Collaborative Research: EDGE FGT: Genome-wide knock-out mutant libraries for the microcrustacean Daphnia

突变感兴趣基因的DNA序列是了解它们在不同生物体中的生物学功能的有价值的方法。与已建立的模型系统相比，大量的基因敲除突变体在新兴的模式生物中并不广泛存在。这一限制极大地削弱了新兴模型系统做出新的科学发现的能力。为了解决这一问题，该项目将利用CRISPR基因编辑技术在三种淡水微甲壳动物Daphnia(D.Magna，D.Pulex和D.Pulicaria)中开发大量基因敲除突变体，这是生态基因组学、进化基因组学和环境基因组学中的一个新兴模式系统。水蚤基因敲除突变体将为科学界提供前所未有的机会来表征这些基因的生物学功能。该项目还将通过免费研讨会传播CRISPR基因编辑的技术专业知识，对象是处于不同职业阶段的生物学家，包括本科生、毕业生、博士后研究人员和教师。此外，该项目将开发一门创新的基因组学课程，使学生能够在课堂上动手研究，并增强不同本科生的教育体验。该计划是敲除水蚤的基因，包括许多候选基因，这些基因可能在这些物种的适应、适应和进化中发挥关键作用。将产生单独的敲除突变系，并在实验室中进行维护，并与研究社区免费分享。将对每个突变株系进行全基因组测序，以确定其基因组背景。该项目将与全球水蚤研究界合作，在基因组和生物水平上评估基因敲除突变体的功能变化，以验证基因在各种环境中的作用，并推断基因相互作用和基因调控网络。最后，将开发一个免费的、面向公众的数据库，以存储突变品系的基因组和表型数据，促进和管理水蚤突变体的分布，并分发实验方案和试剂。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。

Mutating the DNA sequences of genes of interest is a valuable approach for understanding their biological functions in diverse organisms. In contrast to established model systems, large collections of gene knock-out mutants are not widely available in emerging model organisms. This limitation greatly diminishes the power of emerging model systems for making novel scientific discoveries. To address the issue, this project will develop a large collection of gene knock-out mutants using CRISPR gene editing in three species of the freshwater microcrustacean Daphnia (D. magna, D. pulex, and D. pulicaria), which is an emerging model system in ecological genomics, evolutionary genomics, and environmental genomics. The Daphnia knock-out mutants will provide unprecedented opportunities for the scientific community to characterize the biological functions of the genes. The project will also disseminate technical expertise for CRISPR gene editing in Daphnia through free workshops, targeting biologists at different career stages including undergraduates, graduates, postdoctoral researchers, and faculty. Furthermore, the project will develop an innovative genomics curriculum to enable hands-on research in the classroom and enhance the education experience of diverse undergraduate students. The plan is to knock out genes in Daphnia, including many candidate genes that may play critical roles in acclimation, adaptation, and evolution of these species. Individual knock-out mutant lines will be generated, maintained in the laboratory, and freely shared with the research community. Each mutant line will be whole-genome sequenced to characterize its genomic background. In collaboration with the global Daphnia research community, this project will evaluate the functional changes in knock-out mutants at both genomic and organism levels to validate the role of the genes in various environmental settings and to infer gene interaction and gene regulation networks. Lastly, a free, public-facing database will be developed to host the genomic and phenotypic data of the mutant lines, to promote and manage the distribution of Daphnia mutants, and to distribute experimental protocols and reagents.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.