Gene-drive system for efficient chloroplast transformation

Plants contain three genomes, the nuclear, the chloroplast and the mitochondrial. The nuclear genome is the largest and typically encodes in excess of 27,000 genes using from 120 million to several thousand base pairs depending on plant species. In contrast, the chloroplast typically encodes only 120 genes using just 150 thousand base pairs. However, a leaf cell can contain in excess of 100 chloroplasts each with 100 copies of the chloroplast genome. Thus despite representing just 0.1% of the sequence complexity of the cell, chloroplasts can contribute up over 10% of the DNA content. In part because of this, genes located on the chloroplast genome can produce much higher levels of protein than an equivalent single copy gene located on the nuclear genome (up to 300 fold higher). In addition, chloroplasts are excluded from pollen and the chloroplast DNA is only inherited from the pollinated and not the pollinating crop plant. This has made chloroplasts very attractive as "green factories" for producing novel high value proteins, metabolites and bio-polymers where high levels of gene expression are required. However, generating plants containing the introduced DNA sequence in their chloroplast rather than their nuclear genome is challenging, and selecting plants in which every chloroplast genome in every chloroplast in every cell is modified (referred to as homoplastomic) is time consuming and inefficient. Making sure that plants are homoplastomic is important, as if this is not done, the wild type chloroplast genomes tend to outcompete and displace the transgenic ones when the plant is removed from selective media and placed in soil.
Transformation of chloroplasts was first achieved nearly three decades ago, yet high-throughput, multi-species plastid transformation methods are still missing. Several plants species have been successfully used as green cell factories for the production of high value molecules such as vaccines, antimicrobials and other biopharmaceuticals. The technology has now reached the point where some of these are in commercial production in glasshouses in several European facilities. Other high value products such as designer "fish oils" are currently being evaluated in UK field trials of genetically modified oilseed crops. Chloroplast engineering has the potential to dramatically increase yields of such products, but this technology has been held back by the technical challenges described above that will be addressed in this project.
We will develop a novel transformation system using gene editing tools (CRISPR/Cas9) that will increase the efficiency of the initial transformation event and that will result in the introduced DNA rapidly spreading and replacing wild type chloroplast genomes, even in the absence of ongoing growth on selective media. This "gene-drive" mechanism will dramatically reduce the bottleneck in producing chloroplast engineered plants. Unlike other gene-drive systems that have been proposed (eg for eliminating malaria), the system here will be split so that the rapid spread is dependent upon the presence of a previously engineered nuclear background.

Plastid expression systems provide high levels of transgene expression, transgene containment (maternal inheritance in most crops), site-specific integration, and lack of gene silencing. However, chloroplast transformation is inefficient due to three issues; 1) the small size of the chloroplast as a biolistic target, 2) initial poor selection of transformation events when the introduced DNA constitutes the minority of the chloroplast sequences in a cell, 3) obtaining plants where all wild type plastid genomes are eliminated.
We will develop and test a two-component gene-drive system for efficient integration and amplification of plastid transgene constructs. In this system a nuclear encoded Cas9 protein will be imported into the chloroplast and combine with a guide RNA from the plastid transformation cassette to direct cleavage at the site of integration. After insertion, the integrated DNA will itself become a driver, initiating further rounds of cleavage of the wild type chloroplast DNA, which will either be repaired by homologous recombination with the driver or will be destroyed. We will also utilise plant FtsZ sequences whose over expression we have previously shown to give rise to plants with cells that contain a single large chloroplast. Such chloroplasts will provide a larger target for the initial biolistic transformation, further increasing transformation efficiency. Both the Cas9 and the FtsZ sequences causing large chloroplasts will be removed from the transplastomic plants following crossing with wild type individuals (as the pollen donor). Together, these approaches will directly address the three factors that have limited the use of plastid transformation in the past.

Transgenic plants are now are being used commercially to make high value products such as antibodies, antigens and other therapeutic proteins, and transgenic plant production of metabolites such as "fish-oils", waxes and novel carotenoids are in field evaluation stages. Plant derived edible vaccines, particularly for animal husbandry and aquaculture are also receiving increasing attention and several are well advanced towards commercialisation. For many similar applications, chloroplast transformation has been shown to give higher yields, greater stability of transgene expression and also provides a level of gene containment due to the absence of plastid DNA in the pollen generative cell. However, despite first being reported nearly 30 years ago, the protracted, inefficient and species-limited process of chloroplast transformation has meant that the technology has not been widely adopted. Our approach will make chloroplast transformation accessible to research groups, SMEs and larger companies involved in exploiting plant systems for synthetic biology and molecular pharming type projects. It will therefore directly impact on the way in which these groups go about their research and development and could also impact global food security through healthier livestock and fish production with fewer conventional inputs.
Many of the therapeutic products being developed by research groups around the world are focused on providing a cheap source of vaccines and medicines for developing countries. The uptake of this technology should result in higher yields of such products and this will have a positive impact by reducing costs to the end users.
In addition, the proposed technology can be readily adapted for addressing fundamental research questions where specific mutations to, or replacement of, endogenous chloroplast located genes is required. Thus it would impact the ability of research groups carrying out comparative studies of photosynthesis and photosynthetic efficiency to design and carry out a wide range of new experiments.
Our principal goal is to develop and exemplify the technology in a model plant (tobacco). However, successful, the technology could readily be expanded to other crop species. Ultimately, it may allow the stable transformation of monocot species such as maize, rice and barley which remains the Holy Grail of plastid transformation.
We are committed to communicate the benefits of this research to the public and government agencies and policy makers, and to the wider scientific community. An important impact of this research on the wider academic community will be through knowledge exchange via conferences, seminars and research papers published in high impact journals, demonstrating research originality, significance and impact. The project will also foster the development of a PDRA in a multi-disciplinary environment, facilitating career readiness in preparation for the multi-faceted bio-economy.