title: Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.
abstract: 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.
authors.affiliation: South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zhou
authors.forename: Jige
authors.initials: J
authors.lastname: Xin
authors.forename: Nana
authors.initials: N
authors.lastname: Fan
authors.forename: Qingjian
authors.initials: Q
authors.lastname: Zou
authors.forename: Jiao
authors.initials: J
authors.lastname: Huang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Ouyang
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.forename: Bentian
authors.initials: B
authors.lastname: Zhao
authors.forename: Zhaoming
authors.initials: Z
authors.lastname: Liu
authors.forename: Sisi
authors.initials: S
authors.lastname: Lai
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Yi
authors.forename: Lin
authors.initials: L
authors.lastname: Guo
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Esteban
authors.forename: Yangzhi
authors.initials: Y
authors.lastname: Zeng
authors.forename: Huaqiang
authors.initials: H
authors.lastname: Yang
authors.forename: Liangxue
authors.initials: L
authors.lastname: Lai
mesh_terms: D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013552:Swine / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems