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.
Highlights
Recent advances in genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) enable production of gene knock-out animals and rapidly [1,2,3]
To simplify germline genome editing, we developed a method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD), which does not require isolation of zygotes or their ex vivo handling for microinjection and subsequent transfer to recipient females [10]
We injected 1.0–1.5 μl of a solution containing enhanced green fluorescent protein messenger RNA (1 μg/μl) and trypan blue into oviduct lumens and performed in vivo electroporation using a BTX T820 instrument under previously described conditions [10]
Summary
Recent advances in genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) enable production of gene knock-out animals and rapidly [1,2,3]. CRISPR animal genome engineering methods include three broad steps: mating of super-ovulated females and isolation of zygotes, microinjection of genome editing components into the zygotes, and transfer of microinjected zygotes into the oviducts of pseudopregnant females [1, 2]. These steps require (1) a very high level of technical expertise by the technicians who perform these procedures and (2) expensive apparatus, including micromanipulators. The ovaries and oviducts are returned to their
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