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

被引:29
|
作者
Zhou, Yan [1 ]
Liu, Yong [1 ]
Hussmann, Dianna [1 ]
Brogger, Peter [1 ]
Al-Saaidi, Rasha Abdelkadhem [2 ,3 ]
Tan, Shuang [1 ,4 ]
Lin, Lin [1 ]
Petersen, Trine Skov [1 ]
Zhou, Guang Qian [4 ]
Bross, Peter [2 ,3 ]
Aagaard, Lars [1 ]
Klein, Tino [6 ]
Ronn, Sif Groth [7 ]
Pedersen, Henrik Duelund [7 ]
Bolund, Lars [1 ,5 ,8 ]
Nielsen, Anders Lade [1 ]
Sorensen, Charlotte Brandt [2 ,3 ]
Luo, Yonglun [1 ,7 ,8 ]
机构
[1] Aarhus Univ, Dept Biomed, Wilhelm Meyers Alle 4, DK-8000 Aarhus C, Denmark
[2] Aarhus Univ, Dept Clin Med, Res Unit Mol Med, DK-8200 Aarhus N, Denmark
[3] Aarhus Univ Hosp, DK-8200 Aarhus N, Denmark
[4] Shenzhen Univ, Hlth Sci Ctr, Shenzhen Key Lab Antiaging & Regenerat Med, Shenzhen 518060, Peoples R China
[5] BGI Shenzhen, Shenzhen 518083, Peoples R China
[6] Gubra AS, Dept Histol, DK-2970 Horsholm, Denmark
[7] Novo Nordisk AS, Dept Incretin & Obes Res, DK-2760 Malov, Denmark
[8] Aarhus Univ, DREAM, Aarhus, Denmark
关键词
Dual-fluorescent surrogate reporter; TALENs; CRISPR/Cas9; Gene targeting; Genome engineering; Single-strand annealing; Homologous recombination; PLURIPOTENT STEM-CELLS; HOMOLOGOUS RECOMBINATION; TARGETED MUTAGENESIS; GENE KNOCKOUT; BREAK REPAIR; TALEN; CRISPR-CAS9; SPECIFICITY; GENERATION; EFFICIENCY;
D O I
10.1007/s00018-015-2128-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:2543 / 2563
页数:21
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