Extended Data Fig. 3: epegRNA or RS sequence optimizations to improve editing efficiency.
From: Precise integration of large DNA sequences in plant genomes using PrimeRoot editors
a, Overview of pU3-epegRNA and pGS-epegRNA construct architectures. b, Dual-ePPE editing efficiencies mediated by a pU3 or pGS promoter driving epegRNA expression across five endogenous genomic sites in rice protoplasts as measured using high-throughput sequencing; Values and error bars represent the mean and standard error of mean for three independent biological replicates. c, Dual-ePPE editing efficiencies of varying insertion or deletion sizes mediated by a pU3 or pGS promoter driving epegRNA expression at the OsCDC48 genomic site in rice protoplasts as measured using high-throughput sequencing; Values and error bars represent the mean and standard error of mean for three independent biological replicates; P values were obtained using the two-tailed Student’s t-test: ***P < 0.001, ****P < 0.0001. d, Sanger sequencing traces of dual-ePPE editing at OsCDC48 mediated by a pU3 or pGS promoter to drive epegRNA expression; The red line and arrow represent the point of insertion and insertion direction, respectively. e, Dual-ePPE editing efficiencies to generate larger DNA donor insertions mediated by the pGS promoter driving epegRNA expression at the OsCDC48 genomic site in rice protoplasts as measured using high-throughput sequencing; Values and error bars represent the mean and standard error of mean for three independent biological replicates. f, FRT recombinase site truncation and engineered variants. tFRT1 (tF1) represents a truncated form of FRT1 (F1), * identifies key residues recognized by FLP; the red bases represent mutated residues in each variant. g, Percent GFP positive plant protoplast cells reflective of overall insertion efficiencies as evaluated using the all-in-one reporter and measured using flow cytometry; Each bar represents a unique pair of recombinase sites evaluated using the PrimeRoot; Values and error bars represent the mean and standard error of mean for three independent biological replicates. h, GFP insertion efficiencies at OsALS in rice protoplasts as measured using ddPCR; Each bar represents a unique pair of recombinase sites evaluated using the PrimeRoot; Values and error bars represent the mean and standard error of mean for three independent biological replicates.
