Development of a Positive Selection High Throughput Genetic Screen in Dictyostelium discoideum

Abstract

The cellular slime mold Dictyostelium discoideum is a powerful model organism that can be utilized to investigate human health and disease. One particular strength of Dictyostelium is that it can be utilized for high throughput genetic screens. For many phenotypes, one limitation of utilizing Dictyostelium is that screening can be an arduous and time-consuming process, limiting the genomic depth one can cover. Previously, we utilized a restriction enzyme-mediated integration screen to identify suppressors of polyglutamine aggregation in Dictyostelium. However, due to the time required to perform the screen, we only obtained ∼4% genome coverage. Here we have developed an efficient screening pipeline that couples chemical mutagenesis with the 5-fluoroorotic acid counterselection system to enrich for mutations in genes of interest. Here we describe this new screening methodology and highlight how it can be utilized for other biological systems.

CRISPR/Cas9-based genome-wide screening of Dictyostelium

“…Moreover, tenfold more plates must be prepared to analyse tenfold more mutants; hence, it is important to improve the efficiency of selection methods. For selection of REMI and NTG-treated mutants in Dictyostelium , various powerful strategies were applied, including adhesion selection on 10-cm plates, electrotaxis experiments with barcoded microplates, high-throughput growth and development assays that affect lithium and valproic acid, and counterselection with 5-fluoroorotic acid to enrich genes that suppress polyglutamine aggregation 28 , 54 – 56 . Because these techniques allow for the screening of numerous cells, they are easily adaptable to CRISPR mutant pools with millions of mutants.…”

“…The major considerations to genetic screening are saturation of mutation in the mutant pool and ease of identifying mutation sites. It has been indicated that whole-genome sequencing of multiple strains is not cost effective approach and that loss-of-function mutants identified via REMI rarely result in gain-of-function mutations in genes that suppress the original phenotype 26 – 28 . Thus, the use of a variety of approaches is advantageous for the genetic screening of Dictyostelium .…”

Dictyostelium discoideum as a Model for Investigating Neurodegenerative Diseases

“…One caveat of this screen was that it did not approach genome-wide coverage and additional suppressors of polyQ aggregation in Dictyostelium likely exist. The development of novel screening pipelines will enable genome-wide coverage in future screens, fully elucidating the genes that are essential for suppressing polyQ aggregation in Dictyostelium (Williams et al, 2021).…”