Calarco JA, Norris AD
Bio Protoc 2018 Mar 05; 8(5):
Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and (Schuldiner ., 2005; Butland ., 2008; Costanzo ., 2010), but similar large-scale interaction studies using targeted reverse-genetic deletions in multi-cellular organisms have not been feasible. We developed a CRISPR/Cas9-based method for deleting genes in and replacing them with a heterologous fluorescent reporter (Norris ., 2015). Recently we took advantage of that system to perform a large-scale, reverse genetic screen using null alleles in animals for the first time, focusing on RNA binding protein genes (Norris ., 2017). This type of approach should be similarly applicable to many other gene classes in . Here we detail the protocols involved in generating a library of double mutants and performing medium-throughput competitive fitness assays to test for genetic interactions resulting in fitness changes.
Gracida X, Dion MF, Harris G, Zhang Y, Calarco JA
Cell Rep 2017 Dec 12; 21(11):3089-3101
Neuromodulatory cells transduce environmental information into long-lasting behavioral responses. However, the mechanisms governing how neuronal cells influence behavioral plasticity are difficult to characterize. Here, we adapted the translating ribosome affinity purification (TRAP) approach in C. elegans to profile ribosome-associated mRNAs from three major tissues and the neuromodulatory dopaminergic and serotonergic cells. We identified elc-2, an Elongin C ortholog, specifically expressed in stress-sensing amphid neuron dual ciliated sensory ending (ADF) serotonergic sensory neurons, and we found that it plays a role in mediating a long-lasting change in serotonin-dependent feeding behavior induced by heat stress. We demonstrate that ELC-2 and the von Hippel-Lindau protein VHL-1, components of an Elongin-Cullin-SOCS box (ECS) E3 ubiquitin ligase, modulate this behavior after experiencing stress. Also, heat stress induces a transient redistribution of ELC-2, becoming more nuclearly enriched. Together, our results demonstrate dynamic regulation of an E3 ligase and a role for an ECS complex in neuromodulation and control of lasting behavioral states.
Norris AD, Gracida X, Calarco JA
Elife 2017 07 18; 6():
Genetic interaction screens have aided our understanding of complex genetic traits, diseases, and biological pathways. However, approaches for synthetic genetic analysis with null-alleles in metazoans have not been feasible. Here, we present a CRISPR/Cas9-based Synthetic Genetic Interaction (CRISPR-SGI) approach enabling systematic double-mutant generation. Applying this technique in , we comprehensively screened interactions within a set of 14 conserved RNA binding protein genes, generating all possible single and double mutants. Many double mutants displayed fitness defects, revealing synthetic interactions. For one interaction between the MBNL1/2 ortholog and the ELAVL ortholog , double mutants displayed a severely shortened lifespan. Both genes are required for regulating hundreds of transcripts and isoforms, and both may play a critical role in lifespan extension through insulin signaling. Thus, CRISPR-SGI reveals a rich genetic interaction landscape between RNA binding proteins in maintaining organismal health, and will serve as a paradigm applicable to other biological questions.