Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here, we report a method to edit the C. elegans genome using the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) RNA-guided Cas9 nuclease followed by homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites, and that these breaks can be efficiently repaired by homologous recombination. By supplying engineered homologous repair templates, we generated GFP knock-ins and targeted mutations. Together, our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP-SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step.KEYWORDS CRISPR/Cas9; homologous recombination; gene tagging; Caenorhabditis elegans; self-excising cassette A common goal in biological and biomedical research is to visualize the localization of a protein of interest within a cell or organism. This is often accomplished by fusing GFP or another fluorescent protein (FP) to the protein of interest. In the nematode Caenorhabditis elegans, GFP fusions were historically generated by injecting plasmids into the gonad of the adult hermaphrodite worm, resulting in the formation of extrachromosomal arrays (Mello et al. 1991). However, the resulting fusion proteins were typically strongly overexpressed in somatic tissues and silenced in the germline. Microparticle bombardment allowed the generation of low-copy transgenes that in some cases more closely recapitulated endogenous expression levels (Praitis et al. 2001;Sarov et al. 2012), but this technique is inefficient, time consuming, and difficult and requires expensive equipment and materials. More recently, we and others have reported CRISPR/Cas9-based approaches that together can be used to make essentially any desired change to the C. elegans genome, including insertion of GFP into endogenous loci Dickinson et al. 2013;Lo et al. 2013;Chiu et al. 2013;Cho et al. 2013;Katic and Großhans 2013;Tzur et al. 2013;Waaijers et al. 2013;Chen et al. 2013;Zhao et al. 2014;Kim et al. 2014;Arribere et al. 2014;Paix et al. 2014;Ward 2015;Farboud and Meyer 2015). The resulting GFP knock-in strains express 100% labeled protein under the control of all native regulatory elements, resulting in endogenous levels and patterns of expression in all cases reported to date (Dickinson et al. 2013;Kim et al. 2014).Our publis...

Summary Regulated protein-protein interactions are critical for cell signaling, differentiation and development. To study dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here, we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined timepoints and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during C. elegans zygote polarization, which takes place in less than 20 minutes. PAR complex dynamics are linked to the cell cycle by polo-like kinase 1, and govern movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo.

The advent of genome editing techniques based on the clustered regularly interspersed short palindromic repeats (CRISPR)–Cas9 system has revolutionized research in the biological sciences. CRISPR is quickly becoming an indispensible experimental tool for researchers using genetic model organisms, including the nematode Caenorhabditis elegans. Here, we provide an overview of CRISPR-based strategies for genome editing in C. elegans. We focus on practical considerations for successful genome editing, including a discussion of which strategies are best suited to producing different kinds of targeted genome modifications.

Summary A polarized epithelium in the non-metazoan Dictyostelium discoideum requires α-catenin and β-catenin but not classical cadherins, polarity proteins or Wnt signaling. A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex containing a classical cadherin, the Wnt-signaling protein β-catenin and the actin-binding protein α-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an α-catenin ortholog that binds a β-catenin-related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion and proper multicellular morphogenesis. Thus the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins.