Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD

Channabasavaiah B Gurumurthy, Masahiro Sato, Ayaka Nakamura, Masafumi Inui, Natsuko Kawano, Md Atiqul Islam, Sanae Ogiwara, Shuji Takabayashi, Makoto Matsuyama, Shinichi Nakagawa, Hiromi Miura, Masato Ohtsuka

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)2452-2482
Number of pages31
JournalNature protocols
Volume14
Issue number8
DOIs
StatePublished - Aug 1 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
Zygote
Genes
Nucleic Acids
Genome
Guide RNA
Embryonic Structures
Microinjections
Ribonucleoproteins
Single-Stranded DNA
Electroporation
Oviducts
Surgery
Animals
Proteins
Repair
DNA Repair
Embryonic Development
Messenger RNA
Research Personnel

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. / Gurumurthy, Channabasavaiah B; Sato, Masahiro; Nakamura, Ayaka; Inui, Masafumi; Kawano, Natsuko; Islam, Md Atiqul; Ogiwara, Sanae; Takabayashi, Shuji; Matsuyama, Makoto; Nakagawa, Shinichi; Miura, Hiromi; Ohtsuka, Masato.

In: Nature protocols, Vol. 14, No. 8, 01.08.2019, p. 2452-2482.

Research output: Contribution to journalArticle

Gurumurthy, CB, Sato, M, Nakamura, A, Inui, M, Kawano, N, Islam, MA, Ogiwara, S, Takabayashi, S, Matsuyama, M, Nakagawa, S, Miura, H & Ohtsuka, M 2019, 'Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD', Nature protocols, vol. 14, no. 8, pp. 2452-2482. https://doi.org/10.1038/s41596-019-0187-x
Gurumurthy, Channabasavaiah B ; Sato, Masahiro ; Nakamura, Ayaka ; Inui, Masafumi ; Kawano, Natsuko ; Islam, Md Atiqul ; Ogiwara, Sanae ; Takabayashi, Shuji ; Matsuyama, Makoto ; Nakagawa, Shinichi ; Miura, Hiromi ; Ohtsuka, Masato. / Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. In: Nature protocols. 2019 ; Vol. 14, No. 8. pp. 2452-2482.
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