i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats

Sci Rep. 2018 Aug 13;8(1):12059. doi: 10.1038/s41598-018-30137-x.

Abstract

Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus, i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / administration & dosage
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems / genetics*
  • Electroporation / methods*
  • Embryo, Mammalian
  • Fallopian Tubes*
  • Female
  • Gene Editing / methods*
  • Male
  • Mutation
  • PAX6 Transcription Factor / genetics
  • Pregnancy
  • RNA, Guide / administration & dosage
  • RNA, Guide / genetics
  • Rats
  • Rats, Inbred Lew
  • Rats, Sprague-Dawley
  • Rats, Transgenic

Substances

  • PAX6 Transcription Factor
  • Pax6 protein, rat
  • RNA, Guide
  • CRISPR-Associated Protein 9