Andreou, I. | bioRxiv Science

bioRxiv2026

Pre-analytical delay as a dominant confounder in blood RNA-seq: Rapid ex vivo gene expression changes in EDTA blood

Although the impact of delayed processing on gene expression in EDTA blood has been well documented using targeted assays and microarray platforms, the emergence of next-generation RNA sequencing (RNA-seq) has not yet been leveraged to systematically compare these effects against stabilized whole blood collection systems. Notably, no study has performed a time course RNA-seq analysis with human bulk RNA of matched EDTA and PAXgene blood RNA samples drawn from the same subjects. EDTA is still widely used for gene expression analysis studies. Yet, the genome-wide dynamics by which EDTA blood transcriptomes deviate from a stabilized reference over time remain poorly defined. This represents an important methodological gap, given the increasing reliance on RNA-seq for biomarker discovery, clinical transcriptomics and diagnostics.

Molecular Biology↗

bioRxiv2025

Systematic comparison of dCas9-based DNA methylation epimodifiers over time indicates efficient on-target and widespread off-target effects

CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design. We systematically compared specific targeting of the BACH2 gene promoter and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and, interestingly, also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNA caused pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerged as the most efficient tool for stable targeting of the BACH2 promoter, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations. Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.

molecular biology↗