Synthetic Genetic Interaction (CRISPR-SGI) Profiling in Caenorhabditis elegans.

John Calarco,Adam Norris

doi:10.21769/bioprotoc.2756

John Calarco, Adam Norris

Open Access

https://doi.org/10.21769/bioprotoc.2756

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Abstract

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 E. coli (Schuldiner et al., 2005; Butland et al., 2008; Costanzo et al., 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 C. elegans and replacing them with a heterologous fluorescent reporter (Norris et al., 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 et al., 2017). This type of approach should be similarly applicable to many other gene classes in C. elegans. 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.

Highlights

[Background] Large-scale genetic interaction screens using reverse-genetic null alleles have not previously been feasible in animals
We developed a method for efficient editing of the C. elegans genome (Norris et al, 2015) and recently expanded upon that method to enable large-scale genetic interaction profiling in animals using null alleles for the first time
We focused our initial efforts on neuronally-expressed RNA binding protein genes, which have been shown in a number of cases to act combinatorially (Gracida et al, 2016; Norris et al, 2014)

Summary

Introduction

[Background] Large-scale genetic interaction screens using reverse-genetic null alleles have not previously been feasible in animals. RNAi has been used to study genetic interactions in C. elegans by knocking down expression of a large number of different genes in the presence of a single mutant background (Baugh et al, 2005; Lehner et al, 2006). 1. Platinum Wire for worm pick, 30 gauge 0.254 mm diameter (e.g., Genesee Scientific, catalog number: 59-30P6)

Results

Conclusion