运用CRISPR/Cas9技术构建PCSK9点突变家兔模型

目的：运用CRISPR/Cas9基因编辑技术构建前蛋白转化酶枯草溶菌素9（PCSK9）点突变家兔模型。方法：根据PubMed基因蛋白数据对人和兔的PCSK9蛋白功能区进行Blast分析，发现人PCSK9基因的386S（丝氨酸）氨基酸功能区与兔PCSK9基因的485S同源。根据家兔PCSK9基因的485S对应的碱基替换位置及序列分析结果设计3条单链向导RNA和1条单链寡核苷酸供体模板。将合成的单链向导RNA、Cas9 mRNA和单链寡核苷酸供体共同注射入家兔受精卵细胞质内并将胚胎移植入待孕母兔体内。对获得的F0代兔进行PCR、TA克隆、脱靶检测以鉴定PCSK9^S386A是否突变成功。利用获得的PCSK9^S386A基因点突变家兔进行繁殖，扩大群体。结果：共获得15只F0代兔，其中1只为PCSK9^S386A点突变纯合子，2只为PCSK9^S386A点突变杂合子，且该突变可以稳定遗传。结论：利用CRISPR/Cas9技术成功构建了PCSK9^S386A点突变家兔模型，为探究PCSK9功能减弱的分子机制，开发可靠、有效的诊断和治疗措施提供了良好的动物模型。

Construction of PCSK9 point mutation rabbits using CRISPR/Cas9

Objective:To establish a rabbit model of proprotein convertase subtilisin/kexin type9 (PCSK9) point mutation with CRISPR/Cas9 gene editing technique. Methods: 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 PCSK9 gene was homologous to the 485S of rabbit PCSK9 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 PCSK9 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^S386A mutation was successful. Results: Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9^S386A point mutation homozygote and two of them were PCSK9^S386A point mutation heterozygotes, and the mutation could be stably inherited. Conclusion: The rabbit model of PCSK9^S386A 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.