Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity [RNA-Seq]

While CRISPR/Cas9 holds therapeutic promise, broader application demands understanding complications in vast non-coding regions. We found that CRISPR/Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated ATAC/RNA approach (AR-seq) and identified editing-induced chromatin accessibility change, with its scale varying by cell types. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and ultimately loss of stemness identity. To minimize chromatin perturbations and preserve cell identity we refined gene editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for safer and broader application of genome editing technologies. Overall design: To define the transcriptional consequences of genome editing at regulatory-adjacent non-coding loci, we designed an RNA-centered framework that prioritizes spatially resolved, mechanism-informative readouts rather than endpoint differential expression alone. Specifically, we used the RNA arm of AR-seq to quantify locus-proximal and distal expression perturbation after editing, and formalized these effects as a distance-aware metric (?RNA/?RNA span) to test whether transcriptional disruption propagates outward from the cut site. This design was coupled to matched chromatin measurements and perturbation controls (including p53 inhibition and repair-pathway modulation) to separate generic stress responses from editing-linked regulatory rewiring. We further integrated 3D genome context to evaluate whether genes proximal in chromatin contact space are preferentially affected relative to genes closer only in linear genomic distance. In aggregate, this study design enables a causal interpretation in which editing-induced transcriptional changes are modeled as structured, distance- and architecture-dependent regulatory perturbations, rather than as nonspecific global expression drift.