EAGER: Developing High-Throughput CRISPR/Single-cell RNA-seq Screening in Maize

该项目旨在结合两种强大的新方法来加速适应环境压力的作物育种，为应对农业挑战提供新工具，如尽量减少干旱或高温造成的损失。该项目中使用的一种技术方法（简称CRISPR）创造基因变异的方式与自然中随机发生的方式大致相同，但速度更快、更容易、目标更精确。第二种技术被称为单细胞rna测序，它允许研究人员测量单个细胞中每个基因的活性。这两种方法共同实现了育种方法的“小型化”——测试许多作物变种，以找到具有有用农业性状的品种。使用CRISPR可以产生数百万个不同的单细胞变体，这些变体受到只有最适合的细胞才能存活的压力，并使用单细胞rna测序分析这些细胞的精确变化，以使它们能够存活。这种方法与传统育种方法结合使用，可以为植物育种家和遗传学家提供一种资源，帮助他们了解对稳定作物生产至关重要的性状的遗传调节因子。在该项目的教育目标中，研究人员将扩展一个成功的项目，培养对科学事业感兴趣的有才华的纽约市高中生。更具体地说，该项目旨在开发玉米原生质体的CRISPR/单细胞RNA-seq筛选，为植物育种和遗传分析引入一种新的方法。利用CRISPR和相关的内切酶，如Cas9，将进行大规模的基因筛选，其中将一组不同的引导rna转染到数百万个细胞中。然后对这些细胞进行选择；幸存者识别有益的扰动，而池中代表性不足的基因型识别生存压力所必需的基因。与单细胞RNA-seq相结合，这种方法提供了遗传通路的敏感读数，并已在动物细胞中成功使用。该方法尚未应用于植物，不仅代表了作物筛选规模的增加，而且还揭示了以前被遗传冗余遮蔽的植物育种遗传靶点。由于能够筛选数百万个细胞，文库也可以以滴度转染，导致每个细胞有多个向导，有效地允许快速轻松地筛选高阶突变体。已知许多重要的胁迫耐受性性状是在细胞水平介导的，标记辅助选择允许筛选调节组织水平性状的靶向途径。该项目将利用生长素反应标记作为选择工具，在玉米细胞中试实验中评估CRISPR的效率，以生长素信号通路为目标。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 project seeks to combine two powerful, new approaches to accelerate breeding of crops adapted to environmental stresses, providing new tools to deal with agricultural challenges such as minimizing loss from drought or heat. One of the technical approaches used in the project, known by its acronym CRISPR -- creates genetic variation much the same way it occurs randomly in nature but with much greater speed, ease and target precision. The second technique, known as single-cell RNA-sequencing, allows researchers to measure activity of, potentially, every gene in a single cell. Together, the two methods allow a "miniaturization" of the breeding approach -- testing many crop variants to find ones with useful agricultural traits. Different, single cell variants can be generated by the millions using CRISPR, subjected to a stress in which only the fittest cells survive, and those cells analyzed for the precise changes that allowed them to survive using single-cell RNA-sequencing. This method, used in conjunction with classical breeding approaches, can serve as a resource for plant breeders and geneticists working to understand the genetic regulators of traits that are important for stable crop production. In the education goals of the project, the investigators will extend a successful program that trains talented New York City high student interested in a career in science.More specifically, this project aims to develop pooled CRISPR/single-cell RNA-seq screens in maize protoplasts to introduce a novel approach to plant breeding and genetic analysis. Using CRISPR and associated endonucleases like Cas9, large-scale genetic screens will be carried out in which a diverse set of guide-RNAs is transfected into a pool of millions of cells. The cells are then subjected to a selection; survivors identify beneficial perturbations while underrepresented genotypes in the pool identify genes necessary to survive the stress. Coupled with single-cell RNA-seq, this approach provides a sensitive readout of genetic pathways, and has been used successfully in animal cells. The method has not yet been applied to plants and could not only represent an increase in the scale of crop screening but also reveal genetic targets for plant breeding that have previously been obscured by genetic redundancy. With the ability to screen millions of cells, libraries can also be transfected at titers that lead to multiple guides per cell, effectively allowing higher order mutants to be screened quickly and easily. Many important stress tolerance traits are known to be mediated at the cell level, and marker assisted selection allows screening for targeted pathways that regulate tissue level traits. The project will assess the efficiency of CRISPR in maize cells in pilot experiments that target the auxin signaling pathway using auxin response markers as a selection tool.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.