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

Genome Biology
Masato OhtsukaChannabasavaiah B Gurumurthy

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.

References

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Citations

Apr 3, 2018·BMC Biotechnology·Tomoe KobayashiMakoto Matsuyama
Dec 7, 2018·Experimental Dermatology·Matthew R JohnsonRicardo Mallarino
Jun 15, 2019·Development, Growth & Differentiation·Masato Ohtsuka, Masahiro Sato
Jun 21, 2019·Journal of Cellular Biochemistry·Vahideh AhmadzadehHabib Zarredar
Jul 26, 2019·Nature Protocols·Channabasavaiah B GurumurthyMasato Ohtsuka
Sep 17, 2019·Toxicologic Pathology·Kyathanahalli S JanardhanRamesh C Kovi
Jul 6, 2018·Archives of Pharmacal Research·Masahiro SatoChannabasavaiah B Gurumurthy
Jan 11, 2019·Disease Models & Mechanisms·Channabasavaiah B Gurumurthy, Kevin C Kent Lloyd
Aug 5, 2020·Marine Biotechnology·Changqing ZhangZhiyuan Gong
Oct 3, 2020·Genes & Development·James F ClarkPhilippe Soriano
Jan 5, 2021·ELife·Masahito YamagataJoshua R Sanes
Oct 28, 2020·Nature Protocols·Channabasavaiah B GurumurthyMasato Ohtsuka
Dec 18, 2020·Journal of Controlled Release : Official Journal of the Controlled Release Society·Yuichi SuzukiHideyoshi Harashima
Apr 8, 2019·Current Opinion in Biotechnology·Hideki UkaiHiroki R Ueda
Mar 21, 2021·Molecular Psychiatry·Gali UmschweifPaul Greengard
Mar 26, 2021·The Journal of Experimental Medicine·Maria FerezLuis J Sigal
Apr 17, 2021·Cellular and Molecular Gastroenterology and Hepatology·Yen-Ting HoKatsuto Tamai
May 8, 2021·Frontiers in Genetics·Vanessa ChenouardIgnacio Anegon
Aug 26, 2021·Development, Growth & Differentiation·Masumi NambaMakoto Matsuyama
Oct 23, 2021·Scientific Reports·Masumi NambaMakoto Matsuyama
Nov 28, 2021·Nature Reviews. Genetics·Daniel Andergassen, John L Rinn

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Methods Mentioned

BETA
gene knock-out
targeted mutation
genotyping
PCR
in vitro transcription
dissecting
nucleic acid purification

Software Mentioned

GONAD

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