Fig. 1

Overview of the strategy for tracking and retrieving the ancestral clones within a heterogeneous population. a Tracking clonal response to selection (e.g., ± drug) using a lentiviral sgRNA-barcode library. Clonal fitness profiles can be estimated from barcode enrichment across replicates within each condition. b Clones of interest may be retrieved from the ancestral (untreated) population using a retrieval vector containing a targeting region matched to the clone sgRNA-barcode. Nuclease activity at the target region activates a fluorescent marker that can be detected with FACS. c Diagram of the frameshift retrieval vector. In cells from the clone of interest, where the sgRNA-barcode and barcode targets are matched, spCas9-mediated cleavage can induce a − 1/+ 2 frameshift, activating reporter expression and inactivating mCherry expression. GFP+/mCherry- cells can be isolated by FACS. Additional reporter genes enable pre-enrichment such as antibiotic selection (e.g., zeocin) or affinity selection (e.g., H2K surface epitope) prior to FACS