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

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.