Multi-Allelic Precision Editing of the Complex Sugarcane Genome By Homology Directed Repair of CRISPR/Cas9 Induced DNA Breaks

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 require 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 will present data that supported efficient HDR mediated precision editing of multiple alleles of the acetolactate synthase (ALS) gene in the highly polyploid sugarcane and conferred herbicide resistance.