Development of artificial fungal chromosomes

Ferdinand Kirchner
Department of Bioengineering, Danish Technical University, Copenhagen, Denmark

Artificial chromosomes are potentially a valuable tool in the development of stable cell factories. The genetic information is stored in the native manner and propagates in the same way as the original chromosomes. Since chromosomes store a large amount of genetic information it is possible to reconstitute large metabolic pathways. Although there are clear fields of application for artificial chromosomes, to this date there are no vectors in filamentous fungi established that contain the three functional units of chromosomes: A centromere, telomeres and origins of replications. The main problem in the creation of artificial chromosomes in filamentous fungi is, that the structure of the centromere is poorly understood. This makes the de novo assembly of chromosomes difficult; another strategy for artificial chromosome creation is to make large scale deletion in existing chromosomes. In order to facilitate these large chromosomal deletion we use a bipartite marker integrated close to the centromere and telomere, and CRISPR-Cas9 to induce double strand breaks. This allows to delete large parts of chromosomes in a controlled manner with strong selection pressure.