RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing system has been broadly adopted for high-throughput genetic screens. However, the application of genome-wide single guide RNA (sgRNA) libraries can be challenging. We generated a custom sgRNA library, an order of magnitude smaller than genome-wide alternatives, to facilitate the genetic screening of RNA binding proteins (RBPs). We demonstrated the utility of our reagent in a genetic screen for RBPs that conveyed cellular resistance or sensitivity to oxidative stress induced by paraquat. This identified that CSDE1 and STRAP, proteins that interact with each other, convey sensitivity to oxidative stress and that Pumilio homologues (PUM1 and PUM2) convey resistance. Targeting eIF4-E1 and -A1 protected cells from high-dose paraquat, whereas eIF4E2 targeted cells did less well. We also found that G3BP1 promoted sensitivity to a low dose of paraquat but protected cells at a higher dose. Our study highlights the use of genetic screens to identify roles of RBPs and identifies novel genes regulating sensitivity to oxidative stress.

Reduced gene templates for supervised analysis of scale-limited CRISPR-Cas9 fitness screens

“…Focused (or scale-limited) CRISPR-Cas9 knockout screens, employing single-guide RNA (sgRNA) libraries of relatively small size, represent a variation of the aforementioned systematic genome-wide approaches in which only a portion of genes are targeted: typically functionally related genes, components of the same biological pathway, or genes coding for druggable proteins (Birsoy et al, 2015;Roesch et al, 2018;Tarumoto et al, 2018;Girardi et al, 2020;S1abicki et al, 2020;Su et al, 2020;Wheeler et al, 2020;Williams et al, 2020;Zhang et al, 2020;Condon et al, 2021;Parrish et al, 2021;Turner and Turner, 2021;Zhu et al, 2021).…”

Time-resolved proteomic profiling reveals compositional and functional transitions across the stress granule life cycle

“…Much effort has been focused on understanding the functions and regulation of SGs, including the identification of their regulators and constituents. Genetic screens using siRNA- and CRISPR-Cas9-based approaches led to the identification of a large panel of SG regulators 5 , 35 – 38 . In addition, SG constituents have been profiled at proteomic and transcriptomic levels using either purified SGs 24 , 39 or through proximity labeling with SG proteins, such as G3BP1 40 , FMR1 41 , and eIF4A1 42 .…”

Reduced gene templates for supervised analysis of scale-limited CRISPR-Cas9 fitness screens

“…Focused (or scale-limited) CRISPR-Cas9 knockout screens, employing single-guide RNAs (sgRNAs) libraries of relatively small size, represent a variation of the aforementioned systematic genome-wide approaches in which only a portion of genes are targeted: typically functionally related genes, part of the same pathway or genes coding for druggable proteins (Birsoy et al, 2015;Condon et al, 2021;Girardi et al, 2020;Parrish et al, 2021;Roesch et al, 2018;Słabicki et al, 2020;Su et al, 2020;Tarumoto et al, 2018;Turner and Turner, 2021;Wheeler et al, 2020;Williams et al, 2020;Zhang et al, 2020;Zhu et al, 2021). These scaled-limited screens are commonly used to test narrower biological hypotheses and they require an initial pooled population of cells of significantly smaller size compared to genome-wide screens, in order to obtain an appropriate library representation and multiplicity of infection following transfection and selection (Doench, 2018;Gonçalves et al, 2021;Miles et al, 2016;Peets et al, 2019).…”

“…Focused (or scale-limited) CRISPR-Cas9 knockout screens, employing single-guide RNAs (sgRNAs) libraries of relatively small size, represent a variation of the aforementioned systematic genome-wide approaches in which only a portion of genes are targeted: typically functionally related genes, part of the same pathway or genes coding for druggable proteins (Birsoy et al, 2015; Condon et al, 2021; Girardi et al, 2020; Parrish et al, 2021; Roesch et al, 2018; Słabicki et al, 2020; Su et al, 2020; Tarumoto et al, 2018; Turner and Turner,2021; Wheeler et al, 2020; Williams et al, 2020; Zhang et al, 2020; Zhu et al, 2021).…”

“…fitness genes) at a genome-scale to support the discovery of new cancer dependencies and therapeutic targets (Behan et al 2019; Tzelepis et al 2016; Tsherniak et al 2017; Meyers et al 2017), while also widely improving our understanding of human biology (Shalem, Sanjana, and Zhang 2015; Zhou et al 2014). Focused (or scale-limited) CRISPR-Cas9 knockout screens, employing single-guide RNAs (sgRNAs) libraries of relatively small size, represent a variation of the aforementioned systematic genome-wide approaches in which only a portion of genes are targeted: typically functionally related genes, part of the same pathway or genes coding for druggable proteins (Parrish et al 2021; Slabicki et al 2020; Su et al 2020; Williams et al 2020; Roesch, OhAinle, and Emerman 2018; Birsoy et al 2015; Tarumoto et al 2018; Condon et al 2021; Girardi et al 2020; Zhang et al 2020; Zhu et al 2021; Wheeler et al 2020; Turner and Turner 2021).…”