Molecular therapy. Nucleic acidsJun 17, 2026

Detailed evaluation of intended and unintended DNA changes from CRISPR gene editing delivered by fat-based particles

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 18, 2026

Abstract

On-target sequencing in mouse muscle identified insertions primarily from host genomic sequences, with no detectable AAV or transgene integration.

An 'indel cluster' method using high-depth whole-genome sequencing was developed to differentiate between genome editing-induced insertions and background variants.
Integration of prediction tools, cleavage sites, and whole-genome sequencing identified 11 high-confidence off-target candidates, mainly linked to one guide RNA.
The prediction tool Cas-OFFinder was found to be the most sensitive, though its precision was low and correlation with other data was modest.
Reproducibility of off-target detection across different conditions was limited, with many candidate sites overlapping repetitive or low-mappability regions.
The study highlights both the effectiveness and current challenges in assessing off-target risks associated with CRISPR-Cas9 genome editing.

Simplified

Ensuring the safety of CRISPR-Cas9 genome editing requires comprehensive assessment of off-target mutagenesis, particularly for therapeutic applications under regulatory review. DNA-free lipid nanoparticle (LNP) delivery is expected to minimize insertional risks compared to adeno-associated virus (AAV) vectors, but experimental validation has been limited. Here, on-target amplicon sequencing in mouse muscle demonstrated insertions largely derived from host genomic sequences, with no detectable AAV or transgene integration. Benchmarking 13prediction tools identified Cas-OFFinder as the most sensitive, though precision remained low, and correlation withCIRCLE-seq data was modest. To strengthen off-target detection, we employed karyotypically normal human iPSCs and developed an "indel cluster" method using high-depth whole-genome sequencing, enabling discrimination between clustered genome editing-induced indels and background variants. Integration ofpredictions, CIRCLE-seq cleavage sites, andWGS clusters yielded 11 high-confidence off-target candidates, predominantly associated with one gRNA. While sensitivity was maximized, reproducibility across conditions remained limited, and many candidate sites overlapped repetitive or low-mappability regions. Our multilayered framework demonstrates both the utility and current limitations of off-target risk assessment, providing a practical foundation for the safety evaluation of genome editing therapies. in silico in vitro in silico in cellulo

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Funding

Competing interests

The authors declare the following competing interests: N.I. is an employee of Takeda Pharmaceutical Company. Y.M. was an employee of Takeda Pharma during the project and is now an employee of Axcelead Inc. N.F., Y.M., and A.H. have filed patent applications regarding the formulation of LNP. The other authors declare no competing interests.

PubMed

Detailed evaluation of intended and unintended DNA changes from CRISPR gene editing delivered by fat-based particles

Abstract

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Competing interests

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