Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 28 (5-6), 525-535

Introducing Gene Deletions by Mouse Zygote Electroporation of Cas12a/Cpf1

Affiliations

Introducing Gene Deletions by Mouse Zygote Electroporation of Cas12a/Cpf1

Charles-Etienne Dumeau et al. Transgenic Res.

Abstract

CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to introduce large genomic deletions in the Ubn1, Ubn2, and Rbm12 genes in mouse embryonic stem cells (ESC). Here, we have optimized the conditions for electroporation of the same Cas12a RNP pairs into mouse zygotes. Using our protocol, large genomic deletions can be generated efficiently by electroporation of zygotes with or without an intact zona pellucida. Electroporation of as few as ten zygotes is sufficient to obtain a gene deletion in mice suggesting potential applicability of this method for species with limited availability of zygotes.

Keywords: CRISPR-Cas; Cas12a; Cpf1; Electroporation; Gene deletion; Mouse embryo; Mutation.

Conflict of interest statement

We do not have any conflicting financial, personal, or professional interests that are related to this manuscript.

Figures

Fig. 1
Fig. 1
Experimental setup and overview of the procedure for zygote electroporation. a Scheme of the experimental strategy for engineering gene deletions by zygote electroporation. Scale bar, 200 µm. b Setup of the work area for zygote electroporation and placement of a NEPA21 electroporation system and stereomicroscope. c Picture of the electroporation chamber used for electroporation in 5 µl volume and electrical connections. d Placement of mouse zygotes between the electrodes just prior to electroporation using a handling pipette under the stereomicroscope. Scale bar, 1 mm
Fig. 2
Fig. 2
Schematic representation of the strategies for engineering deletions in the Ubn1, Ubn2, and Rbm12 genes. The structure of the gene locus is shown along with the locations of crRNAs (red full arrows), predicted cleavage sites (dashed red arrows), and PCR primers (black arrows) for aUbn1, bUbn2, and cRbm12. (Color figure online)
Fig. 3
Fig. 3
Analysis of gene deletions in ESCs established after zygote electroporation. Representative images of agarose gel electrophoresis of PCR products using primers specific for deletions in the aUbn1, bUbn2, and cRbm12 genes. Lanes with positive control sample (+), wild type control (WT), no template control (−), inversion positive control (inv+), molecular weight marker (M), and samples from ESC lines derived from electroporated embryos (numerical lane labels) are shown. No inversion positive control are available for Ubn1 and Rbm12. The positions of the 500 bp (black arrow head) and 1 kb (white arrow head) size marker fragments are indicated. Primer pairs used are indicated for each gel. The genotype is given for each cell line (below); alleles are categorized for deletion (Del), inversion (Inv), wild type (WT) and uncharactized event (?). Annotation: (*) potential complex rearrangement, (**) potential complex rearrangement or polyclonal ESC line, (***) potential unspecific fragment detected in samples
Fig. 4
Fig. 4
Efficiency of gene deletions by electroporation of Cas12a RNP complexes into zygotes. Embryo development and gene deletions were determined for zygotes that were used for electroporation either with intact zona pellucida (black lines and markers) or whose zona had been partially removed by treatment with acidic Tyrode's solution (orange line and markers). Percentages are calculated relative to the number of zygotes used for the experiment and plotted for each gene (markers) and combined for all 3 genes (lines). (Color figure online)

Similar articles

See all similar articles

References

    1. Boroviak K, Fu B, Yang F, Doe B, Bradley A. Revealing hidden complexities of genomic rearrangements generated with Cas9. Sci Rep. 2017;7:12867. doi: 10.1038/s41598-017-12740-6. - DOI - PMC - PubMed
    1. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. doi: 10.1126/science.1231143. - DOI - PMC - PubMed
    1. Dong D, Ren K, Qiu X, Zheng J, Guo M, Guan X, Liu H, Li N, Zhang B, Yang D, et al. The crystal structure of Cpf1 in complex with CRISPR RNA. Nature. 2016;532:522–526. doi: 10.1038/nature17944. - DOI - PubMed
    1. Fonfara I, Richter H, Bratovic M, Le Rhun A, Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 2016;532:517–521. doi: 10.1038/nature17945. - DOI - PubMed
    1. Hai T, Teng F, Guo R, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res. 2014;24:372–375. doi: 10.1038/cr.2014.11. - DOI - PMC - PubMed

Publication types

LinkOut - more resources

Feedback