中国学者利用CRISPR/Cas9技术建立基因敲除克隆猪

　　

　中国科学院广州生物医药与健康研究院研究员、吉林大学教授赖良学博士的研究团队利用最新的CRISPR/Cas9技术成功地培育出两种基因敲除克隆小型猪，即酪氨酸酶基因敲除猪和PARK2和PINK1双基因敲除猪，建立了人类白化病和帕金森综合征两种猪模型，该研究成果于10月2日在线发表在Cellular and Molecular Life Sciences上。

　　课题组杨化强博士、博士研究生周小青和信吉阁，针对猪的酪氨酸酶、PARK2和PINK1基因的外显子设计了Cas9 打靶质粒，将其分别转染版纳小型猪的胎儿成纤维细胞和巴马小型猪的胎儿成纤维细胞后，获得了纯合的酪氨酸酶基因敲除以及PARK2和PINK1双基因敲除的细胞系。细胞系用于体细胞核移植后获得15头酪氨酸酶基因敲除克隆猪，20头PARK2和PINK1双基因敲除克隆猪。酪氨酸酶基因敲除后，本为黑色的版纳小型猪，变成了白猪，表现出了典型的白化病特征；而缺失PARK2和PINK1双基因的敲除克隆猪可以作为帕金森症大动物模型，用于研究其发生机制和评估相关治疗药物的有效性和安全性。

　　赖良学研究团队长期从事猪等大动物的基因修饰研究，曾率先利用锌指核酸酶、TALEN等基因打靶技术成功制作多种基因修饰猪。本次研究又将新兴的CRISPR/Cas9技术与体细胞克隆相结合，不仅使猪基因打靶效率更高、更为精确，而且在一个世代内首次实现了大动物双基因的等位敲除。该技术路线的建立将大大加速多基因修饰猪制备，对农业而言，可促进猪的生产性能的改良；对于生物医药领域，也可加速大动物疾病模型的建立和异种器官移植研究进展。

 
黑色猪和白色猪

 

 
缺失PARK2和PINK1双基因的敲除克隆猪

原文摘要：

Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer

The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR −/− pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 −/−/PINK1 −/− pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.