title: Establishment of porcine Xist knockout model using CRISPR/Cas9 system.
abstract: Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and two sites (Target 3 and Target 4) whose mutation efficiency were 1% and 3% at the cellular level were selected. We successfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Finally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Guo-ling
authors.initials: GL
authors.lastname: Li
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Cui-li
authors.initials: CL
authors.lastname: Zhong
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Ni
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: De-wu
authors.initials: DW
authors.lastname: Liu
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Geng-yuan
authors.initials: GY
authors.lastname: Cai
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Zi-cong
authors.initials: ZC
authors.lastname: Li
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Hua-qiang
authors.initials: HQ
authors.lastname: Yang
authors.affiliation: National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Zhen-fang
authors.initials: ZF
authors.lastname: Wu
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology; D055786:Gene Knockout Techniques; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism*; D013552:Swine
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D062085:RNA, Long Noncoding; C077885:XIST non-coding RNA