Collaborative Research: EAGER: Development of an Artificial Chromosome System in Chlamydomonas Based on CENH3 Tethering

合作研究：EAGER：基于 CENH3 束缚的衣藻人工染色体系统的开发

• 批准号: 2151106
• 负责人: R Kelly Dawe
• 金额: $ 9.37万
• 依托单位: University of Georgia Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2151106&HistoricalAwards=false
• 关键词: Collaborative; Research; EAGER; Development; Artificial

Some of the most pressing problems in agriculture and biotechnology can only be tackled by large scale changes in crop or host genomes where multiple genes must be introduced and inherited together for successful trait improvement. For example, desirable crop traits such as disease resistance, improved yields and resistance to climate change may require multiple genes introduced into one strain or cultivar, a task that is not practical with current transgenic methods. Artificial chromosomes have the potential for solving this problem, but the technology for creating and deploying plant artificial chromosomes remains inadequate. Under this proposal a green alga and established research organism that is related to land plants, Chlamydomonas reinhardtii, will be used to accelerate the design and implementation of artificial chromosomes. Because of its easy cultivation and rapid generation time (4 hours versus 3+ months for most plants) Chlamydomonas artificial chromosomes can be developed much faster than in land plants. The lessons learned from successful implementation of artificial chromosomes in Chlamydomonas can be used to fast-track the creation of artificial chromosomes in land plants and will have important immediate benefits by enabling the modification and improvement of algae as sources of biomass, biofuel, and high value pharmaceuticals and nutraceuticals which cannot be easily or cheaply produced using existing biotechnology.This proposal aims to address the most significant challenge in any effort to design a functional synthetic chromosome in eukaryotes, the centromere. Centromeres in all eukaryotes are defined by the presence of the histone H3 variant CENP-A/CENH3. The Dawe laboratory has demonstrated the feasibility of activating new plant centromeres using a simple tethering approach based on the DNA binding domain of the LexA repressor to its operator, LexO, which serves as a synthetic centromere organizing center. In this method a host strain expressing a native CENH3 sequence fused to LexA is generated. Next, an array of LexO binding sites is incorporated into a small synthetic chromosome containing telomeric sequences and selectable markers on both arms. Preliminary data from the Dawe laboratory shows that in plants the LexA-CENH3 protein binds to the LexO array and recruits additional CENH3 to create functional centromeres. Under this proposal a similar method will be adopted for the alga Chlamydomonas as a potentially game-changing advance in synthetic biology. This proposal combines the established expertise of the Dawe laboratory in plant artificial chromosomes and the Umen laboratory in algal genetics and molecular biology to test and establish an artificial chromosome system in Chlamydomonas. Under this proposal 1. Chlamydomonas centromeres will be fully sequenced and validated using long-read sequencing methods, and formally validated/defined using chromatin immunoprecipitation with a centromere marker protein, CenH3. 2. Transgenic Chlamydomonas strains expressing its native CenH3 paralogs as fusion proteins to LexA will be created and validated. 3. Chlamydomonas artificial chromosomes of different sizes and configurations (e.g., linear, circular) and containing LexO arrays for centromere nucleation will be built and tested for centromere assembly, mitotic/meiotic segregation, stability, and gene expression.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

农业和生物技术中一些最紧迫的问题只能通过作物或宿主基因组的大规模变化来解决，其中必须引入多个基因并一起遗传以成功地进行性状改良。例如，理想的作物性状，如抗病性，提高产量和对气候变化的抗性可能需要将多个基因引入一个品系或栽培品种，这是目前转基因方法不切实际的任务。人工染色体具有解决这一问题的潜力，但创建和部署植物人工染色体的技术仍然不足。根据这一提议，一种与陆地植物有关的绿色植物和已建立的研究生物体，莱茵衣藻，将用于加速人工染色体的设计和实施。由于其易于培养和快速的世代时间（4小时对大多数植物3个月以上）衣原体人工染色体可以开发得比陆地植物快得多。从衣原体人工染色体的成功实施中吸取的教训可用于快速跟踪陆地植物中人工染色体的创建，并将通过使藻类作为生物质，生物燃料，以及利用现有生物技术不能轻易或廉价生产的高价值药物和营养品。这项建议旨在解决最重要的问题，在真核生物中设计功能性合成染色体的任何努力都是一个挑战，即着丝粒。所有真核生物中的着丝粒由组蛋白H3变体CENP-A/CENH 3的存在来定义。Dawe实验室已经证明了使用基于莱克萨阻遏物的DNA结合结构域与其操纵子LexO的简单拴系方法激活新植物着丝粒的可行性，LexO充当合成的着丝粒组织中心。在该方法中，产生了表达与莱克萨融合的天然CENH 3序列的宿主菌株。接下来，将LexO结合位点的阵列并入在两臂上含有端粒序列和选择性标记的小合成染色体中。来自Dawe实验室的初步数据显示，在植物中，LexA-CENH 3蛋白与LexO阵列结合，并招募额外的CENH 3来创建功能性着丝粒。根据这一提议，一种类似的方法将被用于衣原体，作为合成生物学中潜在的改变游戏规则的进步。该提案结合了Dawe实验室在植物人工染色体方面的专业知识和Umen实验室在藻类遗传学和分子生物学方面的专业知识，以测试和建立衣原体的人工染色体系统。根据这项建议，1。衣原体着丝粒将使用长读序测序方法进行完全测序和验证，并使用染色质免疫沉淀与着丝粒标记蛋白CenH 3进行正式验证/定义。2.将创建并验证表达其天然CenH 3旁系同源物作为与莱克萨的融合蛋白的转基因衣原体菌株。3.不同大小和构型的衣原体人工染色体（例如，该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。