Au, Vinci et al. (2021) International Worm Meeting "Crispr-ing C. elegans genes conserved to humans"

Au, Vinci, Moerman, Donald, Rougvie, Ann, Edgley, Mark, Doell, Claudia, Vargas, Marcus, Palma, Wilber, Hutter, Harald, Park, Heenam, Knott, Julie, Sternberg, Paul, Fernando, Lisa, Juciute, Viktorija, Li-Leger, Erica, Flibotte, Stephane, Martin, Kiana

[International Worm Meeting, 2021]

We are continuing our community resource project to generate gene deletions of high value to those interested in human biology and disease. Our highly coordinated three-site knockout (KO) production strategy has branches in California, Minnesota, and British Columbia, with the Canadian branch transitioning from the University of British Columbia to Simon Fraser University upon the retirement of KO legend Don Moerman. We employ a dual-pipeline strategy to generate KOs, with the Minnesota and Canadian sites using a scheme based on the Calarco Lab's dual selection cassette method. It deletes all or most of the target gene, effectively eliminating function. In addition, it replaces the gene with a fluorescent reporter, allowing simple tracking of the allele in cases where the mutation results in homozygous lethality or sterility, or fails to cause a discernible phenotype, facilitating genetic manipulation of the alleles. Alternatively, the California site developed and uses a "STOP-IN" cassette that places stop codons in all three reading frames near the beginning of the coding region, eliminating activity. This method does not allow visual tracking of alleles but has the advantage in that it inserts a unique guide RNA recognition site, enabling reversion of the locus to wild-type allowing the phenotype to be reassessed. This feature is desired by some users, for example those performing metabolomics experiments requiring quantitation of phenotypes that are sensitive to strain background effects. Each gene edit is carefully confirmed, and validated strains are grossly phenotyped and promptly deposited into the Caenorhabditis Genetics Center (CGC) strain collection along with detailed strain information. To date, 1,040 of our KOs are available through the CGC. Going forward, we plan to target an additional 2500 C. elegans orthologs of human genes, prioritizing known or suspected human disease genes, druggable gene classes, as well as understudied genes that are conserved to humans but that have no actionable information. Finally, we will consider community requests to prioritize specific genes.

Au, Vinci et al. (2017) International Worm Meeting "Applying CRISPR-Cas9 Technology and Whole Genome/Amplicon Sequencing to the C. elegans Knockout Project."

Moerman, Donald G., Flibotte, Stephane, Li-Leger, Erica, Edgley, Mark L., Chaudhry, Iasha, Au, Vinci, Wang, Su, Fernando, Lisa

[International Worm Meeting, 2017]

The C. elegans Gene Knockout Facility generates null mutations in C. elegans for distribution worldwide, with the goal of obtaining knockouts for every Open Reading Frame (ORF) in the genome. To date, 67% of the ORFs in the genome (13,559 ORFs) are represented by null mutations. Our immediate priority is to complete knockouts for all members of important gene families such as transcription factors, kinases, phosphatases, and GTPases. We are also attempting to generate deletions of homologous gene clusters. These tandem gene knockouts may provide insight into the function of a particular group of related genes when a single gene knockout has no detectable phenotype. We are currently using two different methods to achieve our goals: CRISPR-Cas9 genome editing, and whole genome (WGS) and amplicon sequencing of EMS/ENU-mutagenized worm populations. The CRISPR-Cas9 method allows targeting of specific genes, while WGS/amplicon sequencing, albeit less specific, is quicker and more cost effective. The CRISPR-Cas9 system allows us to simultaneously make targeted deletions and insert selectable markers to track mutations. This method is applicable both for single gene knockouts and for tandem gene knockouts. The amplicon sequencing approach uses populations of mutagenized worms that are harvested at the F3 generation. We do WGS on half of each population to identify SNVs of interest. The other half of the population is singled and used for isolation of the mutation, using PCR amplicons that are barcoded, pooled, and sequenced on an Illumina sequencer. This identifies single sub-populations that are homozygous or heterozygous for the SNV of interest. We have generated 308 mutagenized F3 isolates, and have performed WGS on 73 of these populations. Mutations in 451 genes with no previously known null alleles have been identified in this way. To date, we have successfully isolated new knockouts in 19 genes using the amplicon sequencing method, and 42 genes using the CRISPR-Cas9 approach. Finished knockouts are sent to the Caenorhabditis Genetics Center at the University of Minnesota, from which strains are distributed to labs around the world.

Edgley, Mark et al. (2015) International Worm Meeting "Comprehensive Biology: How do we complete the C. elegans Knockout Project."

Moerman, Donald, Suehiro, Yuji, Motohashi, Tomoko, Taylor, Jon, Au, Vinci, Iwata, Satoru, Miller, Angela, Hori, Sayaka, Flibotte, Stephane, Yoshina, Sawako, Fernando, Lisa, Mitani, Shohei, Dejima, Katsufumi, Raymant, Greta, Edgley, Mark

[International Worm Meeting, 2015]

The primary objective of this project is to provide the research community with severe loss-of-function mutations of all C. elegans genes. To date we, and the community, have identified such mutations for over14,000 of the 20,000 genes in the worm (G3 2012 2:1415-25; Genome Res. 2013 23:1749-63). Strains harboring these mutations are available through the Caenorhabditis Genetics Center and all data pertaining to the mutations is available at WormBase.We are using three strategies to generate and isolate mutations in the remaining 6,000 genes. In the first strategy we will continue to isolate deletion mutants using targeted PCR after UV/TMP mutagenesis. This has been a major method for both nodes to identify mutations in non-essential and essential genes until recently, but will now be performed only at the Tokyo node.In the second strategy we are using random mutagenesis coupled to Whole Genome Sequencing (WGS) to identify new mutations, focusing on mutations that occur in the remaining 6,000 genes that do not have null alleles (Vancouver node). This strategy will allow us to identify mutations in both non-essential and essential genes and to recover the mutant animals. Using WGS we have recently identified nonsense and splicing defects in over 2,000 genes, of which about 500 are among the remaining 6,000. We are currently working to isolate each of these mutations as homozygous lines, or as balanced recessive lethal strains. [Also see poster from the Mitani group on UV/TMP/WGS to identify deletions].In the third strategy we are using CRISPR/Cas9 technology to target specific genes as a complement to the random approach described above. We are particularly interested in using this method to target the remaining members of large multi-gene families, for example kinases and transcription factors. We will also use it to expedite community requests if we do not already have hits through the deletion and WGS projects.

Thompson, Owen et al. (2013) International Worm Meeting "The Million Mutation Project and Beyond."

Strasbourger, Pnina, Adair, Ryan, Ewing, Brent, Turner, Emily, Chaudhry, Iasha, Hillier, LaDeana, Moerman, Donald G., Fernando, Lisa, Shendure, Jay, Shen, Bin, Au, Vinci, Hutter, Harald, Lau, Joanne, Miller, Angela, Thompson, Owen, Edgley, Mark, Waterston, Robert H., Taylor, Jon, Raymant, Greta, Flibotte, Stephane

[International Worm Meeting, 2013]

We have created a library of 2,007 mutagenized C. elegans strains, each sequenced to a target depth of 15X coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 820,000 unique SNVs with an average of eight non-synonymous changes per gene and more than 16,000 indel and copy number changes, providing an unprecedented genetic resource for C. elegans. We supplemented this collection with 40 sequenced wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets shows that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. Comparison with expected events suggests that very few missense alleles are incompatible with life but more than 40% are subject to long term selection. All the strains are available through the CGC; all the sequence changes have been deposited in WormBase and our own interactive web site (http://genome.sfu.ca/mmp/about.html). Loss-of-function alleles are now available for 13,760 of 20,514 protein coding genes. To identify mutations in the remaining genes, including essential genes, we use the following strategy. F1 progeny of mutagenized P0s are minimally propagated before sequencing and analysis. We use gain or loss of restriction sites to facilitate following the mutations through subsequent steps to isolate homozygous viable animals, or to create heterozygous balanced lethals. Pilot experiments have demonstrated the feasibility of this approach; we have identified new nonsense alleles of known essential genes, as well as nonsense and splicing mutations in several genes with no previous null mutations.

Konno, Hiroyuki et al. (2015) International Worm Meeting "Mechanism and significance of the maternal mex-3 mRNA localization in C. elegans."

Kohara, Yuji, Noguchi, Koki, Konno, Hiroyuki

[International Worm Meeting, 2015]

In C. elegans, several maternal mRNA localizations are observed, however, the mechanism and significance are not clear. Here, we studied anterior mRNA localization of maternal mex-3 mRNA. mex-3 mRNA localizes to anterior half of embryo from the late 1-cell stage to the 4-cell stage. Localization pattern of MEX-3 protein is similar to that of mex-3 mRNA, raising the hypothesis that mex-3 mRNA localization contributes to MEX-3 protein localization. We identified a CCCH-type zinc finger protein MEX-5 as a trans-acting element. Transgene assay revealed that 54-nucleotide sequence on mex-3 3'-untranslated region is the cis-acting element for the mRNA localization. This sequence is AU-rich and fulfills the feature of the MEX-5 binding sites. We made mex-3 mutant which lacks the cis-acting element by genome editing technology. Interestingly, the localization of not only mex-3 mRNA but also MEX-3 protein were not observed in this mutant. We are going to discuss the significance of mex-3 mRNA localization.

Gogonea CB et al. (1997) International C. elegans Meeting "EXPRESSION OF THE REPORTER GENE MEC-12::LACZ IN LOCOMOTORY AND CHEMOSENSORY MUTANTS OF C. ELEGANS"

Siddiqui ZK, Siddiqui SS, Gogonea CB

[International C. elegans Meeting, 1997]

Mutations in close to 100 genes affect locomotion. These mutants show uncoordinated locomotion, ranging from slow movement, poor backing, colier, kinkier, and paralyzed phenes (J. Hodgkin, 1997: Appendix in C. elegans II). Previously it was shown that mutants in at least 25 genes harbor defects in the axonal growth and process placement of the six touch receptor neurons (Siddiqui, 1990). A reporter gene mec-12::lacZ has been shown to express in the set of six touch receptor neurons (Fukushige et al., 1997). So far, a variety of cellular defects in the process placement, axonal guidance and axonal growth of the touch cells and the PVR in 15 genes (unc-4, unc-5,unc-30, unc-33, unc-44, unc-51, unc-53, unc-55, unc-61, unc-71, unc-76, unc-83, unc-86, and unc-119) have been observed. Defects include ectopic branching of axons, fore- shortened processes, misplaced cell body positions, extra commissures, and a lack of staining. In addition, we have examined the reporter gene expression in the background of various mechano- sensory mutations (Chalfie and Au, 1989). These results will be summarized. We thank the Caenorhabdtis Genetics Center, and Martin Chalfie for various mutant strains.

Yang, Zhe et al. (2015) International Worm Meeting "A C. elegans model for Human Antigen R (HuR)."

Yang, Zhe, Buechner, Matthew

[International Worm Meeting, 2015]

Human antigen (Hu) proteins HuB, HuC, HuD, and HuR are a family of RNA-binding protein that stabilize mRNA by binding to AU-rich elements (ARE) in the 3'UTR. HuR is universally expressed in human cells, and stabilizes many tumor-related genes such as bcl-2 and XIAP. HuR is upregulated in many cancer cell types involved in breast and colon cancer. EXC-7 is the sole homologue of the Hu proteins in C. elegans. EXC-7 maintains the single-cell tubular canals of the excretory cell by stabilizing sma-1 mRNA, encoding betaH-spectrin. Mutants in exc-7 exhibit small fluid-filled cysts throughout shortened excretory canals, as well as a misshapen tailspike. Both EXC-7 and HuR contain 3 RNA-recognition motifs (RRMs), with a region between the 2nd and 3rd RRM that appears to be needed for intracellular transport. We are investigating the functional equivalence of these proteins, by determining whether human HuR can functionally replace EXC-7 in C. elegans. If so, then C. elegans could be developed as a model for studying HuR in vivo, especially for screens for chemicals that interfere with HuR function, using excretory canal phenotype as a measure of HuR activity.We have injected the hur coding region driven by various promoters to express HuR within the excretory canals. HuR appears to affect both the length and diameter of the excretory canal lumen. We plan to attach GFP to HuR to visualize the position and movement of this protein within the long excretory canals, and to make worm/human chimeric proteins to investigate domain activity of these proteins.

Lundquist EA et al. (1995) International C. elegans Meeting "mec-8 ENCODES A PUTATIVE RNA-BINDING PROTEIN AND AFFECTS SPLICING OF unc-52 TRANSCRIPTS"

Rogalski TM, Shaw JE, Moerman DG, Herman RK, Lundquist EA

[International C. elegans Meeting, 1995]

mec-8 mutants are defective in mechanosensation (Chalfie and Au, 1989), dye-filling of amphid and phasmid neurons (Perkins et al., 1986) and attachment of body wall muscle to hypodermis (Lundquist and Herman, 1994). The DNA sequence of the mec-8 gene indicates that it can encode a protein containing two copies of an 80-amino acid RNA recognition motif. We propose that MEC-8 is a trans-acting pre-mRNA processing factor. mec-8 mutations enhance viable unc-52 mutations: the mec-8; unc-52(viable) double mutant resembles the unc-52 null mutant, which is paralyzed and arrested at the twofold stage of elongation, with severe body wall muscle defects (Williams et al. 1994). unc-52 produces many alternatively spliced transcripts that encode proteoglycans found in the basement membrane adjacent to muscle cells (Rogalski et al., 1993). We have used RT-PCR to assay the patterns of alternative splicing of unc-52 transcripts. The concentration of a subset of splice products is clearly reduced in mec-8 animals. We propose that the muscle defects of mec-8 animals and the synthetic lethality of mec-8; unc-52(viable) animals are the result of defects in the generation of certain unc-52 alternatively-spliced transcripts. We have obtained evidence that mec-8 also regulates the processing of its own pre-mRNAs. We propose that MEC-8 controls the splicing of transcripts of other loci that are involved in mechanosensory and chemosensory neuron function. Finally, we suggest that two loci identified by mutations that suppress mec-8 defects, smu-1 and smu-2, may also be involved in pre-mRNA processing.

Kuersten S et al. (1995) International C. elegans Meeting "IN VITRO PROCESSING OF MONO- AND POLYCISTRONIC TRANSCRIPTS IN A C. ELEGANS WHOLE CELL EXTRACT."

Kuersten S, Blumenthal TE

[International C. elegans Meeting, 1995]

Pre-mRNA splicing in C. elegans occurs in three distinct, but related, forms: Cis-splicing (intron removal) and SL1 or SL2 trans-splicing. In trans-splicing, 22 nt leaders are spliced onto the 5' ends of mRNAs. SL1 trans-splicing is specified by an AU-rich intron-like sequence (an "outron") followed by a functional 3' splice site, present at the 5' end of the pre-mRNA. In contrast, SL2 trans-splicing occurs at 3' splice sites present between genes in polycistronic transcription units. In order to determine the signals that mediate trans-splicing specificity, we have developed a whole cell embryo extract capable of at least partially reproducing these processing events in vitro. This extract is capable of performing at least four distinct pre-mRNA processing events: Cis-splicing, SL1 trans-splicing, SL2 trans-splicing and cleavage/polyadenylation. However, the extract is relatively inefficient; RT-PCR is required to detect spliced products. When the extract is incubated with a transcript derived from the rol-6 gene, which is normally trans-spliced to SL1, a trans-spliced product is observed. The presence of this product is ATP-dependent and the transcript is spliced to SL1. In addition, an intron is also removed but more ATP appears to be required for cis-splicing. Both capped and uncapped rol-6 RNAs are spliced to SL1 in the extract, indicating that, at least in vitro, the presence of a 5' cap on the pre-mRNA isn't required for SL1 trans-splicing. Processing of a polycistronic transcript also occursi n vitro, including both cleavage/polyadenylation and trans-splicing at the correct internal site between genes. The downstream mRNA is spliced to both SL2 and SL1, in about equal proportions, even though this mRNA receives exclusively SL2 in vivo. Thus, the in vitro system only partially recapitulates the in vivo specificity. This in vitro system should allow us to dissect the signals that specify SL2 trans-splicing.

Lawry, S.Terese et al. (2019) International Worm Meeting "Using the Million Mutation Project strains to obtain a range of touch insensitive mutants."

Chalfie, Martin, Walker, Matt, Lawry, S.Terese

[International Worm Meeting, 2019]

The Million Mutation Project (MMP; Thomson et al., Genome Res. 23: 1749-1762, 2013) has become an outstanding resource for mutations in many C. elegans genes. More recently Timbers et al. (PLoS Genet. 12: e1006235, 2016) used a subset of MMP strains to identify genes that could be mutated to give a dye-filling (Dyf) phenotype. Because most of the mutations in the MMP strains are homozygous, we were intrigued about the possibility that the MMP strains could be used to identify mutations having subtle phenotypic effects. We also wanted to assess how complete a screen utilizing the MMP strains would be. To this end we have undertaken a screen of the MMP strains to identify those with extreme to slight defects in the gentle touch response. Classical forward genetic screens (Chalfie and Sulston, 1981; Chalfie and Au, 1989) identified 18 genes necessary for gentle touch sensitivity, although others have also been found (e.g., among genes whose null phenotype is lethality). We have currently screened about 1700 of the 2007 MMP strains. 111 of these strains have an abnormal gentle touch response. 73 of these strains have mutations affecting the protein sequence, UTRs, or splice sites of genes previously known to mutate to touch insensitivity. In particular, we identified strains with mutations in all but one gene (mec-17) found in our initial forward mutageneses. mec-17 is extremely difficult to mutagenize to touch insensitivity; all four mutations in MMP strains failed to make the animals touch insensitive. Thus, the MMP collection, at least for viable phenotypes, appears to be essentially complete. Using MMP defects in the known touch genes that did and did not produce touch sensitivity, we were able to look for important domains within the encoded proteins. We are now identifying causative mutations in the remaining 38 strains, starting with the strains that exhibit a more extreme phenotypic defect. Because the MMP strains are sequenced, identifying the causative mutation should be relatively quick using genetic mapping. We are conducting several other screens using this resource and strongly recommend it to our colleagues.