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A Versatile Protocol to Generate Translocations in Yeast Genomes Using CRISPR/Cas9

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Yeast Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2196))

Abstract

Genomic engineering methods represent powerful tools to examine chromosomal modifications and to subsequently study their impacts on cellular phenotypes. However, quantifying the fitness impact of translocations, independently from base substitutions or the insertion of genetic markers, remains a challenge. Here we report a rapid and straightforward protocol for engineering either targeted reciprocal translocations at the base pair level of resolution between two chromosomes or multiple simultaneous rearrangements in the yeast genome, without inserting any marker sequence in the chromosomes. Our CRISPR/Cas9-based method consists of inducing either (1) two double-strand breaks (DSBs) in two different chromosomes with two distinct guide RNAs (gRNAs) while providing specifically designed homologous donor DNA forcing the trans-repair of chromosomal extremities to generate a targeted reciprocal translocation or (2) multiple DSBs with a single gRNA targeting dispersed repeated sequences and leaving endogenous uncut copies of the repeat to be used as donor DNA, thereby generating multiple translocations, often associated with large segmental duplications (Fleiss, et al. PLoS Genet 15:e1008332, 2019).

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Acknowledgments

This work was supported by the Agence Nationale de la Recherche [ANR-16-CE12-0019]. We thank Allyson Holmes and Samuel O’Donnell for reading the manuscript.

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Authors and Affiliations

  1. Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, Laboratory of Computational and Quantitative Biology, Paris, France

    Nicolas Agier, Stéphane Delmas & Gilles Fischer

  2. Synthetic Biology Group, MRC London Institute of Medical Sciences, London, UK

    Aubin Fleiss

  3. Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK

    Aubin Fleiss

Authors
  1. Nicolas Agier
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  2. Aubin Fleiss
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  3. Stéphane Delmas
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  4. Gilles Fischer
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Corresponding author

Correspondence to Gilles Fischer .

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Editors and Affiliations

  1. Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, College of Life Sciences, Capital Normal University Beijing, China, Saskatoon, SK, Canada

    Wei Xiao

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Agier, N., Fleiss, A., Delmas, S., Fischer, G. (2021). A Versatile Protocol to Generate Translocations in Yeast Genomes Using CRISPR/Cas9. In: Xiao, W. (eds) Yeast Protocols. Methods in Molecular Biology, vol 2196. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0868-5_14

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  • DOI: https://doi.org/10.1007/978-1-0716-0868-5_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0867-8

  • Online ISBN: 978-1-0716-0868-5

  • eBook Packages: Springer Protocols

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