Advances in Genome Editing of Sugarcane Using als Genes as a Model

We have investigated CRISPR/Cas9 mediated editing of als genes in sugarcane. To achieve this, two strategies were followed using editing vectors encoding the Cas9 enzyme, one of three specific sgRNAs targeting segments of the sugarcane als gene and three specific ssDNA templates. First, we approached editing the target site through expressing stably integrated editing vectors after biolistic co-delivery into sugarcane calli alongside the specific ssDNA template and an nptII marker for tissue culture selection. Second, we have sought to edit the target site by transiently expressing the editing components CRISPR/Cas9 and sgRNA with an ssDNA template into sugarcane calli. Transgene integration was confirmed using PCR, and target edition was assessed using PCR/RE and sequencing. Stable integration of the pEG_G1 vector was confirmed in four geneticin-selected, independently transformed calli, while the pEG_G2 vector was inserted into one transformed callus. nptII was inserted in all transformants. Sequencing PCR fragments, including the editing site from three transformed calli, reveals distinct 16-19 base deletions of the target fragment including the PAM site required for dsDNA breakage, but not the desired modification of the target codon. Transient-expression experiments resulted in 74 independent putatively transformed calli selected on bispyribac, but the expected mutations were not observed. We have demonstrated that DNA editing occurs in sugarcane after stable integration of editing vectors including Cas9 and sgRNA genes. Editing resulted in base deletions near the target site. Further experiments are required to understand the conditions leading to the editing of the targeted mutation.