Switchable gene drives

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.

We have shown in vitro that synthetic amino acids may be introduced into recombinant proteins in mammalian cells by orthogonal aminoacyl tRNA synthetase/tRNA pairs. The system harnesses pyrrolysyl-tRNA synthetase (PylRS) and Pyl tRNA to introduce the synthetic lysine analogue, Lys(Boc), referred to as BOC, encoded by a rare amber stop codon, UAG. Translation is terminated at the UAG codon when BOC is absent, but when present, BOC is incorporated to produce full-length recombinant protein. We propose to adapt this system to Cas9 in vivo to produce a switchable gene drive in the mouse. In preliminary data, we demonstrated the proof-of-principle by generating live, healthy mice expressing BOC-inducible eGFP. Using our empirical data and the Cas9 crystal structure, we selected 20 Cas9 Lys residues and will change the corresponding codons to UAG. The resulting 20 Cas9-BOC mutants will be characterised for activity with or without BOC in our in vitro cell culture assay system. Inducible Cas9-BOC activity will be confirmed in a developmental system and transgenes generated encoding Pyl tRNA, and PylRS and Cas9-BOC driven by promoters active around the time of fertilisation. Transgenic founders with tissue-restricted, BOC-inducible Cas9-BOC expression will be generated to confirm that full-length, functional Cas9-BOC expression can be induced in vivo. This will pave the way for a Cas9-BOC gene drive cassette that targets the single-copy black coat-colour gene for tyrosinase, Tyr, to cause insertional inactivation. BOC-inducible Cas9-BOC expression will be verified in transgenic mice carrying the Cas9-BOC gene drive cassette and the mice evaluated for their ability to transmit the gene drive in crosses with wild type animals. Control crosses (in which the gene drive is switched off) should produce 100% black offspring, but the gene drive should produce 100% white offspring. The result is a post-transcriptionally switchable gene drive.

1. Knowledge transfer
The Universities at Bath and Cardiff are top national teaching institutions. The applicants communicate cutting-edge research to students at all levels. Non-academic audiences such as charity workers, opinion and policy makers will be affected by this work, which will also benefit UK business and industry exploiting the multiple commercial applications of switchable protein expression and gene drives in vivo. The IP generated in this work will be tightly protected.

2. Research impact
The switchable protein system is not target- or species-limited and will impact national and international, commercial and academic researchers. It will rapidly impact synthetic biology, cell biology, stem cells, tissue engineering, systems research and development. It will enable the development of animals with more and different synthetic components and will impact tissue engineering and organ culture research, for example via switchable targeted chromatin remodeling. All of these will refine and reduce animal experiments, in accordance with the 3Rs.

3. Improved large animal genetics
By 2050 world food production will be required to increase by 70% to feed the population, placing a pressing need on new methods to engineer livestock. BOC-inducible gene drives will enhance livestock genome engineering - which is at present challenging, expensive and unreliable - by accelerating trait propagation within and between individuals and herds. For example, because BOC is cheap (an active serum concentration in pigs can be obtained for ~£1), BOC-inducible drives could be activated by commercial breeders to generate lines homozygous for disease resistance, thereby increasing food security.

4. Veterinary and human biomedicine
The general switchable system can be applied to essential functions in animal pathogens in vivo, so that they cannot grow in the absence of BOC. For example, high biosafety level pathogens containing a BOC codon introduced into an essential replication gene will require BOC for survival, rendering them non-viable outside the containment facility. In gene therapy, switchable gene drives will enable viral vectors to deliver repairing integration through a gene drive that stops once BOC is withdrawn. The drive rationale is applicable to large breeds containing extended human genome segments for improved disease modeling and xenotransplantation.

5. Gene drive control
A stringently switchable gene drive system will address many public concerns about gene drives. Gene drives will potentially combat disease, foster sustainable agriculture and eradicate invasive pests. For example, they could control malaria, zika virus, dengue and others by altering vectors species so that they are no longer spread and enable sustainable agriculture by reversing pesticide and herbicide resistance, for example, by making resistant weeds vulnerable to the broad-spectrum herbicide, glyphosate. Gene drives could also eradicate invasive species or reduce their costs; invasive species cause enormous ecological and environmental damage (an estimated $138 billion annually in the U.S.) and the top 100 introduced invasive pest species worldwide include at least 14 mammalian species. However, gene drives are perceived to be poorly understood and many are nervous about gene drive containment, especially in flying insects (eg mosquitoes and the diamondback moth), in which even limited-scale field trials are challenging and it is difficult in open releases to ensure that the drive cannot spread. A recent House of Lords report that broadly recognises the potential benefits of recombinant insects (HL Paper 68) alludes to these issues. In order to reap the potential benefits of gene drives, it will be necessary to demonstrate control. The current proposal directly addresses this concern in a switchable system.