EDGE CT: Developing transgenic and lineage tracing tools in planarians

涡虫吸引了几代科学家的想象力，今天在我们理解发育和再生的努力中发挥着关键作用。这些小扁虫可以从小的组织碎片中再生整个身体。由于当前测序技术的迅猛发展，在过去二十年中，人们从多种角度了解涡虫生物学取得了巨大进展，包括但不限于再生、干细胞、动物机体计划和进化、种系、神经系统、代谢、先天免疫、信号通路、调控rna和基因组稳定性。然而，由于缺乏转基因表达和基因组编辑工具，整个领域都受到了限制。与此同时，对哺乳动物细胞进行转染和基因编辑的技术创新迅猛发展。特别是，纳米技术方法可以非常局部地破坏细胞膜并直接注入分子货物，提供低细胞毒性和高传递效率。在这个项目中，研究人员将使用新型纳米技术“纳米吸管”将遗传物质输送到涡虫细胞中，并为转基因表达和基因组编辑提供一种简单有效的手段。该项目还旨在通过促进实验室之间的数据、协议和材料共享，以及组织培训研讨会，迅速传播新技术。此外，由于涡虫具有吸引人的再生能力、孩童般的可爱和易于在实验室中饲养，研究人员将促进涡虫在教学、教育和推广活动中的使用，以吸引非科学家对现代生物学研究的兴趣。涡虫是研究组织再生和干细胞生物学的有力动物模型。它们具有独特的能力，可以利用多能体细胞干细胞从微小的组织碎片中再生整个身体。在这个过程中，他们重置了身体的轴线，并以适当的比例重建了所有的器官。尽管有广泛的基因组和转录组学信息，但由于缺乏转基因工具，解决涡虫基因型-表型因果关系的进展受到严重阻碍，尽管数十年的尝试。由于研究者实验室最近取得的技术进步，包括细胞培养和移植方法、在难以转染的细胞类型中传递基因的新型纳米技术以及在虫细胞中成功表达发光报告基因，他们的目标是解决这一长期存在的挑战。本研究项目旨在发展和传播涡虫转基因的方法和资源。具体而言，该项目的目标包括：(1)开发用于在涡虫细胞中传递转基因的纳米级电穿孔方法，(2)演示使用CRISPR/Cas9进行基因组编辑，以及(3)加强涡虫研究界的合作、培训和多样性，以解决分子、细胞、有机体和进化生物学方面的广泛问题。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Planarians have captured the imagination of generations of scientists, and today play a critical role in our efforts to understand development and regeneration. These small flatworms can regenerate their entire body from small tissue fragments. As a result of the current surge in the sequencing technology, tremendous progress has been made over the past two decades to understand planarian biology from a variety of perspectives, including but not limited to regeneration, stem cell, animal body plan and evolution, germline, nervous system, metabolism, innate immunity, signaling pathways, regulatory RNAs, and genome stability. Yet the entire field has been limited by the lack of tools for transgene expression and genome editing. At the same time, technological innovation has exploded for transfection and gene editing of mammalian cells. In particular, nanotechnological approaches can very locally disrupt the cell membrane and inject molecular cargo directly, providing both low cell toxicity and high delivery efficiencies. In this project, the researchers will use the novel nanotechnology, "nanostraws," to deliver genetic materials into planarian cells, and provide a simple and effective means for transgene expression and genome editing. This project also aims to rapidly disseminate the new technology by facilitating data, protocol, and materials sharing across labs, and organizing training workshops. In addition, because of planarians' appealing regenerative ability, childlike cuteness, and ease to rear in the lab, the researchers will promote the usage of planarians in teaching, education, and outreach activities to engage non-scientists' interests in modern biological research.Planarians have been a powerful animal model to study tissue regeneration and stem cell biology. They have the unique capacity to regenerate the entire body from minute tissue fragments using pluripotent somatic stem cells. During this process, they reset the body axes and rebuild all organs in appropriate proportions. Although extensive genomic and transcriptomic information is available, progress on addressing causal genotype-phenotype relationships in planarians has been severely hindered by a lack of transgenic tools despite decades of attempts. Enabled by the recent technical advances made in the investigators' laboratories, including cell culture and transplantation methods, novel nanotechnology for gene delivery in hard-to-transfect cell types, and successful expression of luminescence reporters in planarian cells, they aim to address this long-standing challenge. This research project aims to develop and disseminate the methodology and resources for planarian transgenesis. Specifically, the goals of this project include (1) to develop nanoscale electroporation methods for transgene delivery in planarian cells, (2) to demonstrate genome editing using CRISPR/Cas9, and (3) to enhance the collaboration, training, and diversity of the planarian research community to address a broad spectrum of questions in molecular, cellular, organismal, and evolutionary biology.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.