I-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats

Shuji Takabayashi,Katsuya Kabashima,Takuya Aoshima,Masato Ohtsuka,Masahiro Sato,Kazushi Aoto

doi:10.1038/s41598-018-30137-x

Shuji Takabayashi, Katsuya Kabashima + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-30137-x

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Abstract

Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus, i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.

Highlights

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems employ the dual RNA-guided DNA endonuclease, Cas[9], to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA1,2
Before testing the possibility of successful improved Genome-editing via oviductal nucleic acid delivery (GONAD) (i-GONAD) in rat embryos, we first examined whether late 1-cell eggs isolated at 0.7 dpc are free from cumulus cells, which act as a barrier to the delivery of exogenous substances into embryos[16]
No fluorescent embryos were detected when intraoviductal instillation of the solution alone was performed (Fig. 1C-d,e and Table 1). These findings indicate that i-GONAD is feasible in 0.7 dpc rats

Summary

Introduction

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems employ the dual RNA-guided DNA endonuclease, Cas[9], to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA1,2 This technology allows the precise manipulation of virtually any genomic sequence specified by a short stretch of guide RNA3. This CRISPR/Cas9-based genome editing system was simplified by development of a cloning-free CRISPR/Cas[9], which allows for convenient genome editing by eliminating the time-consuming engineering of individual guide RNA expression vectors[4] This system uses a synthetic single-guide RNA (sgRNA) that mimics the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. We attempted to use i-GONAD to disrupt the endogenous Pax[6] locus, which is involved in the formation of eyes and nasal structures during embryogenesis[19,20]

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