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

农业和生物技术中一些最紧迫的问题只能通过作物或寄主基因组的大规模变化来解决，在这些基因组中，必须引入并共同遗传多个基因，才能成功地进行性状改进。例如，想要获得抗病、提高产量和抵抗气候变化等理想的作物性状，可能需要将多个基因导入一个品系或品种，这一任务在目前的转基因方法下是不现实的。人工染色体具有解决这一问题的潜力，但创造和部署植物人工染色体的技术仍然不足。根据这项提议，将使用绿藻和与陆地植物相关的现有研究有机体--莱茵衣藻--来加速人工染色体的设计和实施。衣藻人工染色体的发育速度比陆地植物快得多，因为它容易培养，而且世代快(大多数植物只需4小时，而大多数植物是3个多月)。从衣藻人工染色体的成功实施中学到的经验可以用来快速在陆地植物中创建人工染色体，并将通过修改和改进藻类作为生物质、生物燃料和高价值药品和营养食品的来源而产生重要的直接好处，这些资源使用现有的生物技术无法轻松或廉价地生产。这项建议旨在解决在真核生物中设计功能性合成染色体的最重大挑战-着丝粒。所有真核生物中的着丝粒都是由组蛋白H3变异体CENP-A/CENH3定义的。Dawe实验室已经证明了使用一种简单的方法激活新的植物着丝粒的可行性，该方法基于LexA抑制子的DNA结合结构域与其操纵子LexO，LexO作为合成着丝粒组织中心。在该方法中，产生了表达与LexA融合的天然CENH3序列的宿主菌株。接下来，一组LexO结合位点被整合到一个小的合成染色体中，该染色体包含端粒序列和双臂上的可选标记。来自Dawe实验室的初步数据显示，在植物中，LexA-CENH3蛋白与LexO阵列结合，并招募额外的CENH3来创建功能着丝粒。根据这项提议，将对藻类衣藻采用类似的方法，作为合成生物学中潜在改变游戏规则的进步。这项建议结合了DAWE实验室在植物人工染色体方面的现有专业知识和乌门实验室在藻类遗传学和分子生物学方面的专业知识，以测试和建立衣藻属人工染色体系统。根据这项提议1.衣藻着丝粒将使用长读测序方法进行完全测序和验证，并使用着丝粒标记蛋白CenH3的染色质免疫沉淀法进行正式验证/定义。2.将其天然的CenH3蛋白作为LexA融合蛋白的转基因衣藻菌株将被建立和验证。3.衣藻人工染色体的不同大小和配置(例如，线性、环形)和包含着丝粒核的LexO阵列将被建立并测试着丝粒组装、有丝分裂/减数分裂分离、稳定性和基因表达。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.