Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity [ATAC-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 characterize how genome editing reshapes chromatin state at and beyond the cleavage locus, we implemented an ATAC-centered design that emphasizes spatial extent and mechanistic interpretability rather than peak-level changes alone. Using the ATAC arm of AR-seq, we quantified editing-associated accessibility shifts as a distance-resolved signal (?ATAC/?ATAC span), enabling direct evaluation of whether chromatin perturbation decays with increasing genomic distance from the edit site. The framework was structured to distinguish local DNA damage effects from broad regulatory remodeling by integrating matched RNA outputs, repair-pathway perturbations, and orthogonal chromatin assays. In particular, the design tested whether pathway bias toward resection-prone non-cNHEJ states is linked to broader accessibility propagation, and whether these accessibility changes align with disruption of higher-order regulatory architecture.