Epigenetic features drastically impact CRISPR–Cas9 efficacy in plants

Abstract

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.

And … cut! Identifying chromatin features affecting CRISPR–Cas9 activity in plants

“…In the current issue of Plant Physiology , Weiss et al (2022) took an unbiased, genome-wide approach to identify sequence-independent factors that affect CRISPR–Cas9 efficacy and outcome in whole Arabidopsis ( Arabidopsis thaliana ) plants. To eliminate the effect that the gRNA sequence would have on Cas9 efficacy, the authors aimed to target identical short sequences within their specific, natural genomic context.…”

“… Strategy for identifying chromatin features associated with CRISPR–Cas9 efficacy in plants. In the work presented by Weiss et al (2022) , a genome-wide search of the Arabidopsis genome identified over 7 million putative CRISPR target sites (20 nt followed by NGG, the PAM recognition site). Subsequently, Weiss et al filtered out sites with low number of repeats in the genome, with inappropriate GC content, and with target sequences in nonnuclear DNA.…”

“…More unexpectedly, besides controlling the chromatin structure and, thereby, the CRISPR–Cas9 efficacy, histone modifications also affected the outcome of DNA repair once Cas9 generated the DNA cut. By correlating the histone landscape around the gRNA sites with the occurrence of a substitution, deletion, or insertion upon repair, Weiss et al observed that three HISTONE3-related features (H3K4me1, H3.1, and H3.3) are negatively correlated with insertional mutations at the edited site ( Weiss et al, 2022 ).…”

“…Overall, the work of Weiss et al (2022) emphasizes that nonsequence-related elements have a clear impact on the efficacy and outcome of CRISPR–Cas9-mediated mutagenesis in Arabidopsis. Findings in this study will help improve editing efficacy in plants.…”

“…Findings in this study will help improve editing efficacy in plants. For example, lowly edited sites were associated with high DNA methylation and heterochromatin, and Weiss et al (2022) demonstrated that reducing DNA methylation by multiple means can increase the Cas9-mediated mutagenesis frequency at lowly edited sites by at least two-fold. Whether these findings are applicable to other plants with higher economical value remains to be explored.…”