title: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.
abstract: The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.|The Key Laboratory of Adolescent Health Assessment and Exercise Intervention of Ministry of Education, East China Normal University, Shanghai 200241, China.
authors.forename: Liren
authors.initials: L
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Yanjiao
authors.initials: Y
authors.lastname: Shao
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Yuting
authors.initials: Y
authors.lastname: Guan
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Wu
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Fangrui
authors.initials: F
authors.lastname: Chen
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Meizhen
authors.initials: M
authors.lastname: Liu
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Huaqing
authors.initials: H
authors.lastname: Chen
authors.affiliation: Hainan Provincial Key Laboratory for human reproductive medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China.
authors.forename: Yanlin
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Xueyun
authors.initials: X
authors.lastname: Ma
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.|The Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA.
authors.forename: Mingyao
authors.initials: M
authors.lastname: Liu
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
authors.forename: Dali
authors.initials: D
authors.lastname: Li
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D017353:Gene Deletion; D055879:Gene Knock-In Techniques / Q000379:methods*; D056426:Genetic Loci; D051379:Mice; D051381:Rats; D015053:Zygote / Q000166:cytology / Q000378:metabolism*