i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases

Genome Biol. 2018 Feb 26;19(1):25. doi: 10.1186/s13059-018-1400-x.

Abstract

We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.

Keywords: CRISPR; Easi-CRISPR; GONAD; In vivo electroporation; Knock-in; Long ssDNA; Transgenic mouse.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins*
  • CRISPR-Associated Proteins
  • CRISPR-Cas Systems*
  • Electroporation
  • Endonucleases*
  • Female
  • Forkhead Transcription Factors / genetics
  • Gene Editing / methods*
  • Gene Knock-In Techniques
  • Mice, Inbred Strains
  • Mice, Knockout
  • Mice, Transgenic
  • Microinjections
  • Ovary / anatomy & histology
  • Pregnancy
  • Sequence Deletion

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Forkhead Transcription Factors
  • Foxe3 protein, mouse
  • Cpf1 nuclease, Acidaminococcus
  • Endonucleases