High-frequency Wus2-integrated transformation toolkit enhances Cas9 editing efficiency and expands capability with SpRY in sorghum

Sorghum bicolor L. (Moench), the fifth most important cereal crop internationally, is used as food, feed, forage, and fuels. Importantly, sorghums natural tolerance to environmental stresses leads to tolerance to climate variation. To optimize sorghum germplasm for crop improvement requires highly efficient genetic transformation and genome editing; however, sorghum has historically been recalcitrant to these genetic approaches. In this study, we report a high-efficiency engineering toolkit, optimized for genome editing via Agrobacterium-mediated transformation. Using CRISPR/Cas9-based editing machinery, the genetic tools led to editing efficiencies up to 95.7% when monocistronic guide RNAs targeted the phytoene desaturase (SbPDS) gene. We also tested a PAM (protospacer adjacent motif)-broadened Cas9 variant, intronized SpRY (ZmSpRYi), achievling comparable editing efficiencies. Using this toolkit permits exploitation of advanced editing tools, like prime editors, base editors, and targeted knock-in methods. These genetic advances offer new methods for sustainable crop improvement in sorghum and potentially other cereals. Technology readinessThis study demonstrates the development of an integrated genome engineering platform that combines morphogenic gene-assisted transformation with CRISPR/Cas9- and SpRY-mediated mutagenesis for multiplex genome editing, establishing a Technology Readiness Level (TRL) of 4/5. This workflow enables the generation of allelic variants through editing of both functional and regulatory elements in the plant genome. Advancing toward TRL 5 will require additional validation in relevant sorghum genotypes, including elite varieties. Moreover, the PAM-flexible editor SpRY is still under optimization, particularly at non-canonical PAM sites, which may limit its application for the modification of certain cis-regulatory elements. Further improvements in precision genome editing, such as the integration of prime editing or targeted knock-in technologies, will also be necessary to enable desired and predictable modifications. With continued optimization and incorporation of these advanced tools, this robust genome engineering platform has the potential to accelerate plant breeding and enable basic research supporting plant-based solutions to address climate change.