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Reliable CRISPR/Cas9 genome engineering in Caenorhabiditis elegans using a single efficient sgRNA and an easily selectable phenotype

Sonia El Mouridi, Claire Lecroisey, Philippe Tardy, Marine Mercier, Alice Leclercq-Blondel, Nora Zariohi, Thomas Boulin
doi: https://doi.org/10.1101/105718
Sonia El Mouridi
Institut NeuroMyoGène Univ Lyon, Université Claude Bernard Lyon 1 CNRS UMR 5310, INSERM U1217 8 Rue Raphaël Dubois 69100, Villeurbanne, France
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Claire Lecroisey
Institut NeuroMyoGène Univ Lyon, Université Claude Bernard Lyon 1 CNRS UMR 5310, INSERM U1217 8 Rue Raphaël Dubois 69100, Villeurbanne, France
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Philippe Tardy
Institut NeuroMyoGène Univ Lyon, Université Claude Bernard Lyon 1 CNRS UMR 5310, INSERM U1217 8 Rue Raphaël Dubois 69100, Villeurbanne, France
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Marine Mercier
Institut NeuroMyoGène Univ Lyon, Université Claude Bernard Lyon 1 CNRS UMR 5310, INSERM U1217 8 Rue Raphaël Dubois 69100, Villeurbanne, France
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Alice Leclercq-Blondel
Institut NeuroMyoGène Univ Lyon, Université Claude Bernard Lyon 1 CNRS UMR 5310, INSERM U1217 8 Rue Raphaël Dubois 69100, Villeurbanne, France
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Nora Zariohi
Institut NeuroMyoGène Univ Lyon, Université Claude Bernard Lyon 1 CNRS UMR 5310, INSERM U1217 8 Rue Raphaël Dubois 69100, Villeurbanne, France
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Thomas Boulin
Institut NeuroMyoGène Univ Lyon, Université Claude Bernard Lyon 1 CNRS UMR 5310, INSERM U1217 8 Rue Raphaël Dubois 69100, Villeurbanne, France
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  • For correspondence: thomas.boulin@univ-lyon1.fr
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ABSTRACT

CRISPR/Cas9 genome engineering strategies allow the directed modification of the C. elegans genome to introduce point mutations, generate knock-out mutants and insert coding sequences for epitope or fluorescent tags. Three practical aspects however complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an 8-fold increase in fluorescence in a direct comparison.

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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 February 03, 2017.
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Reliable CRISPR/Cas9 genome engineering in Caenorhabiditis elegans using a single efficient sgRNA and an easily selectable phenotype
Sonia El Mouridi, Claire Lecroisey, Philippe Tardy, Marine Mercier, Alice Leclercq-Blondel, Nora Zariohi, Thomas Boulin
bioRxiv 105718; doi: https://doi.org/10.1101/105718
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Reliable CRISPR/Cas9 genome engineering in Caenorhabiditis elegans using a single efficient sgRNA and an easily selectable phenotype
Sonia El Mouridi, Claire Lecroisey, Philippe Tardy, Marine Mercier, Alice Leclercq-Blondel, Nora Zariohi, Thomas Boulin
bioRxiv 105718; doi: https://doi.org/10.1101/105718

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