Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

Transgenic Res. 2019 Dec;28(5-6):525-535. doi: 10.1007/s11248-019-00168-9. Epub 2019 Sep 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics*
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Cas Systems / genetics*
  • Electroporation
  • Endodeoxyribonucleases / genetics*
  • Gene Deletion*
  • Gene Transfer Techniques*
  • Genome / genetics
  • Mice
  • Mouse Embryonic Stem Cells / metabolism
  • Mutation / genetics
  • RNA, Guide / genetics
  • Zona Pellucida / metabolism
  • Zygote / growth & development

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Guide
  • Cas12a protein
  • Endodeoxyribonucleases