Related Experiment Video

Updated: May 16, 2026

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
08:23

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

Genotoxicity profiling reveals distinct platform-and cell type-specific effects in therapeutic gene editing for

Lei Lei1, Masako M Kaufmann2, Jessica Lao3

  • 1Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, 79106 Freiburg, Germany; PhD Program, Faculty of Biology, University of Freiburg, 79106 Freiburg, Germany.

Cell Stem Cell
|May 14, 2026
PubMed

Insights

Cytosine base editing (CBE) precisely corrected a genetic defect in mice, restoring immune function and preventing hyperinflammation. Safety profiling revealed cell-type-specific genotoxicity, crucial for clinical translation of gene editing therapies.

Area of Science:

  • Genetics
  • Immunology
  • Molecular Biology

Background:

  • Base editors offer precise gene correction without DNA double-strand breaks.
  • Genotoxicity of base editing platforms and cell types requires thorough characterization.

Purpose of the Study:

  • To apply cytosine base editing (CBE) to correct a splice-site mutation in the Unc13d gene in a mouse model of FHL3.
  • To evaluate the therapeutic efficacy and genotoxicity of CBE in various cell types.

Main Methods:

  • Cytosine base editing (CBE) was used to target the Unc13d locus in Jinx mice.
  • Editing efficiency was assessed in fibroblasts, T cells, and hematopoietic stem cells (HSCs).
  • Genotoxicity profiling compared CBE with CRISPR-Cas9, analyzing off-target edits and structural variants.

Main Results:

  • Efficient CBE (62%-89%) restored Unc13d splicing and cytotoxic T cell function.
  • Transplantation of edited HSCs protected mice from virus-triggered hyperinflammation.
  • Hyperactive CBE showed broader off-target activity and more structural variants than CRISPR-Cas9, with cell-type-specific translocation stability.

Conclusions:

  • Base editing is a viable therapeutic strategy for familial hemophagocytic lymphohistiocytosis type 3 (FHL3).
  • Context-specific safety profiling is essential for the clinical translation of genome editing technologies.
Keywords:
CAST-seqCRISPR-CasFHL type 3base editingcytosine base editorcytotoxicitygenotoxicityhematopoietic stem cell transplantationhyperinflammationoff-target effects

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