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. 2020 Feb;17(1):45-53. doi: 10.1007/s13770-019-00225-8. Epub 2019 Nov 19.

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.

Keywords: AsCpf1/RNP; CRISPR/AsCpf1; Electroporation; Embryo; Gene targeting; LIF.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Blastocyst / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems
  • Electroporation / methods*
  • Endonucleases
  • Gene Editing / methods*
  • Gene Targeting
  • Leukemia Inhibitory Factor / genetics
  • Leukemia Inhibitory Factor / metabolism*
  • Mice, Knockout
  • Microinjections
  • RNA, Guide / genetics
  • Zygote / metabolism*

Substances

  • LIF protein, human
  • Leukemia Inhibitory Factor
  • RNA, Guide
  • CRISPR-Associated Protein 9
  • Endonucleases