title: Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs.
abstract: Transgenic pigs play an important role in producing higher quality food in agriculture and improving human health when used as animal models for various human diseases in biomedicine. Production of transgenic pigs, however, is a lengthy and inefficient process that hinders research using pig models. Recent applications of the CRISPR/Cas9 system for generating site-specific gene knockout/knockin models, including a knockout pig model, have significantly accelerated the animal model field. However, a knockin pig model containing a site-specific transgene insertion that can be passed on to its offspring remains lacking. Here, we describe for the first time the generation of a site-specific knockin pig model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. We also report a new genomic "safe harbor" locus, named pH11, which enables stable and robust transgene expression. Our results indicate that our CRISPR/Cas9 knockin system allows highly efficient gene insertion at the pH11 locus of up to 54% using drug selection and 6% without drug selection. We successfully inserted a gene fragment larger than 9 kb at the pH11 locus using the CRISPR/Cas9 system. Our data also confirm that the gene inserted into the pH11 locus is highly expressed in cells, embryos and animals.
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.|Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, 130012, PR China.
authors.forename: Jinxue
authors.initials: J
authors.lastname: Ruan
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.|Qingdao Institute of Animal Science and Veterinary Medicine, Qingdao, 266100, PR China.
authors.forename: Hegang
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Kui
authors.initials: K
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Tianwen
authors.initials: T
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Jingliang
authors.initials: J
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Yulian
authors.initials: Y
authors.lastname: Mu
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Shulin
authors.initials: S
authors.lastname: Yang
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, 130012, PR China.
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Ouyang
authors.affiliation: Applied StemCell, Inc., Menlo Park, CA 94025, USA.
authors.forename: Ruby Yanru
authors.initials: RY
authors.lastname: Chen-Tsai
authors.affiliation: State Key Laboratory of Animal Nutrition &Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China.
authors.forename: Kui
authors.initials: K
authors.lastname: Li
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D005347:Fibroblasts; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D023061:Gene Order; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional; D013552:Swine; D019076:Transgenes