Gene-drive system for efficient chloroplast transformation

植物含有三个基因组，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，使用1.2亿至数千个碱基对，具体取决于植物物种。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，叶细胞可以含有超过100个叶绿体，每个叶绿体具有100个叶绿体基因组拷贝。因此，尽管叶绿体仅占细胞序列复杂性的0.1%，但它可以贡献超过10%的DNA含量。部分原因在于，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等效单拷贝基因高得多的蛋白质水平（高达300倍）。此外，叶绿体被排除在花粉之外，并且叶绿体DNA仅遗传自授粉作物而不是授粉作物。这使得叶绿体作为“绿色工厂”非常有吸引力，用于生产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。然而，产生在其叶绿体而不是其核基因组中含有引入的DNA序列的植物是具有挑战性的，并且选择其中每个细胞中的每个叶绿体中的每个叶绿体基因组被修饰（称为同源质体）的植物是耗时且低效的。确保植物是同源的是重要的，如果不这样做，当植物从选择性培养基中取出并置于土壤中时，野生型叶绿体基因组倾向于胜过并取代转基因叶绿体基因组。近三十年前首次实现了叶绿体的转化，但仍然缺少高通量、多物种的质体转化方法。几种植物物种已成功地用作绿色细胞工厂，用于生产高价值分子，如疫苗、抗微生物剂和其他生物药物。这项技术现在已经达到了其中一些在欧洲设施的温室中进行商业生产的程度。其他高价值产品，如设计师“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。叶绿体工程有可能大幅提高此类产品的产量，但该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。我们将使用基因编辑工具（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，并将导致引入的DNA快速传播并取代野生型叶绿体基因组，即使在选择性培养基上没有持续生长的情况下。这种“基因驱动”机制将大大减少生产叶绿体工程植物的瓶颈。与其他已经提出的基因驱动系统（例如消除疟疾）不同，这里的系统将被分割，以便快速传播取决于先前工程核背景的存在。

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.