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Clonal analysis by tunable CRISPR-mediated excision

Anna F. Gilles, Johannes B. Schinko, Magdalena I. Schacht, Camille Enjolras, View ORCID ProfileMichalis Averof
doi: https://doi.org/10.1101/394221
Anna F. Gilles
1Institut de Génomique Fonctionnelle de Lyon (IGFL), École Normale Supérieure de Lyon, 32 avenue Tony Garnier, 69007 Lyon, France
2BMIC graduate programme, Université Claude Bernard / Lyon 1, France
4TriGenes, Biberach University of Applied Sciences, Hubertus-Liebrecht-Str. 35, 88400 Biberach/Riss, Germany
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Johannes B. Schinko
1Institut de Génomique Fonctionnelle de Lyon (IGFL), École Normale Supérieure de Lyon, 32 avenue Tony Garnier, 69007 Lyon, France
3Centre National de la Recherche Scientifique (CNRS), France
4TriGenes, Biberach University of Applied Sciences, Hubertus-Liebrecht-Str. 35, 88400 Biberach/Riss, Germany
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Magdalena I. Schacht
1Institut de Génomique Fonctionnelle de Lyon (IGFL), École Normale Supérieure de Lyon, 32 avenue Tony Garnier, 69007 Lyon, France
5Georg-August University of Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
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Camille Enjolras
1Institut de Génomique Fonctionnelle de Lyon (IGFL), École Normale Supérieure de Lyon, 32 avenue Tony Garnier, 69007 Lyon, France
3Centre National de la Recherche Scientifique (CNRS), France
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Michalis Averof
1Institut de Génomique Fonctionnelle de Lyon (IGFL), École Normale Supérieure de Lyon, 32 avenue Tony Garnier, 69007 Lyon, France
3Centre National de la Recherche Scientifique (CNRS), France
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  • ORCID record for Michalis Averof
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Abstract

Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum. We demonstrate that clone marking frequency can be tuned to generate embryos carrying single marked clones. The Valcyrie approach can be applied to a wide range of experimental settings, for example to modulate clone frequency with existing tools in established model organisms and to introduce clonal analysis in emerging experimental models.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted August 17, 2018.
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Clonal analysis by tunable CRISPR-mediated excision
Anna F. Gilles, Johannes B. Schinko, Magdalena I. Schacht, Camille Enjolras, Michalis Averof
bioRxiv 394221; doi: https://doi.org/10.1101/394221
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Clonal analysis by tunable CRISPR-mediated excision
Anna F. Gilles, Johannes B. Schinko, Magdalena I. Schacht, Camille Enjolras, Michalis Averof
bioRxiv 394221; doi: https://doi.org/10.1101/394221

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