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.

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Publication Details

Journal
Cell Reports Methods
Published
2024-08-01
DOI
https://doi.org/10.1016/j.crmeth.2024.100833
Citations
5
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
1.17

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article

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

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5 citations
Cell Reports Methods
CRISPR and Genetic Engineering
1.17
article

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

Akihiro Kuno, Ryoji Ito, Yuko Yamauchi, K. Mikamo, Kazuto Yoshimi, Saeko Ishida, Riichi Takahashi, Tomoji Mashimo, Kosuke Hattori, Satoru Takahashi, Kohei Takeshita, Hiromi Taniguchi, Ryuya Iida, Motohito Goto
article en
5 citations

Abstract

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.

Cell Reports MethodsVol. 4(8)
University of Tsukuba (JP), Tokyo University of Science (JP), Central Institute for Experimental Animals (JP), SPring-8 (JP), Systems Biology Institute (JP), The University of Tokyo (JP)
Ministry of Education, Culture, Sports, Science and Technology, Japan Society for the Promotion of Science, Japan Science and Technology Agency, Institute of Medical Science, University of Tokyo, Co-creation place formation support program
Zero hunger
Openalex Percentile: Top 22%
CRISPR and Genetic Engineering
1.17
DOIOpen Access PDFOpenAlex
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