i-GONAD

Directly edit the genes of zygotes with 'improved-Genome editing via Oviductal Nucleic Acids Delivery'

  • Edits genome in the oviduct of mice, preventing ex vivo handling of zygotes
  • Uses only 40% or fewer animals than required by traditional methods
  • i-GONAD treated females fully retain reproductive function
  • Process does not require highly sophisticated equipment or specialized skill sets
  • Powerful technique for generating any transgenic animal model

 
Licensing Manager: Catherine Murari-Kanti, Ph.D. catherine.murari@unmc.edu or 402-559-3265
 

Description

Directly edit the genes of zygotes with ‘improved-Genome editing via Oviductal Nucleic Acids Delivery’

Diagrammatic illustration showing the anatomical structures of ovary and oviduct and the surgical equipment used for GONAD procedure. A small amount of solution is injected by direct insertion of a glass micropipette through oviduct wall located at the region between the ampulla and infundibulum. Immediately after injection, in vivo electroporation is performed on the entire oviduct. The oviduct dissected on day 0.7 exhibits shrinkage of the ampulla (arrow), and zygotes isolated from the day 0.7 ampulla have fewer cumulus cells, which will less likely hamper the uptake of exogenous nucleic acids/proteins upon electroporation

Diagrammatic illustration showing the anatomical structures of ovary and oviduct and
the surgical equipment used for GONAD procedure. A small amount of solution is injected by direct insertion of a glass micropipette through oviduct wall located at the region between the ampulla and infundibulum. Immediately after injection, in vivo electroporation is performed on the entire oviduct.
The oviduct dissected on day 0.7 exhibits shrinkage of the ampulla (arrow), and zygotes isolated from the day 0.7 ampulla have fewer cumulus cells, which will less likely hamper the uptake of exogenous nucleic acids/proteins upon electroporation

The i-GONAD method delivers genome editing molecules, directly to E0.7 embryos in the oviducts of mice, via in situ electroporation. This technique generates mouse models that can contain single base changes, kilobase sized deletions and knock-ins. i-GONAD treated females retain reproductive function and are useful for generating germlines in future generations.
 
The University of Nebraska Medical Center’s Channabasavaiah Gurumurthy, Ph.D., collaborated with Japanese researchers Masato Ohtsuka, Ph.D., and Hiromi Miura, Ph.D., of Tokai University’s School of Medicine, invented i-GONAD . Traditionally, generating transgenic mouse-models involves three critical steps: isolation of zygotes from sacrificed females, zygote micromanipulation ex vivo and transfer of these modified zygotes into another set of female mice. This process has remained unchanged for over four decades and is laborious – requiring a high level of expertise, expensive and time-consuming.
 
The i-GONAD technique relives these steps by delivering genome editing nucleic acids and CRISPR components into embryos in situ. The process involves the exposure of the ovaries and oviduct of pregnant mice bearing E0.7 embryos. The genome editing reagents are injected into the oviductal lumen and the entire oviduct is subjected to electroporation using tweezer-type electrodes. These in situ, genome edited embryos are allowed to develop to term and genotyped for the targeted mutation.
 
This technique, when used in combination with Easi-CRISPR, another invention from Dr. Gurumurthy and Dr. Ohtsuka, inserts long single stranded DNA donor format with insertion efficiency as high as 100%. This combination changes the landscape of transgenic animal-model generation and provides a tool that is easy-to-perform and has high efficiency.
 
To discuss licensing opportunities contact Catherine Murari-Kanti, Ph.D., at catherine.murari@unmc.edu or 402-559-3265.
 

Additional Info

Technical Details

i-GONAD deliver genome editing tools, such as Easi-CRISPR, to the zygotes in the oviduct of pregnant mice to generate genetically modified animal models with minimal usage of animals and high efficiencies of genetic modification.
 

Creation of genome edited mouse linesYes
Stage of embryonic day for i-GONADDay 0.7. This stage (late one-cell stage) is critical creating genome edited mice. The Day 0.7 embryos are located in ampulla of the oviduct where they are easier to locate compared to older embryos.
Electroporation conditionsTechnique includes “constant current” mode from Bex electroporators
Retained reproductive function in the GONAD treated animalsDemonstrated using i-GONAD with tremendous implications on gene therapy.
NucleasesCas9 and Cpf1
Cas9 and Cpf1 were supplied as protein.
gRNA was supplied as crRNA/tracrRNA.
EfficiencyHigh (~100% for indel mutation)
Making large deletion, single nucleic acid change, insertion of large fragment by Easi-CRISPR is possible.
 

Publications

News Items

  • http://www.omaha.com/livewellnebraska/molecular-scissors-used-in-gene-editing-receive-an-upgrade-courtesy/article_2ebb9930-07ec-5ad6-8b93-fc87e0059fe9.html
  • http://blog.addgene.org/easi-crispr-generating-knock-in-and-conditional-mouse-models
  • https://www.technologynetworks.com/genomics/articles/easi-crispr-technology-could-revolutionize-animal-testing-299680
  • https://www.genengnews.com/gen-articles/genome-editing-explores-new-depths/5924
  • https://www.aucd.org//template/news.cfm?news_id=13312
  • https://www.technology.org/2018/01/09/easi-crispr-thanks-to-a-new-method-gene-editing-becomes-much-easier/
  • http://www.genengnews.com/gen-articles/toward-a-faster-easier-more-precise-crispr/6224
  • https://www.sciencedaily.com/releases/2017/05/170518134954.htm
  • https://www.genomeweb.com/gene-silencinggene-editing/research-team-creates-crispr-strategy-more-efficient-engineering-animal
  • https://www.technologynetworks.com/genomics/news/an-easi-crispr-recipe-for-creating-the-perfect-mouse-model-295847
  • http://blogs.biomedcentral.com/on-biology/2017/12/15/best-of-2017-genome-biology/
  • https://www.genengnews.com/gen-articles/toward-a-faster-easier-more-precise-crispr/6224
  • https://www.unmc.edu/news.cfm?match=21425
  • http://uofuhealth.utah.edu/research-roundup/2017/05/easicrispr.php