title: CRISPR/Cas9-mediated reporter knock-in in mouse haploid embryonic stem cells.
abstract: Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways.
authors.affiliation: Department of Pathology.|Department of Neurology, Graduate School of Medicine.
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: Department of Pathology.|Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oda
authors.affiliation: Department of Pathology.
authors.forename: Tsunetoshi
authors.initials: T
authors.lastname: Nakatani
authors.affiliation: Department of Pathology.|Department of Biosciences, Kitasato University School of Science, Kanagawa, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Sekita
authors.affiliation: Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Zürich, Hönggerberg, 8049 Zürich, Switzerland.
authors.forename: Asun
authors.initials: A
authors.lastname: Monfort
authors.affiliation: Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Zürich, Hönggerberg, 8049 Zürich, Switzerland.
authors.forename: Anton
authors.initials: A
authors.lastname: Wutz
authors.affiliation: Department of Neurology, Graduate School of Medicine.
authors.forename: Hideki
authors.initials: H
authors.lastname: Mochizuki
authors.affiliation: Department of Pathology.|Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.|JST, CREST.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakano
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D023061:Gene Order; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D006238:Haploidy; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D055748:SOXB1 Transcription Factors; C098693:Sox1 protein, mouse