Precision Nucleotide Substitutions in Sugarcane Following CRISPR/Cas9 and Template Mediated Genome Editing.

Genome editing tools such as CRISPR/Cas9 have been employed in several crop genomes. They enable precise targeting and introduction of double stranded DNA breaks in vivo. Subsequent cellular repair mechanisms, predominantly non-homologous end joining (NHEJ), act as critical steps to endogenous gene editing or correction. However, there is very limited control over these mechanisms, which generate an abundance of random insertions and deletions (indels). Frameshift mutations associated with these indels of unspecified size and sequence might result in loss of function phenotypes of agronomic importance. Gain of function mutations, on the other hand, generally requires precise nucleotide substitutions in the target locus. This can be accomplished with the aid of a homologous repair template and involves the cellular homology directed repair (HDR) mechanism. We are presenting an efficient HDR mediated genome editing approach conferring herbicide resistance in the highly polyploid sugarcane. We will also discuss the impact of alternative guide RNAs.