Reversible perturbations of gene regulation after genome editing in Drosophila cells

The prokaryotic phage defense CRISPR/cas-system has developed into a versatile toolbox for genome engineering and genetic studies in many organisms. While many efforts were spent on analyzing the consequences of off-target effects, only few studies addressed side-effects that occur due to the targeted manipulation of the genome. Here, we show that the CRISPR/cas9-mediated integration of an epitope tag in combination with a selection cassette can trigger an siRNA-mediated, epigenetic genome surveillance pathway in Drosophila melanogaster cells. After homology-directed insertion of the sequence coding for the epitope tag and the selection marker, a moderate level of siRNAs covering the inserted sequence and extending into the adjacent transcripts was generated in a Dcr-2 dependent manner. The response affected protein levels less than two-fold and was persisten, as it remained even after single cell cloning. Yet, removal of the selection cassette abolished the siRNA generation, demonstrating that this response is reversible. Consistently, marker-free genome engineering did not trigger the surveillance mechanism. These two observations indicate that the selection cassette we employed induces an aberrant transcriptional arrangement and ultimately sets off the siRNA production. There have been prior concerns about undesirable effects induced by selection markers, but fortunately we are able to show that at least one of the epigenetic changes reverts as the marker gene is excised. Thus, we recommend that when selection markers are used during genome editing, a strategy for their subsequent removal should always be included.