Department of Plant Pathology, Kansas State University, Manhattan, KS, USA.
Wheat Genetic Resources Center, Kansas State University, Manhattan, KS, USA.
Plant Biotechnol J. 2022 Dec;20(12):2332-2341. doi: 10.1111/pbi.13910. Epub 2022 Sep 7.
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.
不同品种中基因转化和编辑效率低下，阻碍了 CRISPR 技术在作物改良中的广泛应用。基于病毒的 CRISPR-Cas 系统传递方法的发展有望克服这些限制。在这里，我们通过直接接种大麦条纹花叶病毒（BSMV）转录本将指导 RNA（sgRNA）导入表达 Cas9 的小麦中。我们证明，用 BSMV-sgRNA 混合物接种小麦可以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。我们通过标记辅助导入和使用 BSMV-sgRNAs 将高表达的 Cas9 基因座转移到适应的春小麦和冬小麦品种中，并编辑两个农艺基因。我们研究中提出的策略可以应用于任何适应的品种，以创造新的顺式调控多样性或生物途径或 QTL 区域中多个基因的大规模编辑，为作物基因组的有效工程化开辟了可能性，并加速基因发现和性状改良的努力。
