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Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease

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Abstract

Background

The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA.

Methods and results

We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7–20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction.

Conclusions

The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.

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Acknowledgements

The authors thank Reiko Terada, Fumika Yoshida, Mika Douki and Keiko Yamauchi (National Institute for Physiological Sciences) for their assistance with animal care and PCR sample preparations.

Funding

This work was supported by a grant from LEAP-AMED (JP18gm0010002) to M.H. and by the NINS program for Cross-Disciplinary Science study to T.G.

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Author notes

  1. Teppei Goto

    Present address: Laboratory for Comparative Connectomics, RIKEN Center for Biosystems Dynamics Research, 650-0047, Kobe, Hyogo, Japan

Authors and Affiliations

  1. Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, 444-8787, Okazaki, Aichi, Japan

    Teppei Goto, Kyoko Yogo & Masumi Hirabayashi

  2. Faculty of Textile Science and Technology, Shinshu University, 386-8567, Ueda, Nagano, Japan

    Shinichi Hochi

  3. The Graduate University of Advanced Studies, 444-8787, Okazaki, Aichi, Japan

    Masumi Hirabayashi

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Contributions

TG: Conceptualization, Investigation, Data Curation, Writing—Original Draft, Funding Acquisition. KY: Investigation. SH: Data Curation, Validation, Visualization, Writing—Review & Editing. MH: Investigation, Data Curation, Writing—Review & Editing, Funding Acquisition, Supervision, Project Administration.

Corresponding author

Correspondence to Masumi Hirabayashi.

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Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

All procedures for animal experimentation were reviewed and approved by the Animal Care and Use Committee of the National Institutes of Natural Sciences (Date: 2015.3.27/No. 15A102, Date: 2015.1.23/No. P09-070-A).

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Goto, T., Yogo, K., Hochi, S. et al. Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease. Mol Biol Rep 50, 1231–1239 (2023). https://doi.org/10.1007/s11033-022-08054-0

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