Mark L. Edgley

The million mutation project: A new approach to genetics in Caenorhabditis elegans

We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worms more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.

Copy number variation in the genomes of twelve natural isolates of Caenorhabditis elegans

BackgroundCopy number variation is an important component of genetic variation in higher eukaryotes. The extent of natural copy number variation in C. elegans is unknown outside of 2 highly divergent wild isolates and the canonical N2 Bristol strain.ResultsWe have used array comparative genomic hybridization (aCGH) to detect copy number variation in the genomes of 12 natural isolates of Caenorhabditis elegans. Deletions relative to the canonical N2 strain are more common in these isolates than duplications, and indels are enriched in multigene families on the autosome arms. Among the strains in our study, the Hawaiian and Madeiran strains (CB4856 and JU258) carry the largest number of deletions, followed by the Vancouver strain (KR314). Overall we detected 510 different deletions affecting 1136 genes, or over 5% of the genes in the canonical N2 genome. The indels we identified had a median length of 2.7 kb. Since many deletions are found in multiple isolates, deletion loci were used as markers to derive an unrooted tree to estimate genetic relatedness among the strains.ConclusionCopy number variation is extensive in C. elegans, affecting over 5% of the genes in the genome. The deletions we have detected in natural isolates of C. elegans contribute significantly to the number of deletion alleles available to researchers. The relationships between strains are complex and different regions of the genome possess different genealogies due to recombination throughout the natural history of the species, which may not be apparent in studies utilizing smaller numbers of genetic markers.

Rapid High Resolution Single Nucleotide Polymorphism–Comparative Genome Hybridization Mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ∼200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.

Chapter 7 Genetic Balancers

Publisher Summary This chapter discusses genetic balancers. They are genetic constructs or chromosomal rearrangements that allow lethal or sterile mutations to be stably maintained in heterozygotes. In this chapter, the term “balancer” primarily to refers to chromosomal duplications or rearrangements that suppress crossing over. Different genetic constructs have been used to balance lethal mutations, however, they have the same basic characteristics: (1) heterozygotes possess a unique phenotype so that they can be selected reliably; (2) progeny phenotypes allow the investigator to tell when recombination has occurred; (3) close proximity of markers increases the degree of balancing by decreasing the frequency of recombination between lethals and markers; and (4) in the majority of cases, common visible markers linked to the lethals ease all phases of analysis. The chapter describes the features and behavior of each class of balancer currently used in Caenorhabditis elegans, discusses the practical aspects of day-to-day balancer use, and provides an overview of the state of balancing for each chromosome.

Rapid Increase in frequency of gene copy-number variants during experimental evolution in Caenorhabditis elegans

BackgroundGene copy-number variation (CNVs), which provides the raw material for the evolution of novel genes, is widespread in natural populations. We investigated whether CNVs constitute a common mechanism of genetic change during adaptation in experimental Caenorhabditis elegans populations. Outcrossing C. elegans populations with low fitness were evolved for >200 generations. The frequencies of CNVs in these populations were analyzed by oligonucleotide array comparative genome hybridization, quantitative PCR, PCR, DNA sequencing across breakpoints, and single-worm PCR.ResultsMultiple duplications and deletions rose to intermediate or high frequencies in independent populations. Several lines of evidence suggest that these changes were adaptive: (i) copy-number changes reached high frequency or were fixed in a short time, (ii) many independent populations harbored CNVs spanning the same genes, and (iii) larger average size of CNVs in adapting populations relative to spontaneous CNVs. The latter is expected if larger CNVs are more likely to encompass genes under selection for a change in gene dosage. Several convergent CNVs originated in populations descended from different low fitness ancestors as well as high fitness controls.ConclusionsWe show that gene copy-number changes are a common class of adaptive genetic change. Due to the high rates of origin of spontaneous duplications and deletions, copy-number changes containing the same genes arose readily in independent populations. Duplications that reached high frequencies in these adapting populations were significantly larger in span. Many convergent CNVs may be general adaptations to laboratory conditions. These results demonstrate the great potential borne by CNVs for evolutionary adaptation.

High-throughput fluorescence-based isolation of live C. elegans larvae

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.

Using C. elegans Forward and Reverse Genetics to Identify New Compounds with Anthelmintic Activity

Background The lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode Caenorhabditis elegans as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner. Methods/Principal findings Using our high throughput screening platform we have identified 14 new potential anthelmintics by screening more than 26,000 compounds from the Chembridge and Maybridge chemical libraries. Using phylogenetic profiling we identified a subset of the 14 compounds as potential anthelmintics based on the relative sensitivity of C. elegans when compared to yeast and mammalian cells in culture. We showed that a subset of these compounds might employ mechanisms distinct from currently used anthelmintics by testing diverse drug resistant strains of C. elegans. One of these newly identified compounds targets mitochondrial complex II, and we used structural analysis of the target to suggest how differential binding of this compound may account for its different effects in nematodes versus mammalian cells. Conclusions/Significance The challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. C. elegans as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides.

Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT).

Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.

MIP-MAP: High Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans. Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C. elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2. We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures.

Optimizing guide RNA selection and CRISPR/Cas9 methodology for efficient generation of deletions in C. elegans.

The Caenorhabditis elegans Gene Knockout (KO) Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately15,000 ORFs have associated putative null alleles. A directed approach using CRISPR/Cas9 methodology is the most promising technique to complete the task. While there has been substantial success in using CRISPR/Cas9 in C. elegans, there has been little emphasis on optimizing the method for generating large insertions/deletions in this organism. To enhance the efficiency of using CRISPR/Cas9 to generate gene knockouts in C. elegans we have developed an online species-specific guide RNA selection tool (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust, efficient and effective protocol for generating deletions. Debate and speculation in the larger scientific community about off- target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude this modified protocol does not generate off-target events to any significant degree in C. elegans.