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Updated: Feb 28, 2026
Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation
Published on: February 2, 2016
Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity
Ming Zhu1, Junsong Yuan1, Qiuchen Meng2
1IDG/McGovern Institute for Brain Research, State Key Laboratory of Molecular Oncology, MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, Beijing Frontier Research Center for Biological Structure, Tsinghua University, Beijing 100084, China; School of Pharmaceutical Sciences, Tsinghua Medicine, Tsinghua University, Beijing 100084, China.
Abstract:
Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of 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 assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. 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 the safer and broader application of genome-editing technologies.
Insights
CRISPR-Cas9 gene editing can unexpectedly cause stem cells to differentiate by altering distant DNA regions. New strategies are needed to minimize these chromatin perturbations for safer genome editing applications.
Area of Science:
- Genetics
- Molecular Biology
- Stem Cell Biology
Background:
- CRISPR-Cas9 gene editing shows therapeutic potential but faces challenges in non-coding DNA regions.
- Understanding CRISPR-Cas9's off-target effects is crucial for its broader clinical application.
Purpose of the Study:
- To investigate the impact of CRISPR-Cas9 on stem cell differentiation and chromatin structure.
- To identify mechanisms by which CRISPR-Cas9 induces unintended cellular changes.
Main Methods:
- Utilized an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach.
- Examined CRISPR-Cas9 cleavage at distant sites in neural stem cells (in vivo) and mouse embryonic stem cells (in vitro).
Main Results:
- CRISPR-Cas9 caused premature differentiation of stem cells, even with cleavage far from regulatory elements.
- Editing induced significant chromatin accessibility changes, particularly affecting stem cells with perturbations extending over 100 kilobases.
- Local DNA perturbations disrupted chromatin architecture, leading to distal transcriptional rewiring and loss of stemness.
Conclusions:
- CRISPR-Cas9 can disrupt stem cell identity through widespread chromatin alterations.
- Refined gene-editing strategies, including distance-aware sgRNA design and pharmacological interventions, can minimize chromatin perturbations.
- This research advances safer and broader applications of genome-editing technologies.
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