Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system
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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.
KeywordsDual-fluorescent surrogate reporter TALENs CRISPR/Cas9 Gene targeting Genome engineering Single-strand annealing Homologous recombination
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.
Compliance with ethical standards
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.
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