ScienTific RepoRTS | 7: 16554 | DOI:10.1038/s41598-017-16328-y
Generation of gene-edited rats by
delivery of CRISPR/Cas9 protein
and donor DNA into intact zygotes
, Vanessa Chenouard
, Laurent Tesson
, Claire Usal
, Lucas Brusselle
, Jean-Marie Heslan
, Tuan Huan Nguyen
, Jean Merot
, Anne De Cian
, Carine Giovannangeli
, Jean-Paul Concordet
The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection
into zygotes which is inecient, time consuming and demands high technical skills. We report the
optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in
combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between
15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise
knock-in mutations in 25–100% of analyzed animals, comparable to microinjection. Electroporation of
long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation
of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous
electroporation of a large number of intact rat zygotes is a rapid, simple, and ecient method for the
generation of a variety of genome-edited rats.
Engineered nucleases, such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases
(TALEN) and more recently clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)
system, are powerful tools for targeting any sequence directly in mammalian zygotes, and obtaining genetically
modied animals, especially species in which there are no robust embryonic stem cells, such as rats but also farm
animals, zebra sh, xenopus, etc
. ese molecular scissors introduce a double-strand break (DSB) at the target
locus, which could be repaired by non-homologous end joining (NHEJ), introducing insertions or deletions
(indels) that if are not multiple of 3 nt result in frame shi of the coding sequence with generation of premature
stop codons, degradation of the whole mRNA and as a consequence generation of knockout animals. e other
potential repair mechanism in presence of single-stranded DNA (ssODNs) or double-strand DNA (dsDNA)
carrying sequences homologous to the target site is the homology-directed repair (HDR) pathway. Both targeted
genome editing pathways, NHEJ and HDR, have been performed until now by microinjection of these nucleases
and donor DNA directly into every processed zygote. Even if this method is ecient, it requires specic skills,
expensive equipment and is time-consuming.
Electroporation is a simple technique that is widely used for introducing macromolecules e.g proteins, DNA,
mRNA, etc… into cultured cells, living tissues and more rarely and recently into preimplantation embryos
e interest of electroporation is that time is shorter vs. microinjection (for 50 zygotes a few seconds vs. 1 hour,
respectively), requires an equipment less expensive than for microinjection and no special technical skills are
needed. Nevertheless, the eciency of electroporation depends on the electroporation parameters (e.g. electric
Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Université de Nantes, Nantes, France.
Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France.
Platform Transgenic Rats
and ImmunoPhenomics, INSERM UMR 1064-CRTI, F44093, Nantes, France.
Platform GenoCellEdit, INSERM UMR
1064-CRTI, F44093, Nantes, France.
INSERM U1096, F76031, Rouen, France.
Institut du thorax, INSERM UMR
1087, CNRS UMR 6291, F44007, Nantes, France.
INSERM U565, CNRS UMR7196, Museum National d’Histoire
Naturelle, F75005, Paris, France. Séverine Remy and Vanessa Chenouard contributed equally to this work.
Correspondence and requests for materials should be addressed to S.R. (email: firstname.lastname@example.org) or
I.A. (email: email@example.com)
Received: 2 August 2017
Accepted: 6 November 2017
Published: xx xx xxxx