title: Comparison of surrogate reporter systems for enrichment of cells with mutations induced by genome editors.
abstract: Genome editors are powerful tools that allow modification of the nuclear DNA in eukaryotic cells both in vitro and in vivo. In vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their isolation for analysis. Episomal reporters encoding fluorescent proteins can be used for enrichment of modified cells by flow cytometry. Here we compare two surrogate reporters, RGS and SSA, for the enrichment of porcine embryonic fibroblasts containing mutations induced by ZFNs or CRISPR/Cas9. Both systems were effective for enrichment of edited porcine cells with the RGS reporter proving more effective than the SSA reporter. We noted a higher-fold enrichment when editing events were induced by Cas9 compared to those induced by ZFNs, allowing selection at frequencies as high as 70%.
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Zuyong
authors.initials: Z
authors.lastname: He
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Meirui
authors.initials: M
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
authors.forename: Guangjie
authors.initials: G
authors.lastname: Sun
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Edinburgh EH25 9RG, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Proudfoot
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Edinburgh EH25 9RG, UK.
authors.forename: C Bruce A
authors.initials: CB
authors.lastname: Whitelaw
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Edinburgh EH25 9RG, UK. Electronic address: simon.lillico@roslin.ed.ac.uk.
authors.forename: Simon G
authors.initials: SG
authors.lastname: Lillico
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, PR China. Electronic address: chyaosh@mail.sysu.edu.cn.
authors.forename: Yaosheng
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology*; D017930:Genes, Reporter; D016678:Genome; D009154:Mutation; D013552:Swine
keywords: CRISPR/Cas9; SIGLEC-1; Surrogate reporter; ZFN