Lobel Joseph H, Ingolia Nicholas T
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, 94720, USA.
Center for Computational Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, 94720, USA.
Genome Biol. 2025 May 25;26(1):142. doi: 10.1186/s13059-025-03610-w.
Genome-wide CRISPR-Cas9 screens have untangled regulatory networks driving diverse biological processes. Their success relies on interrogating specific molecular phenotypes and distinguishing key regulators from background effects. Here, we realize these goals by optimizing CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) to dramatically improve the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely matched promoters. We use our improved CiBER-seq to accurately capture known components of well-studied RNA and protein quality control systems. These results demonstrate the precision and versatility of CiBER-seq for dissecting cellular pathways.
全基因组CRISPR-Cas9筛选已解开驱动各种生物过程的调控网络。它们的成功依赖于对特定分子表型进行研究，并将关键调节因子与背景效应区分开来。在这里，我们通过优化带有条形码的表达报告基因测序的CRISPR干扰（CiBER-seq）来实现这些目标，从而显著提高全基因组筛选的灵敏度和范围。我们通过针对紧密匹配的启动子对表达报告基因进行标准化，系统地解决了扭曲表型测量的技术因素。我们使用改进后的CiBER-seq准确捕获了深入研究的RNA和蛋白质质量控制系统的已知成分。这些结果证明了CiBER-seq在剖析细胞通路方面的精确性和通用性。
