title: Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.
abstract: Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Dianna
authors.initials: D
authors.lastname: Hussmann
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Brøgger
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and University Hospital, 8200, Aarhus N, Denmark.
authors.forename: Rasha Abdelkadhem
authors.initials: RA
authors.lastname: Al-Saaidi
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.|Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, Shenzhen, 518060, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Tan
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Trine Skov
authors.initials: TS
authors.lastname: Petersen
authors.affiliation: Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, Shenzhen, 518060, China.
authors.forename: Guang Qian
authors.initials: GQ
authors.lastname: Zhou
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and University Hospital, 8200, Aarhus N, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Bross
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Aagaard
authors.affiliation: Department of Histology, Gubra A/S, 2970, Hørsholm, Denmark.
authors.forename: Tino
authors.initials: T
authors.lastname: Klein
authors.affiliation: Department of Incretin and Obesity Research, Novo Nordisk A/S, 2760, Måløv, Denmark.
authors.forename: Sif Groth
authors.initials: SG
authors.lastname: Rønn
authors.affiliation: Department of Incretin and Obesity Research, Novo Nordisk A/S, 2760, Måløv, Denmark.
authors.forename: Henrik Duelund
authors.initials: HD
authors.lastname: Pedersen
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.|BGI-Shenzhen, Shenzhen, 518083, China.|The Danish Regenerative Engineering Alliance for Medicine (DREAM), Aarhus University, Aarhus, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
authors.forename: Anders Lade
authors.initials: AL
authors.lastname: Nielsen
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and University Hospital, 8200, Aarhus N, Denmark.
authors.forename: Charlotte Brandt
authors.initials: CB
authors.lastname: Sørensen
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark. alun@biomed.au.dk.|Department of Incretin and Obesity Research, Novo Nordisk A/S, 2760, Måløv, Denmark. alun@biomed.au.dk.|The Danish Regenerative Engineering Alliance for Medicine (DREAM), Aarhus University, Aarhus, Denmark. alun@biomed.au.dk.
authors.forename: Yonglun
authors.initials: Y
authors.lastname: Luo
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005453:Fluorescence; D000072669:Gene Editing / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D059765:Homologous Recombination; D006801:Humans; D061986:MCF-7 Cells; D017526:Receptor, IGF Type 1; D017451:Receptors, Somatomedin / Q000235:genetics; D013552:Swine; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics / Q000378:metabolism
chemical_list: C000715594:CBX5 protein, human; D002868:Chromosomal Proteins, Non-Histone; C000597236:IGF1R protein, human; D017451:Receptors, Somatomedin; D000090266:Chromobox Protein Homolog 5; D017526:Receptor, IGF Type 1; D000069896:Transcription Activator-Like Effector Nucleases
keywords: CRISPR/Cas9; Dual-fluorescent surrogate reporter; Gene targeting; Genome engineering; Homologous recombination; Single-strand annealing; TALENs