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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P068 | DOI: 10.1530/repabs.1.P068

Single-step generation of rabbits carrying a targeted allele using CRISPR/Cas9

Arata Honda1, Michiko Hirose2, Tadashi Sankai3, Lubna Yasmin3, Kazuaki Yuzawa3, Kimiko Honsho1, Haruna Izu1, Masahito Ikawa4 & Atsuo Ogura2

1University of Miyazaki, Miyazaki, Japan, 2RIKEN BRC, Tsukuba, Japan, 3National Institute of Biomedical Innovation, Tsukuba, Japan, 4Research Institute for Microbial Diseases, Osaka University, Suita, Japan

Introduction: The type II bacterial clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (Cas) have been proven to be an effective gene targeting system. Genome editing of non-rodent mammalian species is a promising strategy for generation of animal models for human diseases. Mashiko et al. reported a high efficacy of direct injection of a plasmid DNA, encoding humanized Cas9 and sgRNA (single-guide RNA) in mice. Here we show successful gene targeting in rabbit via the same strategy.

Materials and methods: To establish a CRISPR/Cas9 system in rabbit, we selected the TYROSINASE gene as a target using Dutch-belted rabbits. Plasmid pX330 was inserted with a candidate gRNA for the evaluation of endonuclease activity by single strand annealing (SSA) assay. Circular plasmid containing a selected gRNA was microinjected into the pronucleus of rabbit fertilized embryos. The embryos were cultured for 24 h and then transferred into the oviducts of pseudo-pregnant Japanese White rabbits.

Result and discussion: Four candidate gRNAs were evaluated by SSA assay and then circular prasmid pX330 containing TYROSINASE CR2 as a gRNA sequence was microinjected into 77 embryos. Of them, 67 (87%) were cleaved and transferred into recipients. Among 9 (13%) pups obtained at term, 2 (3%) had targeted alleles. One heterologous and one homologous mutant pups were obtained, although the latter was still born. This study demonstrated that the CRISPR/Cas9 system by direct injection of plasmid DNA into zygotes can also be potentially applicable to rabbits.

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