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

## TL;DR

Researchers developed an optimized CRISPR-Cas9 system to efficiently edit extrachromosomal DNA (ecDNA) in cancer cells without causing harmful side effects.

## Contribution

The study introduces a safeguard CRISPR-Cas9 system with cytosine-extended sgRNAs that enables efficient and non-disruptive ecDNA engineering.

## Key findings

- The conventional CRISPR-Cas9 system caused severe cytotoxicity and ecDNA loss, while the optimized system reduced these effects.
- The safeguard sgRNA strategy enabled efficient knock-in into multiple ecDNA copies per cell.
- Computational simulations revealed how DNA cleavage patterns influence cell death and ecDNA dynamics.

## Abstract

Extrachromosomal DNA (ecDNA) amplification represents an emerging mechanism underlying oncogene amplification, tumor heterogeneity, and drug resistance in cancer. However, the biology of ecDNA remains poorly understood because tools to engineer ecDNAs and precisely monitor their dynamics are limited. In particular, genome engineering strategies have not been established for ecDNA, which exists in tens to hundreds of copies within a single cell. Here, we report a systematic validation of ecDNA editing using standard CRISPR-Cas9 system and optimized CRISPR-Cas9 system with safeguard single-guide RNAs (sgRNAs), in which the addition of cytosine extensions finely reduces excessive Cas9 activity. The conventional CRISPR-Cas9 system induced severe cytotoxicity and markedly reduced ecDNA copy number, together with frequent micronucleus formation. Knock-in efficiency was remarkably low, highlighting an intrinsic difficulty in editing ecDNA. In contrast, the safeguard sgRNA strategy not only alleviated cytotoxicity and ecDNA loss in a cytosine-length–dependent manner but also enabled efficient knock-in into multiple ecDNA per cell. Computational simulations suggested that the degree and temporal patterns of multiple DNA cleavage events shape cell death, micronucleus formation, and rapid expansion of knock-in ecDNA. Collectively, optimization of Cas9 activity using safeguard sgRNAs enables efficient and nondisruptive ecDNA engineering, providing a powerful tool to study ecDNA biology.

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