Epigenetic state and gene expression remain stable after CRISPR/Cas-mediated chromosomal inversions

In Arabidopsis thaliana, the chromosome arms are DNA-hypomethylated and enriched in the euchromatin-specific histone mark H3K4me3. In contrast, pericentromeric regions are DNA-hypermethylated and enriched in H3K9me2. In order to investigate how chromosomal location affects epigenetic stability and gene activity, we induced two differently-sized inversions by CRISPR/Cas and introduced heterochromatic, pericentric sequences into an euchromatic chromosomal arm. The epigenetic status of the lines was investigated by whole genome bisulfite sequencing and chromatin immunoprecipitation. Additionally, we studied the effect of the chromosomal inversions on gene expression. Our analysis revealed that both inversions affected neither the global distribution of eu- and heterochromatin-specific histone marks nor the global DNA methylation landscape. However, minor epigenetic changes were found across the entire genome. Importantly, the inverted chromosome regions and their border regions did not change their epigenetic profile. The transcription analysis of both inversion lines revealed that 0.5 - 1 % of genes were differentially expressed across the entire genome. However, the expression activity of those genes encoded by the inverted segments, and located around the cutting sites of CRISPR/Cas was less affected. Thus, gene expression levels and the epigenetic landscape remain preserved following engineered chromosomal restructuring, at least in the following generations.