X shredders: invasive genetic elements that bias a populations' sex ratio towards males
Nestled within the genomes of organisms throughout the tree of life, there exist classes of genetic elements that display a behavior called genetic drive. Driving elements cheat evolution by increasing the probability that they are inherited from one generation to the next, even when the phenotype they confer is costly to their host. Over time, this advantage can lead to their invasion and fixation within a population.
Researchers have stipulated for decades that harnessing genetic drive within efforts aiming to control populations holds vast potential. Among the most influential of these ideas was proposed in the 60s by W.D. Hamilton who stipulated the following thought experiment: [..] suppose a novel allele arose on a Y chromosome giving it the ability to always win against the X-chromosome for fertilizations. A male with such a Y chromosome would have only sons, and at the population level this new Y would rapidly increase in frequency, since its rate of transmission was higher than that of both the X chromosome and the “wild type” Y chromosome. In doing so, this invasive Y chromosome would bias the overall sex ratio of the population towards males, and since the productivity of the population is dependent on the number of females, eventually the population would collapse when the last female mated with a male carrying this mutated Y chromosome . Hamilton’s foresight was that such alleles could be used for the suppression of disease transmitting mosquitoes, but a method to build these sex ratio distorters remained elusive until forty years later, when developments in mosquito genetic engineering and synthetic biology, led Austin Burt to propose that Y-linked endonucleases re-engineered to recognize and cut specifically the X-chromosome and expressed during spermatogenesis could be the basis for such a synthetic allele . Inspired by this proposal, in previous work colleagues and I took this theoretical concept to proof-of-principle demonstration in the malaria mosquito An. gambiae, and called the system “X-shredding”. We targeted the X-specific ribosomal DNA locus of An. gambiae using a naturally-occurring endonuclease, I-PpoI expressed during spermatogenesis from transgenes located on autosomes.
The X-shredding paradigm. An endonuclease (red triangle) recognizes and cleaves the X chromosome during male meiosis. Sperm carrying the X-chromosome are thus eliminated from the gametic pool leading to male only progeny.
Work in our group is now focused on making these X-shredders invasive, so they can drive into a population from low frequencies. This requires that the X-shredder be physically linked to the Y chromosome, providing both the allele itself and the entire Y chromosome which harbors it a competitive advantage in inheritance against the X. Our work until now has revealed that expression of transgenes from the Y chromosome during the right stages of spermatogenesis is made complicated by a highly conserved but not well understood process called meiotic sex chromosome inactivation (MSCI). MSCI leads to chromosome-wide suppression of gene expression from the sex chromosomes during meiotic stages of spermatogenesis. Taking inspiration from the evolution itself, our approach to overcoming MSCI has been to understand if and how endogenous Y-chromosome sequences are able to do this (see Y chromosome research section). We use state of the art genome sequencing technologies, have developed novel computational methods for their analyses and combine this knowledge with our engineering expertise, including most recently the CRISPR system. We have identified Y-linked genes that are expressed during spermatogenesis at the correct stages and we are now building on these new datasets to improve our engineering designs inspired directly from evolutionary data.
Differences between an autosomal X-shredder, which biases offspring sex ratios towards males and is transmitted in an Mendelian fashion, would disappear from the population once seeded, and a Y-linked X-shredder, which benefits directly from its induced male-bias (sex ratio distortion) and behaves as an invasive element once released.
Transferring the X-shredder paradigm for the control of agricultural pests
In comparison to these recent advances using synthetic biology in the field of genetic control of mosquito species, there has been little comparable progress for insects that are agricultural pests. As a result, there is now growing interest and opportunities to transfer the technologies that I have been developing in the malaria mosquito to other agricultural pests. One of the main advantages of the X-shredding approach is that it exploits the near universal role of paternal chromosome inheritance on the eventual sex of an individual. Since sex ratios are effectively manipulated at the level of chromosome transmission to offspring, species-specific knowledge of an organisms sex determination pathway is not needed. Other requirements, including the ability to genetically transform the species and the availability of regulatory elements to allow sperm-specific expression, are either relatively simple to achieve and/or for a number of agricultural pests are already available. What is required for each species however, is knowledge of X-chromosome specific and abundant sequences that can be targeted during spermatogenesis using endonucleases like CRISPR/Cas9. In our previous work in the malaria mosquito we were able to build X-shredders, because we targeted the mosquito’s rDNA genes, which were known to be exclusively located on the X-chromosome in an array of approximately 400 copies. This arrangement is exceptional however, and the vast majority of insects do not to share it. Furthermore, knowledge of naturally occurring multi-copy X-chromosome specific sequences is limited because repetitive DNA sequences are ipso facto excluded from genome assemblies.
To overcome this challenge, Nikolai Windbichler and I have recently developed a bioinformatic pipeline called redkmer, for repeat extraction and detection based on kmers, that can identify suitable sequences for CRISPR X-shredding using only raw whole genome sequencing of male and female samples from any species with a XY male karyotype (Article, Github). With this bioinformatic pipeline, we are now able to identify suitable target X-specific and abundant sequences and build sex ratio distorters in several agricultural pest species. To do this we are generating the necessary genomic datasets to evaluate species suitability, based on the presence of abundant and specific X target sites.
Redkmer output data. Each spot represents a unique 25 bp sequence of the insect genome and the red spots represent X-specific and abundant 25bp sequences selected by redkmer.
We are focusing our efforts for now on fruitflies including D. melanogaster (which is acting as our first non-mosquito model), D. suzukkii, C. capitata (acting as our Tephritid model), B. olea and B. dorsalis. To make simultaneous progress in multiple species the genetic engineering work is being performed as part of an international consortium with four other partners.