Shen, J.; Aregawi, K.; Anwar, S.; Miller, T.; Lemaux, P. G.

Sorghum bicolor L. (Moench) ranks as the fifth most important crop globally, serving diverse purposes such as forage, food, feed, and fuel. Its natural tolerance to various environmental stresses makes it a valuable resource for addressing climate resilience. Harnessing such genetic resources for crop improvement requires highly efficient genetic transformation and genome editing tools. However, sorghum has historically been recalcitrant to genetic transformation, and publicly available engineering tools remain limited. In this study, we report a high-efficiency engineering toolkit, including shuttle and binary vectors, optimized for genome editing via Agrobacterium-mediated transformation. Using this toolkit, we successfully introduced CRISPR/Cas9-based editing machinery to target the phytoene desaturase (PDS) gene, achieving editing efficiencies up to 95.7% with monocistronic gRNA expression cassettes. To further expand the genome editing repertoire, we employed a protospacer adjacent motif (PAM)-flexible Cas9 variant, SpRY (ZmSpRYi), and achieved comparable editing efficiencies. Using our toolkit increases the potential for advanced editing tools to be used in engineering genetic variation of agronomically important quantitative traits. It also paves the way for deploying advanced genome editors, such as prime editors and base editors, to manipulate genetic and genomic resources. These advances provide a robust approach for advancing research in sorghum and other cereals, offering new opportunities for sustainable crop improvement.