Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system
| Autor: | Peter Brøgger, Lars Aagaard, H. D. Pedersen, Peter Bross, Sif Groth Rønn, Tino Klein, Charlotte Brandt Sørensen, Trine Skov Petersen, Rasha Abdelkadhem Al-Saaidi, Yan Zhou, Dianna Hussmann, Yonglun Luo, Yong Liu, Anders Lade Nielsen, Guangqian Zhou, Lin Lin, Shuang Tan, Lars Bolund |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Chromosomal Proteins Non-Histone Swine Golden Gate Cloning Genetic Vectors Computational biology Biology Fluorescence Genome engineering Receptor IGF Type 1 03 medical and health sciences Cellular and Molecular Neuroscience Gene Knockout Techniques Genome editing Genes Reporter Transcription Activator-Like Effector Nucleases CRISPR Animals Humans Clustered Regularly Interspaced Short Palindromic Repeats Homologous Recombination Molecular Biology Gene Cells Cultured Pharmacology Genetics Gene Editing Transcription activator-like effector nuclease Cas9 Gene targeting Receptors Somatomedin Cell Biology Fibroblasts 030104 developmental biology HEK293 Cells Chromobox Protein Homolog 5 MCF-7 Cells Molecular Medicine CRISPR-Cas Systems |
| Zdroj: | 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, vol. 73, no. 13, pp. 2543-63 . https://doi.org/10.1007/s00018-015-2128-3 |
| DOI: | 10.1007/s00018-015-2128-3 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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