Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.

Interactions between tumor cells and thesurrounding microenvironment contribute to tumor progression, metastasis and recurrence 1-4 . Although mosaic analyses in Drosophila have advanced our understanding of such cellular interactions during tumor initiation 5-8 , parallel approaches have remained challenging to engineer in mammalian systems. Here, we present an oncogene-associated, multicolor reporter mouse model, the Red2Onco system, that allows differential tracing of mutant and wild-type cells in the same tissue. Applied to the small intestine, we show that oncogene-expressing mutant crypts alter the cellular organization of neighboring wild-type crypts, driving accelerated clonal drift. Crypts expressing oncogenic KRAS or PI3K secrete BMP ligands that suppress local stem cell activity, while induced changes in PDGFR lo CD81 + stromal cells by crypts with oncogenic PI3K alter the Wnt signaling environment. Together, these results show how oncogenedriven paracrine remodeling creates a niche environment that is detrimental to the maintenance of wild-type tissue, promoting field transformation dominated by oncogenic clones.