Extended Data Fig. 8. Dual donor and target reprogramming enables core reprogramming and robust DNA recombination.

a, Schematic representation of core reprogramming with or without RTG extension. The four positions in the guide loops which bind the first base of the core were mutated along with the first base of the core in the target/donor sequences to test if the core sequence can be reprogrammed. The RTG was programmed to allow 7 base binding with the RT or kept as the WT. b, Schematic representation of base-pairing between a target-binding loop with the WT IS621 RTG. c, Schematic representation of base-pairing between a target-binding loop with a reprogrammed and extended RTG. d, Plasmid recombination GFP reporter assay to assess the impact of extended RTG on core programmability. The canonical CT core was mutated to GT, AT, and TT, and tested with the IS621 WT RTG (4 bp) and with an extended RTG (7 bp). MFI ± SD for three biological replicates shown. e, DNA recombination in E. coli with reprogrammed bridge RNAs. The distribution of FITC-A signal for the cell population is shown, with a representative gating strategy for evaluating the percentage of GFP+ cells. D1-D1, D2-D2, etc., represents bridge RNA specificity and provided donor, respectively. Plots are representative of 3 replicates featured in Fig. 5g.