Use of CRISPR/Cas9 for Targeted Mutagenesis in Sorghum.

Sorghum (Sorghum bicolor) fulfills the demand for bioenergy resources and also provides substantial diet calories to the world's population. Therefore, many biological studies use sorghum as a research model for improvement of the domesticated food and bioenergy crops. Furthermore, leveraging genome editing systems in a plethora of grass plant species has been extensively studied with no exception in sorghum. However, a protocol that details the genome editing strategies using CRISPR/Cas9 and that combines an efficient tissue culture and transformation platform in sorghum based on Agrobacterium-mediated DNA transfer has yet to be reported. This protocol outlines the steps and workflow from design of sorghum CRISPR target sites using BTx623 as a reference genome, construction of sorghum CRISPR/Cas9 plasmids, tissue culture, to Agrobacterium-mediated transformation followed by genotyping of CRISPR/Cas9 induced mutants. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 expression vector to analysis of CRISPR-edited plants Basic Protocol 2: Stable transformation of sorghum Support Protocol: Management of sorghum plants in a greenhouse.