caa a22 4500 606174982 CHVBK 20210128100741.0 cr unu---uuuuu 210128e20150301xx s 000 0 eng 10.1007/s00018-014-1744-7 doi (NATIONALLICENCE)springer-10.1007/s00018-014-1744-7 Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer [Elektronische Daten] [Xiaoqing Zhou, Jige Xin, Nana Fan, Qingjian Zou, Jiao Huang, Zhen Ouyang, Yu Zhao, Bentian Zhao, Zhaoming Liu, Sisi Lai, Xiaoling Yi, Lin Guo, Miguel Esteban, Yangzhi Zeng, Huaqiang Yang, Liangxue Lai] 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. Springer Basel, 2014 Albinism nationallicence Animal model nationallicence Genome engineering nationallicence Parkinson's disease nationallicence Cas : CRISPR-associated nuclease nationallicence CRISPR : Clustered regularly interspaced short palindromic repeats nationallicence KO : Knockout nationallicence OTS : Off-target site nationallicence PFF : Porcine fetal fibroblast nationallicence PINK1 : PTEN-induced putative kinase 1 nationallicence SCNT : Somatic cell nuclear transfer nationallicence sgRNA : Single-guide RNA nationallicence TYR : Tyrosinase nationallicence WT : Wild type nationallicence Zhou Xiaoqing South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Xin Jige Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, 130062, Changchun, China aut Fan Nana South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Zou Qingjian South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Huang Jiao South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Ouyang Zhen South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Zhao Yu South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Zhao Bentian South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Liu Zhaoming South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Lai Sisi South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Yi Xiaoling South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Guo Lin South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Esteban Miguel South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Zeng Yangzhi Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology College, Yunnan Agricultural University, 650201, Kunming, China aut Yang Huaqiang South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Lai Liangxue South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut Cellular and Molecular Life Sciences Springer Basel 72/6(2015-03-01), 1175-1184 1420-682X 72:6<1175 2015 72 18 https://doi.org/10.1007/s00018-014-1744-7 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00018-014-1744-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Zhou Xiaoqing South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Xin Jige Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, 130062, Changchun, China aut NATIONALLICENCE 700 1- Fan Nana South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Zou Qingjian South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Huang Jiao South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Ouyang Zhen South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Zhao Yu South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Zhao Bentian South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Liu Zhaoming South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Lai Sisi South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Yi Xiaoling South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Guo Lin South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Esteban Miguel South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Zeng Yangzhi Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology College, Yunnan Agricultural University, 650201, Kunming, China aut NATIONALLICENCE 700 1- Yang Huaqiang South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 700 1- Lai Liangxue South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 510530, Guangzhou, China aut NATIONALLICENCE 773 0- Cellular and Molecular Life Sciences Springer Basel 72/6(2015-03-01), 1175-1184 1420-682X 72:6<1175 2015 72 18