Abstract
Cells can repair a double-strand break (DSB) by homologous recombination if a homologous sequence is provided as a template. This can be achieved by classical gene conversion (with or without crossover) or by single-strand annealing (SSA) between two direct repeat sequences flanking the DSB. To initiate SSA, single-stranded regions are needed adjacent to the break, extending up to the direct repeats in such a way that complementary strands can anneal to each other to repair the DSB. In the present protocol, we describe a GFP reporter assay in Saccharomyces cerevisiae allowing for the quantification of nuclease efficacy at inducing a DSB, by monitoring the reconstitution of a functional GFP gene whose expression can be rapidly quantified by flow cytometry.
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Acknowledgments
L.P. was supported by a CIFRE PhD fellowship from SANOFI. Work in G.-F. Richard laboratory was generously supported by the Institut Pasteur and the Centre National de la Recherche Scientifique (CNRS).
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Poggi, L., Dumas, B., Richard, GF. (2020). Monitoring Double-Strand Break Repair of Trinucleotide Repeats Using a Yeast Fluorescent Reporter Assay. In: Richard, GF. (eds) Trinucleotide Repeats. Methods in Molecular Biology, vol 2056. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9784-8_7
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DOI: https://doi.org/10.1007/978-1-4939-9784-8_7
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Publisher Name: Humana, New York, NY
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