Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity [Long Read Amplicon Sequencing]

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.