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

Yan Zhou; Yong Liu; Dianna Hussmann; Peter Brøgger; Rasha Abdelkadhem Al-Saaidi; Shuang Tan; Lin Lin; Trine Skov Petersen; Guang Qian Zhou; Peter Bross; Lars Aagaard; Tino Klein; Sif Groth Rønn; Henrik Duelund Pedersen; Lars Bolund; Anders Lade Nielsen; Charlotte Brandt Sørensen; Yonglun Luo

doi:10.1007/s00018-015-2128-3

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

Yan Zhou, Yong Liu, Dianna Hussmann, Peter Brøgger, Rasha Abdelkadhem Al-Saaidi, Shuang Tan, Lin Lin, Trine Skov Petersen, Guang Qian Zhou, Peter Bross, Lars Aagaard, Tino Klein, Sif Groth Rønn, Henrik Duelund Pedersen, Lars Bolund, Anders Lade Nielsen, Charlotte Brandt Sørensen, Yonglun Luo

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

35 Citationer (Scopus)

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.

Originalsprog	Engelsk
Tidsskrift	Cellular and Molecular Life Sciences
Vol/bind	73
Nummer	13
Sider (fra-til)	2543-63
Antal sider	21
ISSN	1420-682X
DOI	https://doi.org/10.1007/s00018-015-2128-3
Status	Udgivet - 1 jul. 2016

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10.1007/s00018-015-2128-3

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Zhou, Y., Liu, Y., Hussmann, D., Brøgger, P., Al-Saaidi, R. A., Tan, S., Lin, L., Petersen, T. S., Zhou, G. Q., Bross, P., Aagaard, L., Klein, T., Rønn, S. G., Pedersen, H. D., Bolund, L., Nielsen, A. L., Sørensen, C. B., & Luo, Y. (2016). Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system. Cellular and Molecular Life Sciences, 73(13), 2543-63. https://doi.org/10.1007/s00018-015-2128-3

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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.",

keywords = "CRISPR/Cas9, Dual-fluorescent surrogate reporter, Gene targeting, Genome engineering, Homologous recombination, Single-strand annealing, TALENs",

author = "Yan Zhou and Yong Liu and Dianna Hussmann and Peter Br{\o}gger and Al-Saaidi, {Rasha Abdelkadhem} and Shuang Tan and Lin Lin and Petersen, {Trine Skov} and Zhou, {Guang Qian} and Peter Bross and Lars Aagaard and Tino Klein and R{\o}nn, {Sif Groth} and Pedersen, {Henrik Duelund} and Lars Bolund and Nielsen, {Anders Lade} and S{\o}rensen, {Charlotte Brandt} and Yonglun Luo",

year = "2016",

month = jul,

day = "1",

doi = "10.1007/s00018-015-2128-3",

language = "English",

volume = "73",

pages = "2543--63",

journal = "Cellular and Molecular Life Sciences",

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number = "13",

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Zhou, Y, Liu, Y, Hussmann, D, Brøgger, P, Al-Saaidi, RA, Tan, S, Lin, L , Petersen, TS, Zhou, GQ, Bross, P , Aagaard, L, Klein, T, Rønn, SG, Pedersen, HD, Bolund, L, Nielsen, AL , Sørensen, CB & Luo, Y 2016, 'Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system', Cellular and Molecular Life Sciences, bind 73, nr. 13, s. 2543-63. https://doi.org/10.1007/s00018-015-2128-3

TY - JOUR

T1 - Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system

AU - Zhou, Yan

AU - Liu, Yong

AU - Hussmann, Dianna

AU - Brøgger, Peter

AU - Al-Saaidi, Rasha Abdelkadhem

AU - Tan, Shuang

AU - Lin, Lin

AU - Petersen, Trine Skov

AU - Zhou, Guang Qian

AU - Bross, Peter

AU - Aagaard, Lars

AU - Klein, Tino

AU - Rønn, Sif Groth

AU - Pedersen, Henrik Duelund

AU - Bolund, Lars

AU - Nielsen, Anders Lade

AU - Sørensen, Charlotte Brandt

AU - Luo, Yonglun

PY - 2016/7/1

Y1 - 2016/7/1

N2 - 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.

AB - 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.

KW - CRISPR/Cas9

KW - Dual-fluorescent surrogate reporter

KW - Gene targeting

KW - Genome engineering

KW - Homologous recombination

KW - Single-strand annealing

KW - TALENs

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U2 - 10.1007/s00018-015-2128-3

DO - 10.1007/s00018-015-2128-3

M3 - Journal article

C2 - 26755436

SN - 1420-682X

VL - 73

SP - 2543

EP - 2563

JO - Cellular and Molecular Life Sciences

JF - Cellular and Molecular Life Sciences

IS - 13

ER -

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

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