Kim Jinyoung, Muller Ryan Y, Bondra Eliana R, Ingolia Nicholas T
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Center for Computational Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
bioRxiv. 2024 Sep 6:2024.09.06.611573. doi: 10.1101/2024.09.06.611573.
Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-κB) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy of CiBER-seq and its potential for dissecting genetic networks in mammalian cells with superior time resolution.
全基因组CRISPR筛选已成为揭示多种生物过程遗传基础的强大工具。精准筛选通常依赖于直接测量由CRISPR介导的基因扰动引发的分子表型，如调控的基因表达变化。在这里，我们通过将CRISPR干扰（CRISPRi）与条形码表达报告测序（CiBER-seq）相结合，对全基因组规模扰动文库中的人类细胞转录反应进行了定量测量。为了在哺乳动物细胞中实现CiBER-seq，我们优化了将高度复杂的条形码sgRNA文库整合到特定基因组背景中的方法。对核因子κB（NF-κB）报告基因进行CiBER-seq分析，描绘了将跨膜TNF-α受体与炎症基因激活联系起来的经典信号级联反应，并突出了该反应中细胞类型特异性因子。重要的是，CiBER-seq仅依靠大量RNA测序来捕获驱动这种快速转录反应的调控回路。我们的工作证明了CiBER-seq的准确性及其在以卓越的时间分辨率剖析哺乳动物细胞遗传网络方面的潜力。
