i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases
Masato Ohtsuka | Masahiro Sato | Hiromi Miura | Shuji Takabayashi | Makoto Matsuyama | Takayuki Koyano | Naomi Arifin | Shingo Nakamura | Kenta Wada | Channabasavaiah 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.
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