Robust CRISPR/Cas9 mediated gene editing of JrWOX11 manipulated adventitious rooting and vegetative growth in a nut tree species of walnut

• Efficient CRISPR/Cas9 based genome editing protocol developed for walnut using multiple gRNAs targeting JrWOX11 gene. • Knockout of JrWOX11 gene resulted in reduced adventitious roots formation and vegetative growth. • Robust gene editing tool pave the way for genetic improvement of walnut, which is economically important nut tree species grown worldwide. 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.