Inheritable CRISPR based epigenetic modification in a fungus.

The CRISPRoff system was recently introduced as a programmable epigenetic memory writer that can be used to silence genes in human cells. The system makes use of a dead Cas9 protein (dCas9) that is fused with the ZNF10 KRAB, Dnmt3A, and Dnmt3L protein domains. The DNA methylation resulting from the CRISPRoff system can be removed by the CRISPRon system that consists of dCas9 fused to the catalytic domain of Tet1. Here, the CRISPRoff and CRISPRon systems were applied for the first time in a fungus. The CRISPRoff system resulted in an inactivation up to 100 % of the target genes flbA and GFP in Aspergillus niger. Phenotypes correlated with the degree of gene silencing in the transformants and were stable when going through a conidiation cycle, even when the CRISPRoff plasmid was removed from the flbA silenced strain. Introducing the CRISPRon system in a strain in which the CRISPRoff plasmid was removed fully reactivated flbA showing a phenotype similar to that of the wildtype. Together, the CRISPRoff and CRISPRon systems can be used to study gene function in A. niger.