KeyBase®: a targeted mutagenesis technology for the improvement of crop species

Natural genetic variation in germplasm of food crops is becoming more and more limiting and will soon become insufficient to answer the needs of the growing world population. Plant breeders are therefore continuously searching for new sources of genetic variation, and are increasingly interested in novel technologies for precise genome engineering. The last decade saw the ascent of genome editing technologies based on nucleases, such as ZFN, TALENs and CRISPR/Cas9 and their application in a broad range of organisms ranging from human to plants.

A complementary genome editing technology that does not rely on nucleases is oligonucleotide-directed mutagenesis or ODM. ODM allows for the creation of single point mutations in the genome of the organism of interest by means of oligonucleotides carrying a single mismatch with respect to the target sequence. The presence of the mismatch in the oligonucleotide drives the conversion of the corresponding nucleotide in the target DNA sequence resulting in a crop with improved characteristics.

KeyGene has developed its own proprietary ODM technology, called KeyBase®, and over the years worked towards improving various aspects of the system to turn the technology into commercial applications.

Using a fluorescent reporter gene stably integrated into the genome of the model plant Arabidopsis and the tobacco BY2 cell suspension cultures, we have elucidated the key requirements for increasing the mutation frequency to a level where commercial applications in crops become possible.

Results obtained in our model systems will be presented and the challenges lying ahead discussed.