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For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that  Wuschel2 (Wus2) -enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum ( Sorghum bicolor L .). Using  Agrobacterium -mediated transformation, we have demonstrated  Wus2 -induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and “altruistic” transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using  Wus2 -enabled transformation, compared to without  Wus2 , across various targeted loci in different sorghum genotypes.

Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum