ArticleNature protocols2019
Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD.
Article in Nature protocols, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 100 papers, 1 of them a synthesis that pooled it.
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Who cites it
100 citing papers in PubMed, 1 synthesis or guideline pooled it, 200 citations in OpenAlex.
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- A simplified protocol for vitrification of hamster embryos.The Journal of reproduction and development · 2025Article
- Effect of gamma-ray exposure on the genome-editing efficiency of improved genome-editing via oviductal nucleic acids delivery (i-GONAD).Experimental animals · 2025Article
- ThebioRxiv : the preprint server for biology · 2025Article
- A mouse model of Jansen's metaphyseal chondrodysplasia for investigating disease mechanisms and candidate therapeutics.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
40 more citing papers are in PubMed but not listed here.
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Authors and funding
12 authors at 7 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.
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