Switchable gene drives

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Research and biotechnology often require that proteins are switched on or off within living organisms, to understand or manipulate their function. In this proposal, we describe how proteins can be rapidly switched on using the mouse as a model system. As a proof-of-principle, we have selected the gene drive, where tight regulation is essential and a switching mechanism would be extremely advantageous. Gene drives duplicate a segment of the genome whether or not they confer any selective advantage and in principle work in any sexually reproducing species so that all offspring inherit the genome segment that is part of the gene drive. The potential of gene drives to combat disease, foster sustainable agriculture and eradicate invasive pests has been widely recognised. However, given their potential to do harm as well as good, gene drives have provoked concerns about their reversibility and control.Here, we propose a switchable protein system that gives exquisitely tight control of a gene drive in mice. We describe a switchable system that can be controlled by the addition of a synthetic amino acid, BOC, not found in mammals. We have already engineered the basics of this system to contain a non-mammalian aminoacyl-tRNA synthetase enzyme so that it adds BOC to a non-mammalian tRNA. This tRNA recognises a stop codon, so that when BOC is present it is incorporated into the target protein at a position that otherwise causes protein synthesis to terminate: in other words, BOC switches production of the protein on. The switchable synthetic protein system works well in cultured mammalian cells but has never been reported in living vertebrates.In a pilot study, we are applying this system to switch on the expression of a fluorescent protein in mice so that they contain fluorescent cells only when their food includes BOC. Although these experiments are preliminary, they have encouraged us to extend our work to gene drives. As containment is a major concern in work on gene drives, development of a gene drive system lends itself well to our proposed mouse model; the mouse minimises containment issues and will have broad and direct research, biomedical and agricultural applications.Our approach will use a specific protein to execute the gene drives. Using a cell culture system, we will identify mutants of the protein that remain active when they contain BOC. This information will also allow us to produce active proteins containing 2 or 3 BOC residues. The genes required for this switchable system will then be introduced into the mouse genome; the system will be set up so that the genes are active only at the time of fertilisation. Our goal here will be to confirm the switchability of the system by showing that functional BOC mutant proteins are only present at the time of fertilisation and when BOC is present in feed or drinkwater.Based on our pilot study, we are confident of success that will lead to the test of a gene drive using the tyrosinase gene, Tyr. Mice with one or two Tyr genes have a black coat colour; those with no Tyr genes (for example, because they are removed by a gene drive) are white. In our experiment, if BOC is absent from the feed (as normal), the offspring are black, but if it is included, they should be white, providing a clear test of whether the inducible gene drive worked.The proposed work aligns with BBSRC strategic priorities in synthetic biology and technology development for biosciences. Application of this system is not restricted to given proteins and may lead to safeguards in pathogen research and artificial protein regulation for developmental analysis. It constitutes a tractable system for transgenerational genome modification that promises to have applications in plants, insects and other animals, including streamlining the generation of disease-resistant livestock.