Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System

We adapted the CRISPR–Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans.     

Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30–60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.     

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.     

CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering

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.     

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.     

Caenorhabditis elegans: A Genetic Guide to Parasitic Nematode Biology

The advent of parasite genome sequencing projects, as well as an increase in biology-directed gene discovery, promises to reveal genes encoding many of the key molecules required for nematode-host interactions. However, distinguishing parasitism genes from those merely required for nematode viability remains a substantial challenge. Although this will ultimately require a functional test in the host or parasite, the free-living nematode Caenorhabditis elegans can be exploited as a heterologous system to determine function of candidate parasitism genes. Studies of C. elegans also have revealed genetic networks, such as the dauer pathway, that may also be important adaptations for parasitism. As a more directed means of identifying parasitism traits, we developed classical genetics for Heterodera glycines and have used this approach to map genes conferring host resistance-breaking phenotypes. It is likely that the C. elegans and H. glycines genomes will be at least partially syntenic, thus permitting predictive physical mapping of H. glycines genes of interest.     

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene ¹. While the creation of libraries of RNAi clones covering most of the C. elegans genome ^2,3 opened the way for true functional genomic studies (see for example ^4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens ⁸. The approach relies on microscopic imaging and image analysis. Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing⁹. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

A Transparent Window into Biology: A Primer on Caenorhabditis elegans

A little over 50 years ago, Sydney Brenner had the foresight to develop the nematode (round worm) Caenorhabditis elegans as a genetic model for understanding questions of developmental biology and neurobiology. Over time, research on C. elegans has expanded to explore a wealth of diverse areas in modern biology including studies of the basic functions and interactions of eukaryotic cells, host–parasite interactions, and evolution. C. elegans has also become an important organism in which to study processes that go awry in human diseases. This primer introduces the organism and the many features that make it an outstanding experimental system, including its small size, rapid life cycle, transparency, and well-annotated genome. We survey the basic anatomical features, common technical approaches, and important discoveries in C. elegans research. Key to studying C. elegans has been the ability to address biological problems genetically, using both forward and reverse genetics, both at the level of the entire organism and at the level of the single, identified cell. These possibilities make C. elegans useful not only in research laboratories, but also in the classroom where it can be used to excite students who actually can see what is happening inside live cells and tissues.     

Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.     

Rapid and Precise Engineering of the Caenorhabditis elegans Genome with Lethal Mutation Co-Conversion and Inactivation of NHEJ Repair

As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8–9 days, using a temperature-sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 bp of homology drive efficient knock-in of point mutations, and that disabling nonhomologous end joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35–80 bp are sufficient for efficient editing and DSBs up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.     

Functional relevance of “seed” and “non-seed” sequences in microRNA-mediated promotion of C. elegans developmental progression

The founding heterochronic microRNAs, lin-4 and let-7, together with their validated targets and well-characterized phenotypes in C. elegans, offer an opportunity to test functionality of microRNAs in a developmental context. In this study, we defined sequence requirements at the microRNA level for these two microRNAs, evaluating lin-4 and let-7 mutant microRNAs for their ability to support temporal development under conditions where the wild-type lin-4 and let-7 gene products are absent. For lin-4, we found a strong requirement for seed sequences, with function drastically affected by several central mutations in the seed sequence, while rescue was retained by a set of mutations peripheral to the seed. let-7 rescuing activity was retained to a surprising degree by a variety of central seed mutations, while several non-seed mutant effects support potential noncanonical contributions to let-7 function. Taken together, this work illustrates both the functional partnership between seed and non-seed sequences in mediating C. elegans temporal development and a diversity among microRNA effectors in the contributions of seed and non-seed regions to activity.     

Pseudomonas fluorescens NZI7 repels grazing by C. elegans,a natural predator

The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, ‘EDB'' (‘edible''). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.     

Rendering the Intractable More Tractable: Tools from Caenorhabditis elegans Ripe for Import into Parasitic Nematodes

Recent and rapid advances in genetic and molecular tools have brought spectacular tractability to Caenorhabditis elegans, a model that was initially prized because of its simple design and ease of imaging. C. elegans has long been a powerful model in biomedical research, and tools such as RNAi and the CRISPR/Cas9 system allow facile knockdown of genes and genome editing, respectively. These developments have created an additional opportunity to tackle one of the most debilitating burdens on global health and food security: parasitic nematodes. I review how development of nonparasitic nematodes as genetic models informs efforts to import tools into parasitic nematodes. Current tools in three commonly studied parasites (Strongyloides spp., Brugia malayi, and Ascaris suum) are described, as are tools from C. elegans that are ripe for adaptation and the benefits and barriers to doing so. These tools will enable dissection of a huge array of questions that have been all but completely impenetrable to date, allowing investigation into host–parasite and parasite–vector interactions, and the genetic basis of parasitism.     

Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans

We have achieved targeted heritable genome modification in Caenorhabditis elegans by injecting mRNA of the nuclease Cas9 and Cas9 guide RNAs. This system rapidly creates precise genomic changes, including knockouts and transgene-instructed gene conversion.     

Mutation of the lbp-5 gene alters metabolic output in Caenorhabditis elegans

Intracellular lipid-binding proteins (LBPs) impact fatty acid homeostasis in various ways, including fatty acid transport into mitochondria. However, the physiological consequences caused by mutations in genes encoding LBPs remain largely uncharacterized. Here, we explore the metabolic consequences of lbp-5 gene deficiency in terms of energy homeostasis in Caenorhabditis elegans. In addition to increased fat storage, which has previously been reported, deletion of lbp-5 attenuated mitochondrial membrane potential and increased reactive oxygen species levels. Biochemical measurement coupled to proteomic analysis of the lbp-5(tm1618) mutant revealed highly increased rates of glycolysis in this mutant. These differential expression profile data support a novel metabolic adaptation of C. elegans, in which glycolysis is activated to compensate for the energy shortage due to the insufficient mitochondrial β-oxidation of fatty acids in lbp-5 mutant worms. This report marks the first demonstration of a unique metabolic adaptation that is a consequence of LBP-5 deficiency in C. elegans. [BMB Reports 2014; 47(1): 15-20]     

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.