Construction of point mutation rabbits using CRISPR/Cas9.

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.