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.