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.
Conflict of interest statement
All authors declare that they have no conflict of interest.
Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.J Biol Chem. 2016 Jul 8;291(28):14457-67. doi: 10.1074/jbc.M116.733154. Epub 2016 May 5. J Biol Chem. 2016. PMID: 27151215 Free PMC article.
Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation.Anim Sci J. 2019 Jan;90(1):55-61. doi: 10.1111/asj.13129. Epub 2018 Oct 25. Anim Sci J. 2019. PMID: 30368976
Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing.Sci Rep. 2015 Jun 11;5:11315. doi: 10.1038/srep11315. Sci Rep. 2015. PMID: 26066060 Free PMC article.
[The new generation tool for CRISPR genome editing: CRISPR/Cpf1].Sheng Wu Gong Cheng Xue Bao. 2017 Mar 25;33(3):361-371. doi: 10.13345/j.cjb.170029. Sheng Wu Gong Cheng Xue Bao. 2017. PMID: 28941336 Review. Chinese.
Nucleic acids delivery methods for genome editing in zygotes and embryos: the old, the new, and the old-new.Biol Direct. 2016 Mar 31;11(1):16. doi: 10.1186/s13062-016-0115-8. Biol Direct. 2016. PMID: 27037013 Free PMC article. Review.