| Autor: |
Zhou, Yan, Liu, Yong, Hussmann, Dianna, Brøgger, Peter, Al-Saaidi, Rasha, Tan, Shuang, Lin, Lin, Petersen, Trine, Zhou, Guang, Bross, Peter, Aagaard, Lars, Klein, Tino, Rønn, Sif, Pedersen, Henrik, Bolund, Lars, Nielsen, Anders, Sørensen, Charlotte, Luo, Yonglun |
| Předmět: |
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| Zdroj: |
Cellular & Molecular Life Sciences; Jul2016, Vol. 73 Issue 13, p2543-2563, 21p |
| Abstrakt: |
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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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