Systemic comparison of dCas9-based DNA methylation editing systems for specificity and stability [RNAseq_secondRun]

Manipulating epigenetic layers during transcriptional regulation poses significant challenges, yet advancements in dCas-based systems offer promising avenues for targeted manipulation. DNA methylation, a widely studied epigenetic mechanism in mammals, has benefited from recent methodological developments enabling comprehensive profiling across various tissues and conditions. While traditional methods for altering methylation levels lacked specificity and often induced pleiotropic effects, the advent of CRISPR technology has revolutionized this landscape, offering precise manipulation of methylation levels at specific loci. However, despite their advantages, these techniques have limitations, underscoring the need for thorough characterization of existing tools and the development of novel approaches. This study aimed to comprehensively compare the on-target/off-target effects of several available epimodifier systems and explore the potential of a multimerization strategy for epimodifier domains. Through our investigations, we provide insights into the methylation-mediated control of human gene expression and the utility of dCas9-based methylation editors. Overall design: Experimental design: HEK293T cells were transfected with plasmids encoding gRNA (BACH2-g8 or NTC) and dCas9-epimodifier-EGFP (3A, 3A3L, 3A3A, 3A-KRAB, mut3A, M.SssI, SunTag) or CRISPRoff (co-transfected with a gRNA-encoding plasmid). Successfully transfected cells were sorted based on GFP or BFP signal 3 days post-transfection (p.t.) using fluorescent activated cell sorting (FACS), GFP/BFP positive cells were cultured and harvested at days 3, 7, 14, 21, and 30 post transfection for methylation (EPIC, TMS and Pyrosequencing) and expression (Total RNAseq, mRNAseq and qPCR) analyses. Please check for more details under the “Samples” section and the “Treatment” column.