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Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

BACKROUND:

CRISPR/Cpf1 is a class II, type V RNA-guided endonuclease that is distinct from the type II CRISPR/Cas9 nuclease, widely used for genome editing. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some limitations of the CRISPR/Cas9 system. The applications of CRISPR to rodent embryos for the production of knock-out (KO) mice have been achieved mainly by microinjection, which requires heavily-equipped instruments with skillful hands. Here, we evaluated the genome editing efficiency between Cpf1/mRNA and Cpf1/ribonuclear protein (RNP) in mouse embryos, and established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system.

METHODS:

The efficiency of electroporation-based delivery of AsCpf1/mRNA and AsCpf1/RNP to target exon 3 of leukemia inhibitory factor (Lif) into mouse zygotes was evaluated. Embryos that developed to the two-cell stage after zygote electroporation were transferred into the oviducts of surrogate mothers to produce AsCpf1-mediated LIF KO mice. The genome editing efficiency of blastocysts and pups was tested using the T7E1 assay and/or DNA sequencing. Congenital abnormalities and reproductive phenotypes in LIF KO mice produced by electroporation with AsCpf1/RNP were examined.

RESULTS:

Survival and two-cell development of electroporated zygotes were comparable between the AsCpf1/mRNA and AsCpf1/RNP groups, whereas genome editing efficiency was relatively higher in the AsCpf1/RNP group (13.3% vs 18.1% at blastocyst and 33.3% vs 45.5% at offspring), respectively. Two mouse lines with a frameshift mutation in exon 3 of the Lif gene were established from the AsCpf1/RNP group. All congenital abnormalities of LIF KO mice produced by AsCpf1/RNP electroporation were observed. AsCpf1-mediated LIF KO mice showed postnatal growth retardation and implantation failure, both of which are major phenotypes of LIF KO mice generated by conventional gene targeting.

CONCLUSION:

Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.

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Acknowledgements

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A6A1A03032888) and from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant No. HI17C1133).

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Author notes

  1. Yeon Sun Kim and Gyeong Ryeong Kim are equally contributed to this work.

Authors and Affiliations

  1. Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea

    Yeon Sun Kim, Gyeong Ryeong Kim, Mira Park, Seung Chel Yang, So Hee Park, Ji Eun Won, Ju Hee Lee, Ha Eun Shin, Haengseok Song & Hye-Ryun Kim

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  1. Yeon Sun Kim
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Correspondence to Haengseok Song or Hye-Ryun Kim.

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All mice used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of CHA University (IACUC approval No. IACUC170174).

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Kim, Y.S., Kim, G.R., Park, M. et al. Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor. Tissue Eng Regen Med 17, 45–53 (2020). https://doi.org/10.1007/s13770-019-00225-8

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