PgmNr W408: CRISPR-mediated synthetic genetic analysis reveals genetic interactions among RNA binding proteins affecting fitness and lifespan.

Authors:
Adam Norris; Xicotencatl Gracida; John Calarco


Institutes
Harvard University, Cambridge, MA.


Keyword: Other ( Post-transcriptional regulation )

Abstract:

Experiments in unicellular organisms such as yeast have revealed widespread synthetic genetic interactions between genes and demonstrated that systematic analysis of genetic interactions can be utilized to assign shared biological functions to genes. As such, the ability to broadly survey genetic interactions with knockout alleles in metazoans would be highly desirable. We recently developed such a system in C. elegans to perform CRISPR/Cas9-based synthetic genetic analysis (CRISPR-SGA) using null mutations in a multicellular organism for the first time. Homologous recombination-mediated repair of double-strand breaks triggered by CRISPR/Cas9 replaces a target gene with one of two heterologous GFP transgenes, allowing reporter fluorescence to be followed as a proxy for the gene deletion. This enables generation of large numbers of double mutants by simply crossing the compatible single mutant animals and identifying double mutants by fluorescence microscopy. We applied CRISPR-SGA to genetic interactions between RNA binding proteins. RNA binding proteins are critical mediators of post-transcriptional regulation, but the majority of metazoan RNA binding proteins have no known molecular function or phenotype. Moreover, RNA binding proteins often act combinatorially to control post-transcriptional regulation, suggesting that single RNA binding protein mutations may lead to subtle or no phenotypes due to genetic redundancy. We deleted 11 non-essential evolutionarily-conserved RNA binding proteins and generated all 55 possible double mutants. Mutants were tested with a quantitative assay that measures competitive fitness compared to wild-type over multiple generations. We identified a number of double mutants with substantially lower fitness than would be predicted based on the additive defects of the two single mutant fitness scores. Follow-up on one such synthetic fitness defect revealed severe lifespan reduction in exc-7;mbl-1 strains, though neither single mutant has a substantial effect on lifespan. The defect appears to be a specific aging phenotype, because developmental viability is not affected and additional markers of organismal health do not begin to decline until adulthood. Current follow-up experiments include RNA-Seq on the mutants as they age to shed light on the targets of the two RNA binding proteins, and tissue-specific rescue to determine the relevant tissue for the phenotype. These experiments demonstrate a substantial degree of synthetic fitness defects among RNA binding proteins and reveal pairs of genes important for maintaining organismal physiology. Moreover, our CRISPR-SGA system should be broadly applicable to anyone interested in metazoan synthetic genetics.