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Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells

Yohei Sugimoto1, Takeru Kachi1, Yu Watanabe1,2

  • 1Division of Molecular Oncology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

Nucleic Acids Research
|January 14, 2026
PubMed

Insights

A new safeguard CRISPR-Cas9 system effectively engineers extrachromosomal DNA (ecDNA), overcoming limitations in cancer research. This optimized tool allows precise ecDNA editing for studying tumor heterogeneity and drug resistance.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • Extrachromosomal DNA (ecDNA) amplification is crucial for oncogene amplification, tumor heterogeneity, and drug resistance in cancer.
  • Understanding ecDNA biology is limited by a lack of tools for engineering and monitoring its dynamics.
  • Genome engineering strategies for ecDNA, present in high copy numbers per cell, are not established.

Purpose of the Study:

  • To systematically validate ecDNA editing using CRISPR-Cas9 systems.
  • To develop and optimize a CRISPR-Cas9 system for efficient and non-disruptive ecDNA engineering.
  • To provide a powerful tool for studying ecDNA biology.

Main Methods:

  • Systematic validation of standard CRISPR-Cas9 and optimized CRISPR-Cas9 with safeguard single-guide RNAs (sgRNAs).
  • Optimization involved modifying sgRNAs with cytosine extensions to reduce Cas9 activity.
  • Computational simulations to understand the impact of DNA cleavage events on cellular outcomes.

Main Results:

  • Conventional CRISPR-Cas9 caused cytotoxicity, reduced ecDNA copy number, and micronucleus formation, with low knock-in efficiency.
  • The safeguard sgRNA strategy reduced cytotoxicity and ecDNA loss in a dose-dependent manner.
  • Efficient knock-in into multiple ecDNA copies per cell was achieved using the optimized system.

Conclusions:

  • Optimization of Cas9 activity using safeguard sgRNAs enables efficient and non-disruptive ecDNA engineering.
  • This optimized system overcomes previous limitations in editing high-copy ecDNA.
  • Provides a powerful new tool for advancing the study of ecDNA biology in cancer.

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