The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.

The Axin family of scaffolding proteins regulates a wide array of developmental and post-developmental processes in eukaryotes. Studies in the nematode Caenorhabditis elegans have shown that the Axin homolog PRY-1 plays essential roles in multiple tissues. To understand the genetic network of pry-1, we focused on a set of genes that are differentially expressed in the pry-1-mutant transcriptome and are linked to reproductive structure development. Knocking down eight of the genes (spp-1, clsp-1, ard-1, rpn-7, cpz-1, his-7, cdk-1, and rnr-1) via RNA interference efficiently suppressed the multivulva phenotype of pry-1 mutants. In all cases, the ectopic induction of P3.p vulval precursor cell was also inhibited. The suppressor genes are members of known gene families in eukaryotes and perform essential functions. Our genetic interaction experiments revealed that in addition to their role in vulval development, these genes participate in one or more pry-1-mediated biological events. Whereas four of them (cpz-1, his-7, cdk-1, and rnr-1) function in both stress response and aging, two (spp-1 and ard-1) are specific to stress response. Altogether, these findings demonstrate the important role of pry-1 suppressors in regulating developmental and post-developmental processes in C. elegans. Given that the genes described in this study are conserved, future investigations of their interactions with Axin and their functional specificity promises to uncover the genetic network of Axin in metazoans.

The Axin family of scaffolding proteins regulates a wide array of developmental and post-developmental processes in eukaryotes. Studies in the nematode, Caenorhabditis elegans, have shown that the Axin homolog, PRY-1, plays essential roles in multiple tissues. To understand the genetic network of pry-1, we focused on a set of genes that are differentially expressed in the pry-1-mutant transcriptome and are linked to reproductive structure development. Eight of the genes (ard-1, rpn-7, cpz-1, his-7, cdk-1, rnr-1, clsp-1, and spp-1), when knocked down by RNA interference, efficiently suppressed the plate-level multivulva phenotype of pry-1 mutants. In every case, other than clsp-1 and spp-1, the ectopic vulval precursor cell (VPC) induction was also inhibited. The suppressor genes are members of known gene families in eukaryotes and perform essential functions. Our genetic interaction experiments revealed that except for clsp-1, the genes participate in one or more pry-1-mediated biological events. While four of them (cpz-1, his-7, cdk-1, and rnr-1) function in VPC induction, stress response, and aging, the other three (spp-1, ard-1, and rpn-7) are specific to one or more of these processes. Further analysis of the genes involved in aging showed that his-7, cdk-1, and rnr-1 also interacted with daf-16/FOXO. The results of genetic epistasis experiments suggested that his-7 functions upstream of daf-16, whereas cdk-1and rnr-1 act downstream of the pry-1-daf-16 pathway. Altogether, these findings demonstrate the important role of pry-1 suppressors in C. elegans. Given that all of the genes described in this study are conserved, future investigations of their interactions with Axin and their functional specificity promises to uncover the genetic network of Axin under normal and disease states.

The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying the 5’ ends of expressed genes, we have discovered operons and paralogs. In summary, our analysis yielded 10,243 unique 5’ end sequence tags with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. These genes included 14 that are exclusively trans-spliced in C. briggsae when compared with C. elegans orthologs. A major contribution of this study is the identification of 492 high confidence operons, of which two-thirds are fully supported by tags. In addition, two SL1-type operons were discovered. Interestingly, comparisons with C. elegans showed that only 40% of operons are conserved. Of the remaining operons, 73 are novel, including 12 that entirely lack orthologs in C. elegans. Further analysis revealed that four of the 12 novel operons are conserved in C. nigoni. Altogether, the work described here has significantly advanced our understanding of the C. briggsae system and serves as a rich resource to aid biological studies involving this species.

The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying 5' ends of expressed genes, the technique has enabled the discovery of operons and paralogs. Application of TEC-RED yielded 10,243 unique 5' end sequences with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. The method also identified 493 operons, including 334 that are fully supported by tags. Additionally, two SL1-type operons were discovered. Comparisons with C. elegans revealed that 40% of operons are conserved. Further, we identified 73 novel operons, including 12 that entirely lack orthologs in C. elegans. Among other results, we found that 14 genes are trans-spliced exclusively in C. briggsae compared with C. elegans. Altogether, the data presented here serves as a rich resource to aid biological studies involving C. briggsae. Additionally, this work demonstrates the use of TEC-RED for the first time in a non-elegans nematode and suggests that it could apply to other organisms with a trans-splicing reaction from spliced leader RNA.