NSF Postdoctoral Fellowship in Biology: Methods for Virus-based Plant Genome Editing

本行动资助2023财年美国国家科学基金会植物基因组生物学博士后研究奖学金。该奖学金支持奖学金获得者在主办实验室的研究和培训计划，该奖学金获得者还提出了扩大生物学参与的计划。该奖学金的研究和培训计划的标题是“基于病毒的植物基因组编辑方法”。该奖学金的主办机构是明尼苏达大学，赞助科学家是Daniel Voytas博士。植物基因组的精确编辑有可能加速作物改良和推进功能基因组学。在这一领域已经取得了相当大的进展，然而，最常用的植物基因编辑方法通常需要使用组织培养。组织培养的缺点是它需要专业技术，时间密集，并且成功仅限于少数基因型。该项目旨在开发一种基于病毒的植物基因组编辑方法，从而有可能显著减少对组织培养的需求。如果成功，开发的方法可以应用于作物物种，并大大加快作物基因编辑的难度，提高育种者创造新性状的能力。这项工作的结果还可以通过简化基因功能研究中产生突变体的方法，对植物生物学产生广泛的影响。培训目标包括外展活动，以促进社区参与植物基因编辑和植物科学，指导本科生，并通过参与美国国家科学基金会资助的植物基因工程网络（PlantGENE）传播植物基因编辑技术。将基因编辑试剂传递到植物细胞的最常用方法是农杆菌介导的转化和生物轰击。为了恢复基因编辑植物，需要组织培养技术从编辑过的体细胞中再生植物。这是一个挑战，原因有几个。首先，它通常需要数月的培训和技术专长的发展。其次，成功的再生通常局限于少数基因型。最后，组织培养过程可能导致体细胞无性系变异。利用病毒对植物进行基因组修饰，有可能缓解组织培养和试剂输送的瓶颈。本项目的研究目标是：(1)提高基于TRV的载体的载货能力，使TRV能够将CRISPR-Cas核酸酶运送到分生组织细胞；(2)测试使用微型CRISPR-Cas核酸酶来克服基于TRV的载体载货能力的限制；(3)鉴定增加sgrna和核酸酶的细胞间流动性以促进向种系运送的RNA序列。这项研究将产生植物基因组修饰方法的知识，可用于植物育种者和研究人员开发新的性状和研究基因功能。生成的数据将通过提交给公共存储库、项目网页和出版物以及在科学会议、研讨会和讲习班上的演讲向社区发布。种质资源将根据要求提供。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。

This action funds an NSF Plant Genome Postdoctoral Research Fellowship in Biology for FY 2023. The fellowship supports a research and training plan in a host laboratory for the Fellow who also presents a plan to broaden participation in biology. The title of the research and training plan for this fellowship to Chandler Meyer is “Methods for Virus-based Plant Genome Editing” The host institution for the fellowship is the University of Minnesota and the sponsoring scientist is Dr. Daniel Voytas.Precision editing of plant genomes has the potential to accelerate crop improvement and advance functional genomics. Considerable progress has been made in this area, however, the most commonly used methods for gene editing in plants often require the use of tissue culture. The drawback of tissue culture is that it requires technical expertise, it is time intensive, and success is limited to a few genotypes. This project aims to develop a viral-based method of genome editing in plants that offers the potential to significantly reduce the need for tissue culture. If successful, the methods developed could be applied to crop species and greatly accelerate the ease of gene editing in crop plants, improving the ability of breeders to create new traits. The results from this work could also have a broad impact on plant biology by streamlining methods for generating mutants for gene function studies. Training objectives include outreach activities to promote community engagement in plant gene editing and plant science, mentoring undergraduate students, and dissemination of plant gene editing techniques by participation in the NSF-funded Plant Genetic Engineering Network (PlantGENE). The most commonly used methods to deliver gene editing reagents to plant cells are Agrobacterium-mediated transformation and biolistic bombardment. To recover a gene-editing plant, tissue culture techniques are needed to regenerate a plant from the edited somatic cells. This is challenging for a few reasons. First, it often requires months of training and development of technical expertise. Second, successful regeneration is often limited to a few genotypes. Lastly, the tissue culture process can result in somaclonal variation. The use of viruses for genome modification in plants offers the potential to ease the tissue culture and reagent delivery bottlenecks. The research objectives of this project are to (1) improve the cargo capacity of TRV-based vectors so that TRV can deliver CRISPR-Cas nucleases to meristematic cells, (2) test the use of miniature CRISPR-Cas nucleases to overcome TRV-based vector cargo capacity limitations, and (3) identify RNA sequences that increase cell-to-cell mobility of sgRNAs and nucleases to promote delivery to the germline. This research will generate knowledge of methods for genomic modification of plants that can be used by plant breeders and researchers to develop novel traits and study gene function. Data generated will be released to the community through submission to public repositories, project web pages, and publications as well as presentations at scientific conferences, seminars, and workshops. Germplasm will be available upon request.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.