Gene-drive system for efficient chloroplast transformation

植物包含三种基因组，即核、叶绿体和线粒体。核基因组是最大的，通常编码超过27,000个基因，根据植物物种的不同，使用从1.2亿到数千个碱基对。相比之下，叶绿体通常只使用15万个碱基对编码120个基因。然而，一个叶细胞可以包含超过100个叶绿体，每个叶绿体基因组有100个拷贝。因此，尽管只占细胞序列复杂性的0.1%，但叶绿体可以贡献超过10%的DNA含量。部分原因是，位于叶绿体基因组上的基因可以产生比位于核基因组上的同等单一拷贝基因(高达300倍)高得多的蛋白质水平。此外，叶绿体被排除在花粉之外，叶绿体DNA只从授粉的作物中遗传，而不是从授粉的作物中遗传。这使得叶绿体成为需要高水平基因表达的新型高价值蛋白质、代谢物和生物聚合物的“绿色工厂”，非常有吸引力。然而，在叶绿体而不是核基因组中产生含有引入的DNA序列的植物是具有挑战性的，并且选择其中每个细胞的每个叶绿体中的每个叶绿体基因组都被修改的植物(称为同源体体)是耗时和低效的。确保植物是同质体是很重要的，因为如果不这样做，当植物从选择性培养基中移出并放置在土壤中时，野生型叶绿体基因组往往会竞争对手，取代转基因植物。叶绿体的转化早在近30年前就已经实现，但高通量、多物种的叶绿体转化方法仍然缺乏。几种植物已被成功地用作绿色细胞工厂，用于生产疫苗、抗菌剂和其他生物制药等高价值分子。这项技术现在已经达到了这样的地步，其中一些已经在欧洲几家工厂的温室里进行了商业生产。其他高附加值产品，如设计师的“鱼油”，目前正在英国转基因油籽作物的田间试验中进行评估。叶绿体工程有可能极大地提高这类产品的产量，但这项技术受到上述技术挑战的阻碍，这些挑战将在本项目中解决。我们将利用基因编辑工具(CRISPR/Cas9)开发一种新的转化系统，该系统将提高初始转化事件的效率，并将导致导入的DNA快速传播和替换野生型叶绿体基因组，即使在没有在选择性培养基上进行生长的情况下也是如此。这种“基因驱动”机制将极大地减少生产叶绿体工程植物的瓶颈。与已经提出的其他基因驱动系统不同(例如用于消除疟疾)，这里的系统将被拆分，以便快速传播依赖于先前设计的核背景的存在。

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.