Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

Abstract

Pooled CRISPR screens based on lentiviral systems have been widely applied to identify the effect of gene knockout on cellular phenotype. Although many screens were successful, they also have the limitation that genes conferring mild phenotypes or those essential for growth can be overlooked, as every genetic perturbation is incorporated in the same population. Arrayed screens, on the other hand, incorporate a single genetic perturbation in each well and could overcome these limitations. However, arrayed screens based on siRNA-mediated knockdown were recently criticized for low reproducibility caused by incomplete inhibition of gene expression. To overcome these limitations, we developed a novel arrayed CRISPR screen based on a plasmid library expressing a single guide RNA (sgRNA) and disrupted 1514 genes, encoding kinases, proteins related to endocytosis, and Golgi-localized proteins, individually using 4542 sgRNAs (three sgRNAs per gene). This screen revealed host factors required for infection by coxsackievirus B3 (CVB3) from Picornaviridae, which includes human pathogens causing diverse diseases. Many host factors that had been overlooked in a conventional pooled screen were identified for CVB3 infection, including entry-related factors, translational initiation factors, and several replication factors with different functions, demonstrating the advantage of the arrayed screen. This screen was quite reliable and reproducible, as most genes identified in the primary screen were confirmed in secondary screens. Moreover, ACBD3, whose phenotype was not affected by siRNA-mediated knockdown, was reliably identified. We propose that arrayed CRISPR screens based on sgRNA plasmid libraries are powerful tools for arrayed genetic screening and applicable to larger-scale screens.

ACBD3 is an essential pan-enterovirus host factor that mediates the interaction between viral 3A protein and cellular protein PI4KB

“…3) elicited PI4KB recruitment in the absence of ACBD3. In agreement with our data, the inhibition of EV-A71 and CVB3 was recently reported in ACBD3 KO cells (30–32). The discrepancy in the role of ACBD3 from KD to KO condition could result from insufficient suppression of ACBD3 function by RNA interference.…”

System-Based Approaches to Delineate the Antiviral Innate Immune Landscape

“…Thus, depending on the experimental design, making the identification of weak phenotypes achievable. This characteristic was demonstrated by Kim et al [ 128 ]. In this study, the authors conducted a direct comparison between a pooled and an arrayed loss-of-function screen using the CRISPR/Cas9 system.…”

A benchmark of algorithms for the analysis of pooled CRISPR screens

“…Arrayed designs can separate individual guides into different pools in the array. Thus, they obtain direct estimates of the genetic effects but are limited in their throughput (hundreds to a few thousands of genes, e.g., [9,10]) and require high-throughput analysis (e.g., imagebased) to measure the association with the desired phenotype [11]. This is an extremely difficult problem, as a multitude of batch effects and extrinsic noise can complicate the analysis (e.g., [12]).…”