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，在这些基因组中，必须将多个基因引入和遗传在一起以成功提高特质。例如，理想的作物特征，例如抗病性，提高的产量和对气候变化的抵抗力可能需要将多个基因引入一个菌株或品种中，这是当前转基因方法不实用的任务。人造染色体具有解决此问题的潜力，但是创建和部署植物人造染色体的技术仍然不足。根据该提案，将使用与土地植物有关的绿色藻类和建立的研究生物，Chlamydomonas renhardtii将用于加速人工染色体的设计和实施。由于其易于种植和快速生成时间（大多数植物的3个月比3个月以上），人造染色体的衣原体可以比土地植物快得多。从成功实施人造染色体中汲取的经验教训可以用于快速跟踪陆地植物中人造染色体的创建，并通过实现藻类的修改和改善作为生物量，生物燃料和高价值的生物学的生物学的源泉，从而使藻类的修改和改善能够轻松地进行生物学，从而获得重要的直接益处。在设计真核生物（Centromere）中设计功能性合成染色体的任何努力中，最重大的挑战。所有真核生物中的centromeres均由组蛋白H3变体CENP-A/CENH3的存在定义。 DAWE实验室证明了使用基于Lexa阻遏物的DNA结合结构域来激活新的植物中心粒子的可行性，该方法是其操作员Lexo，该方法是合成中心的组织中心。在这种方法中，产生表达与Lexa融合的天然CENH3序列的宿主应变。接下来，将一系列雷克萨结合位点纳入两个臂上的小合成染色体中，其中包含端粒序列和可选标记。来自DAWE实验室的初步数据表明，在植物中，Lexa-Cenh3蛋白与Lexo阵列结合，并募集其他CENH3以创建功能性的centromeres。根据该提案，藻类衣原体将采用类似的方法，作为合成生物学的潜在改变游戏规则的进步。该提案结合了DAWE实验室在植物人造染色体中的既定专业知识，以及在藻类遗传学和分子生物学中的UMEN实验室，以测试和建立在衣原体中的人造染色体系统。在此提案1下，将使用长阅读测序方法对衣原体中心粒子进行全面测序并验证，并使用Centromere标记蛋白CENH3正式验证/定义/定义/定义。 2。将创建和验证的转基因衣原体菌株作为融合蛋白作为融合蛋白作为融合蛋白。 3。将建造和测试中央组装，有丝粒组装，有线/减数分裂的稳定性，稳定性，通过评估nsf的Intorditial deem，将建立和测试，并测试具有不同大小和配置（例如，线性，圆形）和包含用于中心成核的Lexo阵列的衣原体人造染色体。优点和更广泛的影响审查标准。