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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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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).

Author information

Correspondence to Haengseok Song or Hye-Ryun Kim.

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All authors declare that they have no conflict of interest.

Ethical statement

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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Keywords

  • CRISPR/AsCpf1
  • Electroporation
  • AsCpf1/RNP
  • Gene targeting
  • Embryo
  • LIF