Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity [Cut&Tag]

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 investigate whether editing-induced chromatin effects involved changes in active regulatory marks, we performed histone CUT&Tag profiling after genome editing as an orthogonal epigenomic readout. In this study, histone CUT&Tag was used to evaluate how active chromatin signatures around edited loci and nearby regulatory elements changed in relation to the observed accessibility and transcriptional shifts. To investigate whether editing could alter higher-order regulatory architecture through boundary-associated factors, we performed CTCF CUT&Tag after genome editing. Here, CTCF CUT&Tag was designed to measure the extent and direction of CTCF occupancy changes around edited sites, and to test whether these changes were consistent with the chromatin and transcriptional patterns observed in matched samples. We used this assay conservatively as an architectural readout, together with other datasets, to assess whether displacement or reduction of CTCF-linked regulation was a plausible contributor to distal expression changes.