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.
02 - 04 Sep 2014
World Congress of Reproductive Biology