Extended Data Fig. 7: Comparing the editing activities of CGBEs and PEs in multiple human cell lines.

a, Schematic of prime editing (PE) used to install a C-to-G substitution. PE fusion protein consists of an SpCas9-H840A nickase fused to an engineered Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The prime editing guide RNA (pegRNA) consists of a standard targetable SpCas9 gRNA that also harbors a 3’ extension containing a primer binding site (PBS) and a reverse transcription template (RTT) that encodes the desired edit. The PE2 system encompasses the prime editor fusion protein and a pegRNA. The PE3 system additionally includes a nicking gRNA (ngRNA). b, Bar plots showing the on-target DNA prime editing frequencies induced by nCas9(H840A), PE2 and PE3 using a pegRNA that targets FANCF site 1 across four human cancer cell lines. Gray overlay bars at top represent deletions at each nucleotide. Editing frequencies of four independent replicates (n = 4) for HEK293T cells or three independent replicates (n = 3) for K562, U2OS, and HeLa cells at each base are displayed side-by-side. Numbering on the bottom indicates the position of the base with 1 being the first nucleotide 3’ of the pegRNA/Cas9-induced nick. Arrowheads indicate guanines that exhibit desired G-to-T prime edits. c, d, Bar and dot plots representing the average on-target DNA prime editing and indel frequencies of PE2 and PE3 targeting FANCF site 1 for G-to-T prime editing (c, data from the same experiment as b) and HEK site 3 for PE-induced CTT insertion (d) in 4 cell lines. Single dots represent individual replicates (n = 4 for HEK293T and n = 3 for K562, U2OS, and HeLa cells). Error bars represent standard deviation (s.d.). Measure of center for the error bars = mean. e, Bar and dot plots showing the average on-target DNA C-to-G base or prime editing frequencies induced by CGBE1, miniCGBE1, PE2 or PE3 on four genomic target loci. Single dots represent individual replicates (n = 4 for HEK293T and n = 3 for K562, U2OS, and HeLa cells). A two-tailed Student’s t-test with p-values adjusted for multiple testing was used to calculate the shown p-values (p = 0.043 for both). Error bars represent (s.d.). Measure of center for the error bars = mean. f, Bar and dot plots representing the average frequency of alleles with indels (%) induced by pegRNAs and nicking gRNAs used in the experiments shown above (and FANCF site 1 +21 ngRNA control, Supplementary Table 9) with wild-type SpCas9 in HEK293T. pegRNAs/ngRNAs designed by Anzalone et al. (left) and by us (right) are separated by the dashed line. Single dots represent individual replicates (n = 3 independent replicates). Error bars represent (s.d.). Measure of center for the error bars = mean. ND, not done.