Genome-editing technologies allow the targeted introduction of mutations at specific DNA sequences within the genome and have proven to be a powerful tool for functional genomics and crop improvement. The recently developed CRISPR-Cas9 system consists of an RNA-guided nuclease capable of causing Double Stranded Breaks (DSBs) at specific target loci. The subsequent repair of the DSBs can be achieved by the non-homologous end joining (NHEJ) or the homology directed repair (HDR) cellular mechanisms. Repair by the NHEJ is error-prone, resulting in the introduction of mutations in the targeted DNA sequence. The CRISPR-Cas9 system has been used in multiple food crops but its application in a highly polyploid and heterozygous genome like that of sugarcane has not been reported. Our efforts to optimize CRISPR-Cas9 in sugarcane have focussed on two target genes. The first is a GUS transgene that is present as two copies in the genome of an established transgenic line. This target was chosen because it avoids the issues of extreme polyploidy and heterozygosity, and, therefore, should allow rapid confirmation of CRISPR-induced mutations. The second target is the endogenous phytoene desaturase (PDS) gene, which was chosen due to the easily observable photo bleaching phenotype characteristic for mutations in this gene. Targeting of the PDS gene allows to determine the feasibility of inducing simultaneous mutations in multiple alleles, an important prerequisite for the practical applications of CRISPR in sugarcane. Progress with these two target genes is presented, along with a discussion of the usefulness of CRISPR-Cas9 genome editing for sugarcane improvement.
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