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Transgenic Res. 2019 Sep 3. doi: 10.1007/s11248-019-00168-9. [Epub ahead of print]

Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1.

Author information

1
Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland. charles-etienne.dumeau@biol.ethz.ch.
2
Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland.
3
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
4
Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, 6708 WE, Wageningen, The Netherlands.
5
Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland. awutz@ethz.ch.

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

PMID:
31482512
DOI:
10.1007/s11248-019-00168-9

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