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

The epigenetic state of chromatin, gene activity and chromosomal positions are interrelated in plants. In Arabidopsis thaliana, chromosome arms are DNA-hypomethylated and enriched with the euchromatin-specific histone mark H3K4me3, while pericentromeric regions are DNA-hypermethylated and enriched with the heterochromatin-specific mark H3K9me2. We aimed to investigate how the chromosomal location affects epigenetic stability and gene expression by chromosome engineering. Two chromosomal inversions of different sizes were induced using CRISPR/Cas9 to move heterochromatic, pericentric sequences into euchromatic regions. The epigenetic status of these lines was investigated using whole-genome bisulfite sequencing and chromatin immunoprecipitation. Gene expression changes following the induction of the chromosomal inversions were studied via transcriptome analysis. Both inversions had a minimal impact on the global distribution of histone marks and DNA methylation patterns, although minor epigenetic changes were observed across the genome. Notably, the inverted chromosomal regions and their borders retained their original epigenetic profiles. Gene expression analysis showed that only 0.5-1% of genes were differentially expressed genome-wide following the induction of the inversions. CRISPR/Cas-induced chromosomal inversions minimally affect epigenetic landscape and gene expression, preserving their profiles in subsequent generations.