Recent advances in genome engineering technologies based on designed endonucleases (DE) allow specific and predictable alterations in plant genomes to generate value-added traits in crops of choice. The EXZACT Precision technology, based on zinc finger nucleases (ZFN), has been successfully used in the past for introduction of precise mutations and transgenes to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here, we describe for the first time, an alternative ZFN delivery method where plant cells are transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allows generation of edited, but not transgenic cells or tissues. For this study, we designed ZFNs targeting the wheat IPK1 locus, purified active ZFN protein from bacterial cultures, complexed with cell-penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos. NGS analysis of ZFN-treated material showed targeted edits at the IPK1 locus in independent experiments. This is the first description of plant microspore genome editing by a ZFN when delivered as a protein complexed with CPP.

中文翻译：

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小麦小孢子和单倍体胚胎中的基因组编辑，由ZFN蛋白和细胞穿透肽复合物的传递介导。

基于设计的核酸内切酶（DE）的基因组工程技术的最新进展允许对植物基因组进行特定且可预测的改变，从而在所选作物中产生增值特性。过去，基于锌指核酸酶（ZFN）的EXZACT Precision技术已成功用于引入精确的突变和转基因，从而在几种作物物种中产生新颖且理想的表型。当前用于将ZFN传递到植物细胞中的方法是基于传统的遗传转化方法，该方法可导致核酸酶在基因组中的稳定整合。在这里，我们首次描述了另一种ZFN传递方法，该方法用ZFN蛋白转染植物细胞，消除了对稳定的核酸酶基因组整合的需求，并允许生成经过编辑的转基因细胞或组织。对于本研究，我们设计了针对小麦IPK1基因座的ZFN，从细菌培养物中纯化的活性ZFN蛋白，与细胞穿透肽（CPP）结合并直接将其转染到小麦小孢子或胚中。对ZFN处理的材料的NGS分析显示，在独立实验中，IPK1基因座具有针对性的编辑。这是当ZFN以与CPP复合的蛋白质形式交付时，对植物小孢子基因组编辑的首次描述。