EAGER: Heritable Gene Editing in Maize using Transient Expression of the CRISPR-Cas9 Endonuclease System

农作物的生长和产量基本上是由DNA决定的。在整个农业历史中，农民和育种者一直在选择DNA序列中的变异，以提高植物的生产力。最近的技术，统称为基因编辑，可以在基因组的特定位点诱导DNA变异。基因编辑可以在作物中产生所需的性状，但目前的玉米基因编辑过程在技术上具有挑战性，而且产量有限。该项目将探索在玉米基因组的特定位点更有效地诱导DNA变异的方法。这项研究将测试短期向玉米干细胞输送基因编辑酶是否会导致DNA变异，这种变异可以从经过处理的植物遗传给下一代。如果成功，该项目将为基因组研究和玉米育种开发一种新的工具，预计将对了解基因功能和开发更高产的玉米品种产生重大影响。该计划亦会让本科生直接参与研究，以支援下一代科学家的教育和专业发展。这项研究的长期目标是开发更有效的方法来编辑玉米基因组。当CRISPR系统作为一个稳定的转基因位点引入时，使用聚类规则间隔短回文重复序列（CRISPR）系统进行基因编辑在产生可遗传突变方面非常有效。本项目将研究在茎尖分生组织细胞中瞬间表达Cas9内切酶和合成的单导RNA （gRNA）是否能产生遗传基因编辑。核心假设是，Cas9和gRNA在植物干细胞中的短暂表达会诱导足够大的突变区产生配子。如果成功，瞬时诱变将产生目标突变，而不需要组织培养或转基因生物的产生。Aim 1将利用CRISPR-Cas9系统确定瘤化农杆菌浸润幼苗芽后是否能在茎尖分生组织中瞬间表达外源DNA，并达到足够水平，从而引起靶向突变。目的2将确定细胞穿透肽是否能够以足够的水平将DNA、蛋白质和gRNA转移到射击干细胞中，从而引起靶向突变。

The growth and productivity of crop plants are fundamentally determined by DNA. Throughout the history of agriculture, farmers and breeders have selected for variants in DNA sequence that improve plant productivity. Recent technologies, collectively known as gene editing, can induce DNA variants at specific sites in the genome. Gene editing can produce desired traits in crops, but the current process for gene editing in corn is technically challenging with limited throughput. This project will explore methods to more efficiently induce DNA variants at specific sites in the corn genome. The research will test whether short-term delivery of gene editing enzymes to corn stem cells will cause DNA variants that can be inherited from the treated plant to the next generation. If successful, the project will develop a new tool for genome research and corn breeding that is expected to have a high impact on understanding gene function and developing more productive corn varieties. The project will also directly involve undergraduates in the research to support the education and professional development of the next generation of scientists. The long-term goal of this research is to develop more efficient methods to edit the maize genome. Gene editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is very effective at generating heritable mutations when the CRISPR system is introduced as a stable transgenic locus. This project will investigate whether transient expression of the Cas9 endonuclease and a synthetic, single guide RNA (gRNA) in shoot apical meristem cells can produce inheritable gene edits. The central hypothesis is that transient expression of Cas9 and gRNA in plant stem cells will induce mutant sectors large enough to contribute to the gametes. If successful, transient mutagenesis will produce targeted mutations without tissue culture or the generation of a transgenic organism. Aim 1 will determine if Agrobacterium tumefaciens infiltration of seedling shoots can transiently express foreign DNA in the shoot apical meristem at sufficient levels to cause targeted mutations with the CRISPR-Cas9 system. Aim 2 will determine if cell penetrating peptides can transfer DNA, protein, and gRNA into shoot stem cells at sufficient levels to cause targeted mutations.