title: Reliable CRISPR/Cas9 Genome Engineering inCaenorhabditis elegansUsing a Single Efficient sgRNA and an Easily Recognizable Phenotype.
abstract: CRISPR/Cas9 genome engineering strategies allow the directed modification of theCaenorhabditis elegansgenome 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 thedpy-10gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as thedpy-10gene. 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, aC. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.
authors.affiliation: Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69622 Villeurbanne, France.
authors.forename: Sonia
authors.identifier: 0000-0002-3674-3151
authors.initials: S
authors.lastname: El Mouridi
authors.affiliation: Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69622 Villeurbanne, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Lecroisey
authors.affiliation: Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69622 Villeurbanne, France.
authors.forename: Philippe
authors.identifier: 0000-0002-4313-2527
authors.initials: P
authors.lastname: Tardy
authors.affiliation: Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69622 Villeurbanne, France.
authors.forename: Marine
authors.identifier: 0000-0003-1508-8845
authors.initials: M
authors.lastname: Mercier
authors.affiliation: Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69622 Villeurbanne, France.
authors.forename: Alice
authors.initials: A
authors.lastname: Leclercq-Blondel
authors.affiliation: Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69622 Villeurbanne, France.
authors.forename: Nora
authors.initials: N
authors.lastname: Zariohi
authors.affiliation: Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, INSERM U-1217, Institut NeuroMyoGène, F-69622 Villeurbanne, France thomas.boulin@univ-lyon1.fr.
authors.forename: Thomas
authors.identifier: 0000-0002-1734-1915
authors.initials: T
authors.lastname: Boulin
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D003094:Collagen / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D049751:Genome, Helminth; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D029742:Caenorhabditis elegans Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C084630:dpy-10 protein, C elegans; D003094:Collagen
keywords: CRISPR/Cas9 genome engineering; Caenorhabditis elegans; coconversion; dpy-10; mScarlet