The traditional approach for the generation of gene-targeted animals is homologous recombination (HR) conducted in embryonic stem (ES) cells followed by chimera technology, such as in mice and rats, or HR in somatic cells combined with nuclear transfer in those animals in which germline-competent ES cells are not available, such as pigs, sheep, goats, and cattle. However, no germline-competent rabbit ES cells are yet available. Furthermore, the efficiency of rabbit cloning is so low that no gene-targeted rabbits have been produced thus far by somatic cell nuclear transfer approaches. Over the past several years, zinc-finger nucleases (ZFNs) technology has been employed to produce gene knockout (KO) animals, such as rats1, pigs2, and rabbits3. Recently, transcription activator-like effector nucleases (TALENs), a new genome-modifying technology, has been employed for in vivo genetic engineering in vertebrates. Unlike ZFNs, TALENs have fewer off-target effects and lower toxicity. Given the success of the TALEN technology in reported species 4,5,6, we attempted to explore the feasibility of producing KO rabbits using this technology. In the current study, we chose recombination activation genes (RAGs), including RAG1 and RAG2, as the first genes of interest. RAG gene product have a crucial function as enzymes that activate or catalyze the V(D)J recombination reaction in primary lymphoid tissues. RAG gene-targeted mice without mature B and T cells are powerful tools for studies of allografts, xenografts, tumors, vaccine development, and infectious diseases7,8. Owing to their suitable size and similar physiology to humans, RAG-deficient rabbits would be valuable models in biomedicine research.

A total of 40 embryos injected with RAG1 TALENs mRNA were transferred into 3 surrogate rabbits. All foster mothers developed to term and gave birth to 18 bunnies. Four of the founders grew up healthily, while others died before sexual maturation, due to unknown reasons. Up to 24 embryos injected with RAG2 TALENs mRNA were transferred into 2 surrogate mothers. One surrogate rabbit developed to term and delivered 4 bunnies (Figure 1C). DNA sequencing analysis revealed that 17 of the RAG1 newborns (Figure 1C and Supplementary information, Figure S2) and all 4 RAG2 newborns (Figure 1C and Supplementary information, Figure S3) carried the expected mutation at the target locus (Supplementary information, Table 2 and Supplementary information, Figure S4). Among the RAG-KO rabbits, a variety of mutations were found at the target loci, including deletions from 1 bp to 108 bp and insertions from 3 bp to 5 bp (Supplementary information, Figures S2 and S3). Most of the rabbits were mosaic animals with at least two different genetic modifications at the RAG locus. Interestingly, we also found that 11 RAG1-KO and all RAG2-KO rabbits were biallelically modified, and two RAG1-KO rabbits and one RAG2-KO rabbit carried the same mutation at both RAG loci. The mutations in the majority of the newborn rabbits were frame-shift mutants.

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