Genetic Improvement of Sugarcane by Targeted Loss- or Gain of Function Mutations using TALEN or CRISPR-Cas9

Programmable endonucleases like RNA-guided nucleases (e.g.CRISPR/Cas9) or transcription activator-like effector nucleases (TALENs) enable precise genome modifications. Targeted mutagenesis for “loss of function” is typically more efficient than gene replacement for “gain of function” since it does not require template mediated homology directed repair (HDR) and instead relies on the more efficient but error prone non-homologous end joining (NHEJ) DNA-repair pathway. However, sugarcane is a highly polyploid species (x=10-13) which may require knock-out of a very large number of alleles/copies for “loss of function” mutants. Suppression of lignin biosynthesis is a prime target for mutagenesis in biofuel feedstocks like sugarcane. Recently we described TALEN induced mutagenesis to suppress one of the lignin biosynthetic genes, caffeic acid O-methyltransferase (COMT), resulting in low lignin and brown-midrib sugarcane phenotypes. Here we will discuss sequencing results of long COMTamplicons from brown-midrib sugarcane, which allowed us to precisely determine the number of copies/alleles which were co-mutated. Data describing the cell wall composition of COMT mutants and their agronomic and conversion performance will be presented. A precision gene-editing approach for allele re-placement conferring “gain of function” will also be discussed, involving a DNA repair template facilitating homology-directed repair (HDR) and CRISPR/Cas9 as programmable endonuclease.