Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation

Satoru Iwata,Hitomi Nakadai,Daisuke Fukushi,Takashi Iwamoto,Mami Jose,Miki Nagahara

doi:10.1038/s41598-019-50900-y

Satoru Iwata, Hitomi Nakadai + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-50900-y

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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (which are much more convenient strategies) have recently been developed. However, it remains unknown whether these electroporation methods are able to manipulate genomes at the chromosome level. In the present study, we used these techniques to introduce chromosomal inversions of several megabases (Mb) in length in mouse zygotes. Using in vitro electroporation, we successfully introduced a 7.67 Mb inversion, which is longer than any previously reported inversion produced using microinjection-based methods. Additionally, using in vivo electroporation, we also introduced a long chromosomal inversion by targeting an allele in F1 hybrid mice. To our knowledge, the present study is the first report of target-specific chromosomal inversions in mammalian zygotes using electroporation.

Highlights

Chromosomal rearrangements are the cause of many hereditary disorders and play a role in the pathogenesis of diseases such as cancer[1,2]
We designed two single-stranded oligodeoxynucleotides that joined the chromosomal breakpoints together; each ssODN had a 100 bp sequence that was homologous to each junction point so that chromosomal inversion would be induced by the homology-directed repair (HDR) process to occur between the targeted regions and the homologous ssODNs (Supplementary Fig. 2)
We attempted to generate a chromosomal inversion via a previously developed in vivo electroporation technique called improved genome editing via oviductal nucleic acid delivery (i-GONAD)[14,15] (Fig. 2a, Supplementary Fig. 1a,c,d)

Summary

Introduction

Chromosomal rearrangements are the cause of many hereditary disorders and play a role in the pathogenesis of diseases such as cancer[1,2]. We present an editing method to generate chromosomal rearrangements of substantial size using in vitro and in vivo electroporation in mouse zygotes. This method resulted in successful genomic rearrangements, generating large-scale inversions and recessive lethal deletion alleles at specific sites.

Results

Conclusion