Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The type I CRISPR system is a powerful tool for genomic modification.
• Its application in zygote editing for creating model animals was previously unestablished.

Purpose of the Study:

• To demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system.
• To establish a versatile method for genetic engineering in model animals.

Main Methods:

• Utilized the type I-E CRISPR-Cas3 system for zygote editing in mice and rats.
• Employed a long-read sequencing-based multiplex genotyping approach for deletion detection.
• Applied zygote electroporation methods for gene editing.

Main Results:

• Achieved efficient generation of deletions (several thousand base pairs) at targeted loci in mice (40%-70% efficiency).
• Demonstrated successful genome editing in both mice and rats, including zygote electroporation.
• Confirmed no off-target mutations in the edited mice.
• Successfully performed SNP exchange and genomic replacement using a donor plasmid in mice.

Conclusions:

• The type I CRISPR-Cas3 system is effective for large-scale genome editing in zygotes.
• This method offers increased flexibility and broader applications in genetic engineering across species.
• The developed technique facilitates the generation of genetically modified model animals.