ABSTRACT The RNA-guided DNA recognition of CRISPR/Cas9 now enables comprehensive genetic screening in mammalian cells and other previously intractable systems. CRISPR/Cas9 provides highly programmable genetic perturbation through DNA cleavage, transcriptional inhibition, or targeted mutagenesis. Measuring the phenotypes associated with these genetic perturbations remains a challenge, and this often represents the greatest barrier to mapping the genetic dependencies of important biological processes. We propose to develop a general approach that links molecular phenotypes with the targeting guide RNAs that induce those effects in a large, pooled cell population. We label distinct guide RNAs with unique nucleotide barcodes that are expressed in an RNA reporter, linking the RNA abundance of the barcode to intracellular processes of interest. Our system can directly monitor transcriptional, post-transcriptional, and post-translational responses, allowing us to couple it with a wide array of intracellular signals. These expression measurements are well suited for epistasis analysis, which can identify genetic pathways and uncover novel gene functions through correlated patterns of genetic interaction from quantitative phenotypic profiles. We will make available our validated reagents for implementing this screening approach in budding yeast and in mammalian cells, providing a broadly useful resource for high-precision genetic profiling.