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

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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.

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Acknowledgments

We are grateful to the FACS CORE facility (with special thanks to Charlotte Christie Petersen) at the Department of Biomedicine, Aarhus University for assistance with flow cytometry and FACS. This work was supported in part by grants from the STAR programme from the R&D Department, Novo Nordisk A/S to YL; and the Danish Research Council for Independent Research (16942) to YL; the Sapere Aude Young Research Talent prize to YL (18382); the Lundbeck Foundation (R173-2014-1105, R151-2013-14439, R126-2012-12448, R100-A9209, R173-2014-993, and R100-A9606) to YL, LB, PB, CBS, and ALN respectively; the China Scholarship Council (CSC) to YZ; the Natural Science Foundation of China (81472126) to ST and GQZ; the Toyota Foundation ALN; and the AUFF AU IDEAS Programme and The Karen Elise Jensen Foundation to CBS.

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    Authors and Affiliations

    1. Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000, Aarhus C, Denmark

      Yan Zhou, Yong Liu, Dianna Hussmann, Peter Brøgger, Shuang Tan, Lin Lin, Trine Skov Petersen, Lars Aagaard, Lars Bolund, Anders Lade Nielsen & Yonglun Luo

    2. Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and University Hospital, 8200, Aarhus N, Denmark

      Rasha Abdelkadhem Al-Saaidi, Peter Bross & Charlotte Brandt Sørensen

    3. Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science Center, Shenzhen University, Shenzhen, 518060, China

      Shuang Tan & Guang Qian Zhou

    4. BGI-Shenzhen, Shenzhen, 518083, China

      Lars Bolund

    5. Department of Histology, Gubra A/S, 2970, Hørsholm, Denmark

      Tino Klein

    6. Department of Incretin and Obesity Research, Novo Nordisk A/S, 2760, Måløv, Denmark

      Sif Groth Rønn, Henrik Duelund Pedersen & Yonglun Luo

    7. The Danish Regenerative Engineering Alliance for Medicine (DREAM), Aarhus University, Aarhus, Denmark

      Lars Bolund & Yonglun Luo

    Authors
    1. Yan Zhou
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    2. Yong Liu
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    3. Dianna Hussmann
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    4. Peter Brøgger
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    5. Rasha Abdelkadhem Al-Saaidi
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    6. Shuang Tan
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    7. Lin Lin
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    8. Trine Skov Petersen
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    9. Guang Qian Zhou
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    10. Peter Bross
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    11. Lars Aagaard
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    12. Tino Klein
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    13. Sif Groth Rønn
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    14. Henrik Duelund Pedersen
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    15. Lars Bolund
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    16. Anders Lade Nielsen
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    17. Charlotte Brandt Sørensen
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    18. Yonglun Luo
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    Corresponding author

    Correspondence to Yonglun Luo.

    Ethics declarations

    Conflict of interest

    YL (2012–2014), SGR, and HD were financed by Novo Nordisk A/S. TK was financed by Gubra ApS. A patent claim is declared to the generation of a diabetes pig model based on genetic modification of the porcine IAPP gene. No other competing interests are declared by the authors.

    Additional information

    Y. Zhou, Y. Liu, D. Hussmann, P. Brøgger contributed equally to the study.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    Additional File 1: Genebank file of the C-Check vector (TXT 12 kb)

    Additional File 2: List of TALEN and CRISPR/Cas9 target sites (XLSX 43 kb)

    18_2015_2128_MOESM3_ESM.pdf

    Additional File 3: Optimization of nucleofection in porcine fibroblasts. Five nucleofection reagents (P1-P5) and 15 nucleofection programs were evaluated (PDF 608 kb)

    Additional File 4: List of oligonucleotides and primers (DOCX 166 kb)

    Additional File 5: Sanger sequencing analysis of one IGF1R potential off-target site (PDF 14353 kb)

    Additional File 6: Sanger sequencing of IGF1R knockout clonogenic cell clones (DOCX 20 kb)

    Additional File 7: Sanger sequencing of CBX5 knockout clonogenic cell clones (DOCX 131 kb)

    Additional File 8: C-Check CRISPR OFF sgRNA design and sequences (XLSX 54 kb)

    Additional File 9: Detailed protocol for construction of the C-Check reporter vector (DOCX 110 kb)

    Additional File 10: C-Check complementary oligonucleotide design (XLSX 41 kb)

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    Cite this article

    Zhou, Y., Liu, Y., Hussmann, D. et al. Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system. Cell. Mol. Life Sci. 73, 2543–2563 (2016). https://doi.org/10.1007/s00018-015-2128-3

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    • DOI: https://doi.org/10.1007/s00018-015-2128-3

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