Gene-drive system for efficient chloroplast transformation

用于高效叶绿体转化的基因驱动系统

• 批准号: BB/R021937/1
• 负责人: Katalin Kovacs
• 金额: $ 19.31万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR021937%2F1
• 关键词: Gene; drive; system; efficient; chloroplast

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.

植物包含三个基因组，分别是核，叶绿体和线粒体。核基因组是最大的，通常用1.2亿至数千对的基因编码超过27,000个基因，具体取决于植物物种。相反，叶绿体通常仅使用15万个碱基对编码120个基因。但是，叶片细胞可以包含超过100个叶绿体，每个叶绿体都有100份叶绿体基因组。因此，尽管仅代表细胞序列复杂性的0.1％，但叶绿体仍可以贡献超过10％的DNA含量。在某种程度上，位于叶绿体基因组上的基因比位于核基因组上的等效单拷贝基因（高达300倍）的基因可以产生更高的蛋白质水平。此外，叶绿体被排除在花粉中，叶绿体DNA仅从授粉而不是授粉的农作物植物遗传。这使叶绿体变得非常吸引人，就像“绿色工厂”，用于产生新的高价值蛋白，代谢物和生物聚合物，其中需要高水平的基因表达。然而，生成含有叶绿体中引入的DNA序列而不是其核基因组的植物具有挑战性，并且选择植物，其中每个细胞中每个叶绿体中每个叶绿体基因组的每个叶绿体基因组都经过修饰（称为同源体）都是耗时且效率不足的。确保植物是同质植物很重要的，就好像未完成一样，野生型叶绿体基因组往往会超过并置于转基因的植物从选择性培养基中去除并放置在土壤中时。叶绿体的转化最初是在将近三十年前实现的，但仍缺少高通量，多物种塑料转化方法。几种植物已成功用作绿细胞工厂，用于生产高价值分子，例如疫苗，抗菌剂和其他生物药物。现在，该技术已经达到了其中一些在欧洲设施中的广阔的商业生产中的地步。目前，正在对基因改良油料作物的英国现场试验进行评估，目前正在对其他高价值产品（例如设计师“鱼油”）进行评估。叶绿体工程有可能显着提高此类产品的产量，但是这项技术因上述技术挑战所阻碍，该技术将在该项目中解决。我们将使用基因编辑工具（CRISPR/CAS9）开发一种新型的转换系统，该系统将提高初始转化事件的效率，并导致引入DNA迅速扩散并取代野生型叶绿体基因组，即使没有持续的选择性培养基的增长。这种“基因驱动”机制将大大减少产生叶绿体工程植物的瓶颈。与已提出的其他基因驱动系统（例如消除疟疾）不同，此处的系统将被拆分，以便快速传播取决于先前设计的核背景。