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...

Fluorescent protein tags are the primary tool for labeling gene products and analyzing their dynamics using live-cell imaging. A quantitative comparison is made of fluorescent protein brightness and photostability in an in vivo animal model system, and tools and recommendations are given for optimal fluorescent protein selection.

Oriented cell divisions are critical to establish and maintain cell fates and tissue organization. Diverse extracellular and intracellular cues have been shown to provide spatial information for mitotic spindle positioning; however, the molecular mechanisms by which extracellular signals communicate with cells to direct mitotic spindle positioning are largely unknown. In animal cells, oriented cell divisions are often achieved by the localization of force-generating motor protein complexes to discrete cortical domains. Disrupting either these force-generating complexes or proteins that globally affect microtubule stability results in defects in mitotic positioning, irrespective of whether these proteins function as spatial cues for spindle orientation. This poses a challenge to traditional genetic dissection of this process. Therefore, as an alternative strategy to identify key proteins that act downstream of intercellular signaling, we screened the localization of many candidate proteins by inserting fluorescent tags directly into endogenous gene loci, without overexpressing the proteins. We tagged 23 candidate proteins in Caenorhabditis elegans and examined each protein’s localization in a well-characterized, oriented cell division in the four-cell-stage embryo. We used cell manipulations and genetic experiments to determine which cells harbor key localized proteins and which signals direct these localizations in vivo. We found that Dishevelled and adenomatous polyposis coli homologs are polarized during this oriented cell division in response to a Wnt signal, but two proteins typically associated with mitotic spindle positioning, homologs of NuMA and Dynein, were not detectably polarized. These results suggest an unexpected mechanism for mitotic spindle positioning in this system, they pinpoint key proteins of interest, and they highlight the utility of a screening approach based on analyzing the localization of endogenously tagged proteins.

Fadero et al. present lateral interference tilted excitation (LITE) microscopy–a tilted light-sheet method to illuminate high-numerical-aperture objectives for fluorescence microscopy. LITE can be implemented unobtrusively on most microscope systems and combines low photodamage with high resolution and efficient detection in imaging fluorescent organisms.

The size of mitotic chromosomes is coordinated with cell size. Through an RNAi screen in Caenorhabditis elegans, Ladouceur et al. identify CENP-A and topo-II as factors affecting chromosome length. Quantitative analyses of protein dynamics suggest that CENP-A and topo-II localize and function independently to provide centromeric chromatin structure and determine the length of holocentric mitotic chromosomes.