I-GONAD: A robust method for in situ germline genome engineering using CRISPR nucleases

Masato Ohtsuka, Masahiro Sato, Hiromi Miura, Shuji Takabayashi, Makoto Matsuyama, Takayuki Koyano, Naomi Arifin, Shingo Nakamura, Kenta Wada, Channabasavaiah B. Gurumurthy

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

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.

Original languageEnglish (US)
Article number25
JournalGenome biology
Volume19
Issue number1
DOIs
StatePublished - Feb 26 2018

Keywords

  • CRISPR
  • Easi-CRISPR
  • GONAD
  • In vivo electroporation
  • Knock-in
  • Long ssDNA
  • Transgenic mouse

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Genetics
  • Cell Biology

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