Dynamic barcoding. (A) Schematic representation of dynamic barcoding with a poly(A) readout. (B) Schematic representation of the experimental setup. Double-positive cells were defined as GFP⁺ (Cas9) and mCherry⁺ (Barcode) cells and were collected by FACS at all sequenced time points (days 0, 2, 4, 8, 10, and 18). In total, 82 replicate-1 libraries with coverage greater than 200 reads were analyzed (40 Cas9 and 42 BE3; see setion Bulk Barcode Analysis in the Methods). (C) Proportion of barcodes based on their mutational profiles for Cas9 (left) and BE3 (right). Barcodes are grouped into three categories: edits on the protospacer with intact PAM motif (active), absence of PAM motif (inactive), and uncut gRNA (original). Data represent the average across all seven gRNAs. (D) Proportion of original barcodes over time across different gRNAs. Each point corresponds to an individual sample. (E) Barcode mean divergence from the original barcode sequence over time, considering mismatches, gaps, and gap extensions across different gRNAs (on average, 449 different barcode sequences were detected per sample). Each point corresponds to an individual sample. (F) Proportion of original barcodes over time for gRNAs with 21 bp spacers (left) or 26 bp spacers (right). Box plots are colored by the mean spacer length at different time points (Cas9 system). Sample size (left to right): for 21 bp spacers, n = 10, 9, 20, 9, 5, and 8; for 26 bp spacers, n = 4, 2, 5, 3, 3, and 4. (G) Mean percentage of the original nucleotide over time, aligned relative to the PAM sequence in the Cas9 system (n = 39). (H) Fraction of C > T mutations over time, considering all different gRNAs classified by Cas9 versions. Sample size (left to right): n = 7, 7, 5, 6, 11, 14, 7, 5, 4, 4, 6, and 6. For all box plots, the boxes represent the interquartile range (IQR), with the horizontal line inside each box indicating the median.