EAGER: A Genome Wide HDR Enhancement Screen in Maize

保护作物免受病害和气候胁迫的有效方法是用抗性品种的性状代替易感作物品种的性状。不同的植物为了应对压力而进化出的各种各样的特征，就可以用来保护食物供应。CRISPR（定期间隔聚集的短回文重复序列）的发现及其在生物学上的应用使得这种性状替换方法成为可能，这种方法是在植物DNA的精确位置用另一段DNA（编码性状的遗传物质）替换一段DNA。然而，依赖于植物自身蛋白质的替代活性在植物中效率非常低，使其应用不现实。为了使CRISPR的替代功能更有效，该项目开始寻找测试许多可能改变作物DNA的方法。在这个项目中，植物和动物研究人员联合起来进行一种被称为筛选的搜索，这种搜索以前在人类细胞中进行，现在在植物细胞中进行。这些筛选是在数百万个自由细胞的准备工作中完成的，这些细胞激活了许多基因，以测试一种使替代更有效的基因。该项目的目标是开发一种方法，使最高产的作物能够接受新的基因，使它们更耐压力。利用crispr介导的同源定向修复（HDR）将性状从一种植物转移到另一种植物的能力，将为开发能够耐受气候变化的新品种提供一种强有力的方法。然而，HDR涉及内源基因的活性，在植物中效率极低，限制了其使用。该项目将在动物中进行的HDR增强筛选应用于植物，筛选提高HDR效率的内源性基因。筛选涉及游离植物细胞（原生质体）悬浮液中的全基因组CRISPR激活（CRISPRa）成分，本例中来自玉米。对CRISPRa成分进行滴定，以激活每个细胞中的一个基因。这些细胞来自一个携带组成表达的绿色荧光蛋白（GFP）的细胞系。一种罕见的“命中”——一种激活能改善HDR的基因——将用青色荧光蛋白（CFP）序列取代绿色荧光蛋白。通过荧光活化细胞分选（FACS）分离cfp阳性细胞，通过聚合酶链反应（pcr）扩增引导RNA盒并测序鉴定介导HDR事件的基因。然后通过独立的测试来验证这些命中，如果它们通过了二次筛选，就会被连接到CRISPR相关的蛋白9酶上，在植物中进行测试。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。

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.