerful new tool for the area-wide control of harmful insects.

Planned Impact

The proposed project aims to tackle one of greatest challenges science has ever faced. By 2050 the world's population will increase to over 9 billion people. Global food production will be required to increase by 70% to feed us all. Insect pests affect food production, storage and transport. For example annual agricultural losses due to insect damage amount to almost 8% in Brazil, a reduction of approximately 25M tons of food, fibre and biofuels, with economic losses reaching US$ 17.7 billion per year. Agricultural losses due to insect damage during food production, translates to empty stomachs for the poorest and economic losses for affected farmers. A changing climate, the spread of invasive species following in its wake as well as the rise of insecticide resistance is predicted to increase the burden of insects on agricultural production and also on human health with half the world's population now at risk from vector-borne disease. According to the WHO, vector-borne diseases are responsible for close to 1 million deaths each year and insecticide-resistant mosquitoes now inhabit more than 60 countries.

New tools for the control of insect pests and disease vectors have the potential to save and improve the lives of millions in disease endemic countries. Genetic control is a form of biological control that has the potential to replace more indiscriminate methods of pest control and thus reduce ecosystem degradation. The successful development of a novel and powerful class of control strategies for agricultural pests and disease vectors, as envisioned here, would benefit those people that are first in line to be affected by these insects and suffer from suffer from bites, disease, poverty and hunger.

Those individuals, organizations and nations that can contribute towards solutions to these mounting global challenges will benefit themselves in turn. The United Kingdom is a world leader in the development of GM technology for genetic control of insects. Based in the UK are a number of the leading academic research groups for example at Imperial College London as well as the Pirbright Institute. Oxitec Ltd. a spin-out from the University of Oxford it is the only company in the world that offers the use of genetic technologies to control insects that spread disease and damage crops. Funding of the proposed project will further strengthen the UK's and Imperial's position in this field. The global challenges laid out above and the opportunities represented by genetic control were recently recognized by the Science and Technology Select Committee of the House of Lords in it's 1st report on genetically modified insects (2015) and the committee has called for further investment in research capabilities in the field of genetic control in the UK.
Description We have established we established the basic toolbox for the endogenous use of CRISPR genome editing in the medfly. We successfully characterized CcU6 promoters and endogenous Cas9 and Cas12a activity in the medfly germline. We achieved near optimal activity against the white eye marker gene and characterized a number of gRNAs for meiotic sex-distortion by X-shredding. We managed to engineer highly biased sex ratios in the medfly. Our progress in developing the CRISPR toolkit has coincided with our identification the Y-linked male-determining factor MoY, showing its essential role in overwriting the default female development in the medfly. We now have a blueprint of the mechanism governing sex determination in the medfly, confirming also that its Y chromosome does not harbour loci necessary for male fertility. Unexpectedly, we found that MoY is highly conserved in other Tephritid flies, a finding that contrasts observations of rapidly evolving sex determination systems in other insects. As part of this work, we also developed a number of relevant genomic and transcriptomic datasets for the medfly that are available for use by us and others.
Exploitation Route These advances lay the principal foundation for developing a number of genetic control strategies, including the generation of sex-distorters, genetic sexing strains, CRISPR based SIT strains (e.g. pgSIT) and suppressive gene drives in Tethritid agricultural pests. These next steps could be achieved by academic researchers of private enterprise.
Sectors Agriculture, Food and Drink,Healthcare

Description IAEA 
Organisation International Atomic Energy Agency
Country Austria 
Sector Charity/Non Profit 
PI Contribution We will sequence Anastrepha ludens and Bactrocera dorsalis to indentify suitable target sites for X-shredding.
Collaborator Contribution The partner Konstantinos Bourtzis representing the IAEA will provide, after performing a series of genetic crosses, tissue samples from male and female Anastrepha ludens and Bactrocera dorsalis.
Impact Anastrepha ludens and Bactrocera dorsalis sequencing data.
Start Year 2017
Description Medfly - Naples 
Organisation University of Naples
Country Italy 
Sector Academic/University 
PI Contribution Using redkmer and the data provided by the collaborator we have provided data regarding the nature of the potential M-factor (male-determining factor) in the medfly. Identification of the M factor could be another avenue for genetic control of the medfly.
Collaborator Contribution We have received next-gen sequencing data from the medlfy Ceratitis capitata in order to determine if it is a suitable species for X-shredding.
Impact We have generated a medlfy X-specific kmer dataset and identified candidates for the M factor in ceratitis. These will be subject of further work.
Start Year 2017
Description Project Partner - Philipos Aris Papathanos 
Organisation University of Perugia
Country Italy 
Sector Academic/University 
PI Contribution Dr. Papathanos is a full partner (project partner) in the project and is involved in planning and execution of every step.
Collaborator Contribution Dr. Papathanos is a full partner (project partner) in the project and is involved in planning and execution of every step.
Impact Dr. Papathanos is a full partner (project partner) in the project and is involved in planning and execution of every step.
Start Year 2017
Description olive fly - Larissa 
Organisation University of Thessaly
Country Greece 
Sector Academic/University 
PI Contribution We have identified, using the data provided by our collaborators Kostas Mathiopoulos and Konstantina Tsoumani suitable target sites to test the X-shredding paradigm in the olive fly Bactrocera olae. This will be the basis for the molecular work.
Collaborator Contribution The collaborators Kostas Mathiopoulos and Konstantina Tsoumani have provided pacbio data of the olive fly that we have analysed. The collaborators will carry out the molecular work to test X-shredding in the olive fly.
Impact Konstantina Tsoumani has received an IKY national fellowship for postdoctoral research on this topic. Dr. Tsoumani has visited the Imperial lab as a visiting researcher from 15th of February 2018 to 19th of March.
Start Year 2017
Title redkmer 
Description We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact We are now able to select gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species with sequencing data, a prerequisite for conducting the molecular work in these species.