CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

To realize the promise of CRISPR-Cas9–based genetics, approaches are needed to quantify a specific, molecular phenotype across genome-wide libraries of genetic perturbations. We addressed this challenge by profiling transcriptional, translational, and posttranslational reporters using CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). Our barcoding approach allowed us to connect an entire library of guides to their individual phenotypic consequences using pooled sequencing. CiBER-seq profiling fully recapitulated the integrated stress response (ISR) pathway in yeast. Genetic perturbations causing uncharged transfer RNA (tRNA) accumulation activated ISR reporter transcription. Notably, tRNA insufficiency also activated the reporter, independent of the uncharged tRNA sensor. By uncovering alternate triggers for ISR activation, we illustrate how precise, comprehensive CiBER-seq profiling provides a powerful and broadly applicable tool for dissecting genetic networks.