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Cell Mol Life Sci. 2016 Jul;73(13):2543-63. doi: 10.1007/s00018-015-2128-3. Epub 2016 Jan 11.

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Author information

1
Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark.
2
Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and University Hospital, 8200, Aarhus N, Denmark.
3
Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, Shenzhen, 518060, China.
4
Department of Histology, Gubra A/S, 2970, Hørsholm, Denmark.
5
Department of Incretin and Obesity Research, Novo Nordisk A/S, 2760, Måløv, Denmark.
6
BGI-Shenzhen, Shenzhen, 518083, China.
7
The Danish Regenerative Engineering Alliance for Medicine (DREAM), Aarhus University, Aarhus, Denmark.
8
Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark. alun@biomed.au.dk.
9
Department of Incretin and Obesity Research, Novo Nordisk A/S, 2760, Måløv, Denmark. alun@biomed.au.dk.
10
The Danish Regenerative Engineering Alliance for Medicine (DREAM), Aarhus University, Aarhus, Denmark. alun@biomed.au.dk.

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.

KEYWORDS:

CRISPR/Cas9; Dual-fluorescent surrogate reporter; Gene targeting; Genome engineering; Homologous recombination; Single-strand annealing; TALENs

PMID:
26755436
DOI:
10.1007/s00018-015-2128-3
[Indexed for MEDLINE]

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