Genome-wide loss of CHH methylation with limited transcriptome changes in Setaria viridis DOMAINS REARRANGED METHYLTRANSFERASE (DRM) mutants.

The DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element (TE) content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. Double mutant (drm1ab) plants exhibit some morphological abnormalities but are fully viable. Whole-genome methylation profiling provided evidence for the widespread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for the locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified genes with altered expression in the drm1ab mutants. However, the majority of genes with high levels of CHH methylation directly surrounding the transcription start site or in nearby promoter regions in wild-type plants do not have altered expression in the drm1ab mutant, even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of TEs identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons are likely to require active RdDM for the maintenance of transcriptional repression.

Epigenetic features drastically impact CRISPR-Cas9 efficacy in plants.

CRISPR-Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR-Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR-Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR-Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.