Search

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.

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.

CRISPR-Cas9 mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies considered DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence revealed the significant role of chromatin context. Nonetheless, most of the prior studies were 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 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 significant variations in mutagenesis efficiency by up to 250 folds. 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 variations in CRISPR-Cas9 mutation profiles reflected by the 1 bp insertion rates. Our findings provided strong evidence that specific chromatin features could have significant and lasting impacts on both CRISPR-Cas9 mutagenesis efficiency and DNA double strand break repair outcomes.

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 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. The analysis of drm1ab double mutant plants revealed limited morphological consequences for the loss of RdDM. Whole-genome methylation profiling provided evidence for wide-spread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified a limited number of genes with altered expression in the drm1ab mutants. The majority of genes with elevated CHH methylation directly surrounding the transcription start site or in nearby promoter regions 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 transposable elements identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons likely require active RdDM for maintenance of transcriptional repression.Significance statementMethylation profiling of Setaria viridis plants that lack functional Domains Rearranged Methyltransferase genes reveal widespread loss of DNA methylation in the CHH sequence context. Transcriptome analysis reveals a small set of genes and transposons that are silenced by RNA-directed DNA methylation.

Memory CD8+ T cells populate non-lymphoid tissues (NLT) and can protect against malignancies, but little is known about the establishment of tumor-specific tissue resident memory CD8+ T cells (TRM) by therapeutically initiated immune responses. We used a transplantable mouse model of prostate carcinoma and MHCI tetramers to track and quantify tumor-specific CD8+ T cells. Tumor challenge led to systemic expansion of endogenous, naïve tumor antigen-specific CD8+ T cells and established a small population of TRM (CD44hi, CD62Llo, CD69+, CD103+) in several NLTs. Previously, we demonstrated that debulking primary tumors using irreversible electroporation (IRE) induced expansion of tumor-specific T cells. We hypothesized that IRE would cooperate with checkpoint blockade to increase antigen-specific TRM and promote tumor clearance in vivo. Combination treatment with IRE followed by α-CTLA4 promoted a robust 50-fold expansion of proliferating (Ki67+) tumor-specific T cells in blood, a 10-fold increase of TRM in NLT, and was associated with complete remission of residual tumor. Conversely, monotherapy allowed tumor outgrowth and failed to boost T cell numbers, resulting in small populations of PD-1hi tumor-specific CD8+ T cells. Similarly, anti-PD-1 alone or following IRE was also ineffective, suggesting that combined IRE/α-CTLA4 therapy targets newly primed T cells. IRE also enhanced antigen presentation by XCR1+ dendritic cells, promoting T cell priming in regional lymph nodes. These results indicate that combining α-CTLA4 checkpoint immunotherapy with focal tumor ablation, such as IRE, capable of releasing high quantities and qualities of endogenous tumor antigens may serve as a potent in situ tumor vaccination strategy.