CRISPR/Cas9, a powerful genome-editing tool, has been widely adopted in model organisms but its application in walnut (Juglans regia L.), an economically important nut tree species grown worldwide, has been limited to date. To develop capacity for CRISPR/Cas9 technology in walnut, multiplexing CRISPR/Cas9 vectors expressing three single-guide RNAs (sgRNAs) targeting the walnut WUSCHEL‐related homeobox 11 (WOX11) gene were tested in a model somatic embryo (SE) using Agrobacterium-mediated genetic transformation. Most resulting mutations were small indels, but large fragment deletions of 26 nt were also generated. Knockout of the JrWOX11 gene resulted in reduced adventitious root (AR) formation and vegetative growth. The results demonstrate successful site-directed mutagenesis in walnut with CRISPR/Cas9 and the usefulness of a model SE culture to validate genome editing systems. This study sheds light on the promising application of genome editing tools for boosting basic and translational research in this vital nut tree species.

CRISPR/Cas9 是一种强大的基因组编辑工具，已在模式生物中广泛采用，但迄今为止，其在核桃（Juglans regia L.）（一种世界范围内种植的经济上重要的坚果树种）中的应用受到限制。为了在核桃中开发 CRISPR/Cas9 技术的能力，使用农杆菌在模型体细胞胚胎 (SE) 中测试了表达三种靶向核桃WUSCHEL相关同源框 11 ( WOX11 ) 基因的单向导 RNA (sgRNA) 的 CRISPR/Cas9 载体-介导的遗传转化。大多数产生的突变是小的插入缺失，但也产生了 26 nt 的大片段缺失。JrWOX11的淘汰赛基因导致不定根（AR）形成和营养生长减少。结果证明了使用 CRISPR/Cas9 对核桃进行成功的定点诱变以及模型 SE 培养对验证基因组编辑系统的有用性。这项研究揭示了基因组编辑工具在促进这一重要坚果树种的基础和转化研究方面的前景广阔的应用。