Fig. 4

Retrieval of hygromycin-resistant clones from a heterogeneous population of HeLa cells. a Workflow to identify resistant clones using a sgRNA-barcode library. (Barcoding) A mixed population of hygromycin-resistant and hygromycin-sensitive HeLa cells was transduced with sgRNA-barcodes. (Selection) The resulting library was bottlenecked to limit barcode complexity, re-expanded, and cryo-preserved to define an early time point (ETP). Cells were then treated with either hygromycin or vehicle control (PBS). Hygromycin-enriched barcodes were determined by NGS. b Hygromycin-resistant barcodes were enriched across hygromycin-treated replicates. Barcode abundance for T1 (hygromycin-sensitive barcode candidate), T2 (hygromycin-resistant barcode candidate), and T3 (hygromycin-resistant barcode candidate). The raw barcode read counts are provided as a CSV file in Additional file 5: Table S7-barcode_counts.csv and the raw histograms for barcode counts are available in Supporting Data 2: Barcode histograms [15]. c Dot plot showing the abundance of each selected barcode across replicates treated with either PBS or hygromycin. Note that this experiment was done separately from the actual retrieval experiment; T4 is under-detected limited in this original ETP population. d Workflow to retrieve resistant clones using the frameshift reporter. (Retrieval vector transduction) Hygromycin-sensitive and resistant candidate barcodes were selected for retrieval, and the matching barcode targets were cloned into the retrieval vector. Cells from the ETP were transduced with barcode-specific retrieval vectors and spCas9 expression was induced. (Clone enrichment and isolation) FACS sorting or zeocin selection was used to enrich for barcodes of interest. Single-cell clones were isolated by FACS. (Characterization) Barcode identification and functional validation. The integrated retrieval vector was sequenced to characterize specific and nonspecific mutations leading to reporter activation. e The ETP abundance of each targeted barcode. f Population-level enrichment of targeted barcodes using selection by FACS (TMv2) or Zeocin selection (TMv2-Zeo). g Fraction of single-cell clones with the targeted barcode. h The hygromycin sensitivity of single-cell clones isolated by FACS corresponded to the sensitivity predicted by clonal tracking