Extended Data Fig. 8: Prime editing by separate RNA modules.
From: A split prime editor with untethered reverse transcriptase and circular RNA template
a, Schematic of proof-of-concept experiment on delivering the RT template separately. The 3’ extension of the pegRNA (the RTT-PBS sequence) was removed from the 3’ of the tracrRNA scaffold and provided separately under the control of a U6 promoter. An sgRNA plasmid was co-transfected to carry out the nicking event in conjunction with the nCas9. b, Illustration of the circularization pathway to generate petRNAs. c, PE efficiency by modular RNA components at the FANCF locus introducing a 3-nt substitution (+2 C to T and +4-5 TG to AC). Plasmids expressing RNAs were co-transfected with Cas9H840A and the split RT, which lacks the MCP domain. Nicking sgRNAs were used for all prime editing. d, Validation of petRNA adaptability to an alternative nickase. The petRNA was designed to target a site at the FANCF locus where SpyCas9 and SauCas9 nickases share the same nick and thus a single petRNA guide/primer/template sequence. The petRNA and the MCP-RT were co-transfected with plasmids encoding SpyCas9H840A-sgRNA or SauCas9N580A. Nicking sgRNAs were used for all prime editing. Two-tailed unpaired Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data and error bars indicate the mean and standard deviation of three independent biological replicates.
