EAGER: A Genome Wide HDR Enhancement Screen in Maize

EAGER：玉米全基因组 HDR 增强屏幕

• 批准号: 2409037
• 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: 2024
• 资助国家: 美国
• 起止时间: 2024-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2409037&HistoricalAwards=false
• 关键词: EAGER; Genome; Wide; HDR; Enhancement

A powerful way to protect crops from disease and climate stress is to replace traits of susceptible crop varieties with traits from resistant varieties. The enormous variety of traits that different plants have evolved to cope with stress then becomes available to protect the food supply. The discovery of CRISPR (clustered regularly interspaced short palindromic repeats) and its application to biology have made such trait-replacement approaches possible by replacing one piece of DNA (the genetic material that encodes traits) with another at a precise location in a plant’s DNA. However, the replacement activity, which relies on the plant’s own proteins, is highly inefficient in plants, making its use impractical. The project embarks on a search to test many possible ways to change crop DNA in order to make the replacement function of CRISPR more efficient. In this project, a plant and animal researcher join forces to conduct the type of searches – called screens – that were done in human cells and now in plant cells. These screens are done in preparations of millions of free cells that activate many genes to test for one that makes replacement more efficient. The goal of the project is to develop a method to make the most productive crops amenable to receiving new genes that make them more stress-tolerant.The ability to transfer traits from one plant to another using CRISPR-mediated homology directed repair (HDR) would offer a powerful approach to develop new varieties that could tolerate climate change. However, HDR, which involves the activity of endogenous genes, is highly inefficient in plants, limiting its use. The project adapts HDR-enhancement screens performed in animals to plants, screening for endogenous genes that improve HDR efficiency. The screen involves a genome-wide CRISPR activation (CRISPRa) component in a suspension of free plant cells (protoplasts), in this case from Zea mays. The CRISPRa components are tittered to activate one gene per cell. The cells come from a line that carries a constitutively expressed green fluorescent protein (GFP). A rare “hit” – a gene whose activation improves HDR – will replace the GFP with a cyan fluorescent protein (CFP) sequence. The CFP-positive cells are separated via Fluorescence Activated Cell Sorting (FACS) and the genes that mediated the HDR events are identified by amplifying the guide RNA cassette by the Polymerase Chain Reaction and sequencing. These hits are then verified by independent tests and, if they pass secondary screening, are tethered to a CRISPR associated protein 9 enzyme for testing in planta.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.

保护农作物免受疾病和气候压力的强大方法是用耐药性变异的特征代替易感作物变化的特征。然后，不同植物发展为应对压力的各种特征增强，然后可以保护食物供应。发现CRISPR（定期间隔短的短质体重复序列）及其在生物学上的应用，通过将一种DNA（编码性状编码性状的遗传材料）与另一个DNA的遗传材料替换为植物DNA中的精确位置，从而使这种性状替代方法成为可能。但是，依赖植物自身蛋白质的替代活动在植物中效率高，因此其使用不切实际。该项目开始进行搜索，以测试许多可能的方法来改变作物DNA，以使CRISPR的替代功能更加有效。在这个项目中，一名动植物研究人员联手进行了在人类细胞和植物细胞中进行的搜索类型（称为筛查）。这些筛选是为了制备数百万个自由细胞的制备，这些细胞激活许多基因以测试使替代效率更有效的基因。该项目的目的是开发一种方法，使最有生产力的农作物可容纳新基因，以使其更具压力耐受性。使用CRISPR介导的同源性维修（HDR）将特征从一种植物转移到另一种植物的能力将提供一种有力的方法来开发新变体，从而可以耐受气候变化。但是，涉及内源基因活性的HDR在植物中效率高，限制了其使用。该项目适应了在动物中进行的HDR-增强筛选，以筛选提高HDR效率的内源基因。屏幕涉及自由植物细胞（原生质体）悬浮液中的全基因组CRISPR激活（CRISPRA）成分，在这种情况下是Zea May。 CRISPRA成分被滴落在每个细胞中激活一个基因。这些细胞来自一条携带组成型表达的绿色荧光蛋白（GFP）的线。一个罕见的“命中”（一种活化可改善HDR的基因）将用青色荧光蛋白（CFP）序列代替GFP。 CFP阳性细胞通过荧光活化细胞分选（FACS）分离，并且通过通过聚合酶链反应和测序来扩增引导RNA盒子来鉴定介导HDR事件的基因。然后，通过独立测试对这些命中进行验证，如果它们通过次要筛选，则将其束缚在planta中的CRISPR相关蛋白9酶进行测试。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识分子优点和更广泛的影响来评估的珍贵的支​​持。