title: Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair.
abstract: Precise genome editing in livestock is of great value for the fundamental investigation of disease modeling. However, genetically modified pigs carrying subtle point mutations were still seldom reported despite the rapid development of programmable endonucleases. Here, we attempt to investigate single-stranded oligonucleotides (ssODN) mediated knockin by introducing two orthologous pathogenic mutations, p.E693G for Alzheimer's disease and p.G2019S for Parkinson's disease, into porcine APP and LRRK2 loci, respectively. Desirable homology-directed repair (HDR) efficiency was achieved in porcine fetal fibroblasts (PFFs) by optimizing the dosage and length of ssODN templates. Interestingly, incomplete HDR alleles harboring partial point mutations were observed in single-cell colonies, which indicate the complex mechanism of ssODN-mediated HDR. The effect of mutation-to-cut distance on incorporation rate was further analyzed by deep sequencing. We demonstrated that a mutation-to-cut distance of 11 bp resulted in a remarkable difference in HDR efficiency between two point mutations. Finally, we successfully obtained one cloned piglet harboring the orthologous p.C313Y mutation at the MSTN locus via somatic cell nuclear transfer (SCNT). Our proof-of-concept study demonstrated efficient ssODN-mediated incorporation of pathogenic point mutations in porcine somatic cells, thus facilitating further development of disease modeling and genetic breeding in pigs.
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Kankan
authors.initials: K
authors.lastname: Wang
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Tang
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Zicong
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zou
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Mengjing
authors.initials: M
authors.lastname: Li
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Hongming
authors.initials: H
authors.lastname: Yuan
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Ouyang
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Huping
authors.initials: H
authors.lastname: Jiao
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, Department of Animal Biotechnology, College of Animal Science, Jilin University, Changchun, PR China.
authors.forename: Daxin
authors.initials: D
authors.lastname: Pang