A large CRISPR-induced bystander mutation causes immune dysregulation.

Area of Science:

• Genetics
• Molecular Biology
• Immunology

Background:

• CRISPR-Cas9 gene editing offers precise DNA modification.
• A persistent concern is the potential for off-target mutations at unintended genomic locations.

Purpose of the Study:

• To investigate the safety and unintended consequences of CRISPR-Cas9 gene editing in mice.
• To identify potential pathogenic mutations arising from on-target DNA repair processes.

Main Methods:

• CRISPR-Cas9 gene editing was used to delete an intronic enhancer in mice.
• Analysis of a founder line exhibiting immune dysregulation.
• Whole-genome sequencing and targeted genotyping assays were employed.

Main Results:

• A founder line displayed significant immune dysregulation.
• This dysregulation was linked to a 24 kb tandem duplication adjacent to the intended on-target deletion.
• The duplication was not detected by routine targeted genotyping.

Conclusions:

• Unintended repair of on-target genomic cuts can generate pathogenic "bystander" mutations.
• These mutations can cause significant physiological effects, such as immune dysregulation.
• Current genotyping methods may fail to detect these complex on-target repair-associated mutations, highlighting a critical safety concern for gene editing applications.