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

EAGER：在玉米中开发高通量 CRISPR/单细胞 RNA-seq 筛选

• 批准号: 1833156
• 负责人: Kenneth Birnbaum
• 金额: $ 30万
• 依托单位: New York University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-15 至 2020-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1833156&HistoricalAwards=false
• 关键词: EAGER; Developing; Throughput; CRISPR; Single

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.

该项目旨在将两种强大的新方法结合起来，加速适应环境压力的作物育种，提供新工具来应对农业挑战，例如最大限度地减少干旱或高温造成的损失。该项目中使用的技术方法之一，其缩写为 CRISPR，产生遗传变异的方式与自然界中随机发生的方式非常相似，但速度、简便性和目标精度要高得多。第二种技术被称为单细胞 RNA 测序，它使研究人员能够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”——测试许多作物变体以找到具有有用农业性状的作物变体。使用 CRISPR 可以产生数以百万计的不同单细胞变体，在压力下只有最适应的细胞才能生存，并使用单细胞 RNA 测序分析这些细胞的精确变化，使它们能够生存。这种方法与经典育种方法结合使用，可以作为植物育种家和遗传学家的资源，帮助他们了解对稳定作物生产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，培养对科学职业感兴趣的纽约市优秀高中生。更具体地说，该项目旨在开发玉米原生质体中的 CRISPR/单细胞 RNA-seq 筛选，以引入一种新的植物育种和遗传分析方法。使用 CRISPR 和相关的核酸内切酶（如 Cas9），将进行大规模遗传筛选，其中将一组不同的向导 RNA 转染到数百万个细胞中。然后对细胞进行选择；幸存者识别出有益的扰动，而池中代表性不足的基因型则识别出在压力下生存所必需的基因。与单细胞 RNA-seq 相结合，这种方法提供了遗传途径的灵敏读数，并已成功应用于动物细胞。该方法尚未应用于植物，不仅可以代表作物筛选规模的增加，还可以揭示以前因遗传冗余而掩盖的植物育种的遗传目标。由于能够筛选数百万个细胞，文库还可以以每个细胞产生多个指导的滴度进行转染，从而有效地快速、轻松地筛选更高阶的突变体。已知许多重要的应激耐受性状是在细胞水平上介导的，标记辅助选择允许筛选调节组织水平性状的目标途径。该项目将在试点实验中评估 CRISPR 在玉米细胞中的效率，这些实验使用生长素反应标记作为选择工具，以生长素信号通路为目标。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。