Generation and Phenotype Identification of Gene Knockout Rabbit by CRISPR/Cas9 System.

Paired-homeodomain transcription factor 4 () gene encodes a transcription factor which plays an important role in the generation, differentiation, development, and survival of insulin-producing β-cells during mammalian pancreas development. is a key diabetes mellitus (DM) susceptibility gene, which is associated with many different types of DM, including T1DM, T2DM, maturity onset diabetes of the young 9 (MODY9) and ketosis prone diabetes. In this study, a novel gene knockout (KO) model was generated through co-injection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) mRNA/sgRNA into rabbit zygotes. Typical phenotypes of growth retardation, persistent hyperglycemia, decreased number of insulin-producing β cells and increased number of glucagon-producing α cells were observed in the homozygous KO rabbits. Furthermore, DM associated phenotypes including diabetic nephropathy, hepatopathy, myopathy and cardiomyopathy were also observed in the homozygous KO rabbits but not in the wild type (WT) controls and the heterozygous KO rabbits. In summary, this is the first gene KO rabbit model generated by CRISPR/Cas9 system. This novel rabbit model may provide a new platform for function study of gene in rabbit and gene therapy of human DM in clinical trails.