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.

中文翻译：

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通过靶向 CRISPR-Cas9 单向导 RNA 文库识别作为氧化应激调节剂的 RNA 结合蛋白

成簇的规则间隔短回文重复序列 (CRISPR)-Cas9 基因组编辑系统已被广泛用于高通量遗传筛选。然而，全基因组单向导 RNA (sgRNA) 文库的应用可能具有挑战性。我们生成了一个定制的 sgRNA 文库，比全基因组的替代方案小一个数量级，以促进 RNA 结合蛋白 (RBP) 的遗传筛选。我们展示了我们的试剂在 RBP 遗传筛选中的效用，该筛选传递了细胞对百草枯诱导的氧化应激的抵抗力或敏感性。这确定了 CSDE1 和 STRAP，相互相互作用的蛋白质，传达了对氧化应激的敏感性，而 Pumilio 同源物（PUM1 和 PUM2）传达了抗性。靶向 eIF4-E1 和 -A1 可保护细胞免受高剂量百草枯的侵害，而 eIF4E2 靶向细胞的效果较差。我们还发现 G3BP1 提高了对低剂量百草枯的敏感性，但在较高剂量下保护细胞。我们的研究强调了使用遗传筛选来确定 RBP 的作用并确定调节氧化应激敏感性的新基因。