mutagenesis frequency identification in foxtail millet protoplasts induced by CRISPR/Cas9

The CRISPR/Cas9 technology is the most effective gene editing system. The mutagenesis efficiency critically depends on the sgRNA used. In this project, we established a reliable methods for assessing the sgRNA activity in foxtail millet protoplasts. We designed two sgRNAs to target each of the five endogenous genes, foxtail millet bran protein (SiFMBP), DNA-binding with one finger 4 (SiDof4), betaine aldehyde dehydrogenase 2 (SiBADH2) genes, granule bound starch synthase 1 (SiGBSS1) and inositol-pentakisphosphate 2-kinase 1 (SiIPK1). The mutagenesis frequency and pattern was analyzed. We also tested three multiplex genome editing systems, including MCTU, Csy4 and tRNA systems and assessed their effective in multiplex genome editing simutaneously.