The low efficiency of genetic transformation and gene editing across diverse cultivars hinder the broad application of CRISPR technology for crop improvement. The development of virus-based methods of CRISPR-Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we perform direct inoculation of wheat leaves with the barley stripe mosaic virus (BSMV) transcripts to deliver guide RNAs (sgRNA) into the Cas9-expressing wheat. We demonstrate that wheat inoculation with the pool of BSMV-sgRNAs could be used to generate heritable precise deletions in the promoter region of a transcription factor and to perform multiplexed editing of agronomic genes. We transfer the high-expressing locus of Cas9 into adapted spring and winter cultivars by marker-assisted introgression and use of the BSMV-sgRNAs to edit two agronomic genes. A strategy presented in our study could be applied to any adapted cultivar for creating new cis-regulatory diversity or large-scale editing of multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes, and accelerating gene discovery and trait improvement efforts.

中文翻译：

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使用基于病毒的引导 RNA 传递系统对小麦进行多重启动子和基因编辑

不同品种间遗传转化和基因编辑的低效率阻碍了CRISPR技术在作物改良中的广泛应用。开发基于病毒的 CRISPR-Cas 系统递送至植物细胞的方法有望克服这些限制。在这里，我们用大麦条纹花叶病毒 (BSMV) 转录本直接接种小麦叶子，将引导 RNA (sgRNA) 传递到表达 Cas9 的小麦中。我们证明，用 BSMV-sgRNA 库接种小麦可用于在转录因子的启动子区域产生可遗传的精确缺失，并进行农艺基因的多重编辑。我们通过标记辅助基因渗入并使用 BSMV-sgRNA 编辑两个农艺基因，将 Cas9 的高表达位点转移到适应的春季和冬季品种中。我们研究中提出的策略可以应用于任何适应的品种，以创造新的生物途径或QTL区域中多个基因的顺式调控多样性或大规模编辑，为作物基因组的有效工程提供了可能性，并加速了基因发现和性状改良工作。