title: Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.
abstract: Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.
authors.affiliation: College of animal science and technology, China Agricultural University, Beijing 100193, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Wu
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Caihong
authors.initials: C
authors.lastname: Wei
authors.affiliation: College of animal science and technology, China Agricultural University, Beijing 100193, China.
authors.forename: Zhengxing
authors.initials: Z
authors.lastname: Lian
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Ruizao
authors.initials: R
authors.lastname: Liu
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Caiye
authors.initials: C
authors.lastname: Zhu
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Huihua
authors.initials: H
authors.lastname: Wang
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Jiaxue
authors.initials: J
authors.lastname: Cao
authors.affiliation: Beijing Biocytogen Co., Ltd, Beijing 100176, China.
authors.forename: Yuelei
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Fuping
authors.initials: F
authors.lastname: Zhao
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: College of life science and technology, Jinan university, Guangzhou 510632, China.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Mu
authors.affiliation: College of life science and technology, Jinan university, Guangzhou 510632, China.
authors.forename: Yayu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of life science and technology, Jinan university, Guangzhou 510632, China.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Wang
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors.forename: Lixin
authors.initials: L
authors.lastname: Du
authors.affiliation: College of animal science and technology, China Agricultural University, Beijing 100193, China.
authors.forename: Chuduan
authors.initials: C
authors.lastname: Wang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D055879:Gene Knock-In Techniques / Q000379:methods*; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010053:Ovary / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012756:Sheep
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems