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

Generally, the epigenetic state of chromatin, the activity of genes and their chromosomal positions are interrelated. 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. To address the role of the chromosomal position of a DNA sequence on epigenetic stability and gene activity, we induced two differently-sized inversions by CRISPR/Cas-based chromosome engineering two differently-sized inversions that both purposely introduced heterochromatic, pericentric sequences into an euchromatic chromosomal arm context each. The epigenetic status of the lines RW290 and RW295, which are characterized by a 5 Mb and 7.5 Mb-large inversion on chromosome III, respectively, was compared to wild type A. thaliana by whole genome bisulfite sequencing (WGBS) and chromatin immunoprecipitation (ChIP). Additionally, we studied the effect of the chromosome 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 (±100 kb from the inversion breakpoints) did not change their epigenetic profile due to induced inversion. 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 were located ±100 Kb up and downstream of CRISPR/Cas-cutting sites was less affected. Thus, gene expression levels and the epigenetic landscape remain preserved following engineered chromosomal restructuring, at least in the following generations.