TRTech-PGR: Rapid Transformation and Editing in Maize

转化和基因编辑是基因组分析的关键步骤，对于未来几年农业跟上养活世界的需求至关重要。在植物中恢复稳定的转基因和基因编辑有两个方面，即将新的基因引入植物细胞，然后随后恢复转化或编辑的细胞。该项目将在玉米中开发一种系统，通过重组反应将转基因放置在基因组的预定位置，而不是随机整合的标准程序。该系统被设计成能够在这些预先确定的位点上继续添加新的基因，并伴随着随后的转化事件。该系统将引入快速开花的小型玉米品种，每年可种植6-7代。该品种不仅发育快，而且比普通玉米小，因此可以在受控环境的生长室内种植。这个品种可以用来诱导和编辑母系植物的单倍体，只有一个基因组拷贝，然后可以翻倍成编辑的二倍体。目标系可以是任何品种的普通玉米，因此使用这种技术可以快速编辑任何玉米系。如果成功，一种非常高效和非常快速的转化和各种基因编辑技术将可用于植物生物界。在两种不同版本的快花迷你玉米中，将接受phiC31整合酶介导的添加物的着陆点放置在5条不同的正常染色体上。B染色体上着陆点的复制将允许基因堆叠在这条多余染色体上，这可以用于增加转基因的拷贝数，或者通过有丝分裂产生两个精子时B的不分离特性去除相同的基因。phiC31整合酶将用于将基因放置在着陆点上，并使用可选择的标记和形态发生调节剂进行回收。选择标记和形态发生调节因子将使用Cre重组酶去除，保留基因货物。多个后续的基因添加可以通过重复过程添加到相同的位点上。在着陆台上进行的基因堆叠将作为将多个基因放入基因组的概念验证。基因编辑已经成为基因组分析的一个组成部分，因此该系统将通过将堆叠线转换为单倍体诱导剂来适应。这些系可以在单倍体基因组中产生编辑，使其加倍以恢复编辑，或者作为雌性杂交以在同一近交系中作为杂合子挽救编辑。一旦为特定的基因目标产生了特定的单倍体诱导编辑系，该砧木可以永续并用于编辑任何系，而不需要组织培养，而不考虑其可转化性。所有项目成果都将方便公众查阅。序列数据可应要求通过NCBI SRA/GEO和生物资源获得，也可通过玉米遗传合作库存中心和Addgene等长期资源库获得。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。

Transformation and gene editing are critical procedures for genomic analysis and will be important for agriculture in the coming years to keep pace with the need to feed the world. There are two aspects to the recovery of stable transgenes and gene edits in plants, namely introducing the new genes into plant cells and then the subsequent recovery of the transformed or edited cells. This project will develop a system in corn for placing transgenes at predetermined sites in the genome in a recombination reaction as opposed to the standard procedure of random integration. The system is designed to be able to continue to add new genes at these pre-determined sites with subsequent transformation events. This system will be introduced into a fast-flowering mini-maize variety that can be grown up to 6-7 generations per year. This variety is not only fast developing but also smaller than normal corn and thus can be grown in growth chambers in controlled environments. This variety can be used to induce and edit haploids of maternal plants with only one copy of the genome that can then be doubled into edited diploids. The target lines can be normal corn of any variety, so the editing of any line of corn can be rapid using this technique. If successful, an extremely efficient and extremely rapid technique for both transformation and gene editing of various sorts will be available to the plant biology community. Landing pads that will accept additions mediated by phiC31 integrase will be placed on five different normal chromosomes of maize in two different versions of fast flowering mini-maize. A copy of the landing pad on the B chromosome will allow gene stacking on this supernumerary chromosome that can be used for increasing the copy number of transgenes or removal of the same via the nondisjunction property of the B at the mitosis that produces the two sperm. The phiC31 integrase will be used to place genes at landing pads and recovered using selectable markers and a morphogenic regulator. The selectable marker and morphogenic regulator will be removed using Cre recombinase, retaining the gene cargo. Multiple subsequent additions of genes can be added to the same sites through a reiterative process. Gene stacking at the landing pads will be conducted as a proof of concept to place multiple genes into the genome. Gene editing has become an integral part of genomic analysis so the system will be adapted by converting the stacking lines to haploid inducers. These lines can produce edits in haploid genomes to be doubled to recover the edits or crossed as females to rescue the edits as heterozygotes in the same inbred line. Once a specific haploid inducing editing line is produced for specific gene targets, the stock can be perpetuated and used to edit any line regardless of its transformability and without the need for tissue culture. All project outcomes will be readily accessible by the public. Sequence data will be available through the NCBI SRA/GEO and biological resources either upon request or through long-term repositories such as the Maize Genetic Cooperation Stock Center and Addgene.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.